Aaron Silva-Sanchez

Troy D Randall

Selene Meza-Perez

Christopher George Mueller

Saba Nayar

David Gardner

Francesca Barone

Christophe Klein

Priyanka Devi-Marulkar

Emma Jones

Awen Gallimore

Ann Ager

Claudia Gutierrez-Chavez

Samantha Knockaert

Marie-Caroline Dieu-Nosjean

Jérémy Goc

Chunyan Gu-Trantien

Soizic Garaud

Edoardo Migliori

Cinzia Solinas

Jean-Nicolas Lodewyckx

Karen Willard

Elisa Corsiero

Lucas Jagemann

Michele Bombardieri

Costantino Pitzalis

Hélène Kaplon

Myriam Lawand

Jean-Luc Teillaud

Lucile Regard

Clémence Martin

Sophie Sibéril

Pierre-Régis Burgel

Anthony B Rodriguez

J David Peske

Victor H Engelhard

Kelli A Connolly

Mursal Nader

Nikhil Joshi

Victor H Engelhard •

Beirne B Carter

vi adapted to the tissue contexture of these structures. Thus, the analysis of B-cell repertoires and the functionality of the corresponding antibodies produced has been performed by generating a large number of recombinant monoclonal antibodies from isolated single B cells (Chapter 10). The development of protocols for the sorting of lymphocyte subpopulations by negative selection or of very rare populations such as follicular-helper T lymphocytes by positive selection will allow the performance of ex vivo functional tests (Chapters 11 and 12).

Finally, Chapters 13, 14, 15, and 16 are entirely dedicated to the development of murine inflammatory models allowing the functional study of TLS in the context of infection or malignancy.

Overall, this book entitled Tertiary Lymphoid Structures: Methods and Protocols is intended to guide our colleagues in the many techniques and methods that can be used to study TLS in pathological situations in patients. Some preclinical models are also depicted in detail in order to show that TLS structure, development, and maintenance can be studied in vivo and targeted. This will undoubtedly increase our knowledge on the immune function and the targeting of these structures paving the way to possible therapeutic applications.

v

The immune system has developed many mechanisms to build up a protective response against pathogens or abnormal cells. Some of them take place in organs (i.e. secondary lymphoid organs, SLO) or structures (such as gut-associated lymphoid tissue (GALT) and mucosa-associated lymphoid tissue (MALT)) that are lifelong and fully dedicated to the elaboration of adaptive immune responses. However, there are also inducible lymphoid structures that develop transiently in response to chronic inflammation. These structures, also known as tertiary lymphoid structures (TLS), exhibit features similar to those of SLO, with the segregation of B, T, and dendritic cells in two distinct B-cell and T-cell rich areas.

TLS are found in upper and even lower vertebrates that emerged more than 500 million years ago, suggesting that the ability to form TLS is anterior to the development of SLO during the evolution of the species. It is intriguing to note that TLS that are transient structures persisted in mammals despite the emergence of SLO and other constitutive lymphoid organizations. The advantage gained in conserving TLS during evolution raises many questions about their role in the setting and maintenance of local immune responses in inflammatory environments. It is through the various chapters that make up this book that different aspects of TLS can be discovered. How they can be studied in preclinical models or in the clinics is also presented.

After an introductory chapter that discusses where TLS stand among all the existing lymphoid organizations (Chapter 1), two chapters (Chapters 2 and 3) detail TLS composition, organization, and location. They also present particular situations such as the presence of TLS in the central nervous system. The next chapters describe original and innovative methods allowing the study of TLS in humans and mice. One of the first questions arising when TLS are studied is how to define a TLS in a pathological context. Are we detecting only lymphocyte aggregates or true TLS? Which marker(s) should be used for TLS identification? What are the criteria that should be taken into account to characterize and quantify TLS for prognostic and predictive studies? Thus, Chapters 4, 5, 6, and 7 describe various methods and tools to detect, image, quantify, and analyze TLS. From single or double immunohistochemical staining performed on serial tissue sections to multiplex staining on a single tissue section, an array of methods that make it possible to investigate TLS structure and function is proposed. Similarly, different strategies are offered to quantify TLS in an automated way using stained tissue sections. In particular, a methodology is proposed to visualize TLS from a number of immunostainings made on serial sections by reconstructing images with an open-source software (Chapter 4).

The investigation of TLS immune functions may require switching from cellular to molecular approaches due to the low number of immune cells that can be isolated from these structures or to study a given area of interest. One method of choice is the use of laser capture microdissection to study functional signatures of TLS from frozen tissues (Chapter 8). The search for TLS presence can be also performed by identifying a gene signature from fixed and paraffin-embedded tissues (Chapter 9).

The study of B and T lymphocyte subsets present in TLS has been made also possible, thanks to the optimization of well-known technologies and the development of protocols

Introduction

Immune responses are initiated in secondary lymphoid organs (SLOs), which collect antigens and cells from surrounding tissues, recruit naïve B and T cells from the blood, and provide an environment that maximizes encounters between antigen-presenting cells and lymphocytes and supports the proliferation and differentiation of effector cells. All SLOs share a similar cellular architecture that includes primary and secondary B-cell follicles organized around a network of follicular dendritic cells (FDCs); a T-cell zone organized around a network of fibroblastic reticular cells (FRCs); macrophages and dendritic cells (DCs) strategically placed to capture antigen and prime T cells; high endothelial venules (HEVs) for lymphocyte extravasation; and lymphatic vessels (except in spleen) for collection of antigens and cells from tissues as well as for the release of cells into the blood [1,2].

SLOs form during embryogenesis at predetermined locations, independently of antigen or inflammation, using a well-defined program of cellular and molecular interactions [3,4]. In contrast, architecturally and functionally similar tissues, known as tertiary lymphoid organs (TLOs) or tertiary lymphoid structures (TLS), form after birth at unpredictable sites, due to persistent local inflammation triggered by infection, autoimmune diseases, malignancy, allograft recognition, and other conditions [5][6][7]. In each of these situations, lymphocytes and other cells that make up TLOs are likely responding to locally produced antigens or autoantigens. In turn, antigen-stimulated lymphocytes provide the signals needed for TLO organogenesis and maintenance. Once antigen is cleared and inflammation resolves, TLOs often disappear [4,8].

The accumulation of lymphocytes and the degree to which they become organized in peripheral, nonlymphoid tissues vary depending on the strength, type, and duration of the inflammatory and antigenic stimuli. As a result, lymphoid aggregates range from loose collections of a few B cells to highly organized tissues with all the hallmarks of TLOs. As a result, investigators have made efforts to classify the spectrum of lymphoid aggregates, under the hypothesis that not all aggregates are functionally equivalent [9][10][11]. Although the minimal attributes needed to form a functional TLO are not known, we will define a TLO as a lymphoid aggregate with differentiated stromal components (FDCs, FRCs, or HEVs) that would not normally reside in that tissue. This type of definition would exclude inflammatory aggregates of B or T cells that lack differentiated stromal compartments.

Development of TLOs

Many of the mechanisms controlling TLO formation are similar to those governing SLO development. Both involve reciprocal interactions between fibroblastic stromal cell types, such as FDCs and FRCs, and lymphocytes such as B cells, T cells, and innate lymphoid cells (ILCs). On one side of this interaction, stromal cells in SLOs and mature TLOs express homeostatic chemokines, such as CCL19, CCL21, CXCL13, and CXCL12 [12], that attract lymphoid and myeloid cells and also express cytokines like RANKL and IL-7 that promote survival and expansion of lymphocytes. On the other side of this interaction, lymphocytes express TNF family members, including lymphotoxin (LT), TNF-α, and LIGHT [4,13,14] that maintain stromal cell differentiation.

Although this model generally applies to both SLOs and TLOs, the details differ considerably. For example, although SLOs typically require specialized ILCs known as lymphoid tissue inducer (LTi) cells to express LT and TNF-α, these same cytokines are provided by B and T cells in the context of TLO formation [15][16][17]. More importantly, SLOs form in the absence of inflammation, whereas the formation of TLOs is triggered by inflammatory cytokines, including IL-1α [18], IL-6 [19], IL-17 [15], and IL-22 [20]. Interestingly, these cytokines are often associated with either the development or the function of Th17 cells, which are often associated with the formation of TLOs in autoimmune, infectious, and inflammatory diseases. Intriguingly, many of these same cytokines are involved in the function of the SLO-inducing LTi cells, suggesting that they may have overlapping functions. Although Th17 cells and IL-17 are often associated with the formation of TLOs, there are additional inflammatory pathways that trigger TLO formation. Regardless of the inflammatory stimuli that trigger TLO formation, the end result still requires LT-triggered stromal cell differentiation and homeostatic chemokine production.

Despite some commonalities in their development, TLOs form under a wide variety of conditions and cytokine milieus that depend on the initiating stimulus (infection, autoimmunity, transplantation, malignancy) and the tissue in which the TLO is forming. Consequently, it is likely that TLOs forming in response to diverse stimuli in different tissues will have unique characteristics that may impart distinctive attributes to immune responses occurring in those TLOs. Thus, it will be important for future studies to identify both the shared and unique features of individual TLO-associated diseases, in order to understand disease progression and to develop therapies that specifically target TLO formation or function.

Role of TLOs in Pathogenic Immune Responses

TLOs are often observed in tissues damaged by autoimmune responses. For example, TLOs are observed in the inflamed synovium of patients with rheumatoid arthritis [21][22][23][24][25], the salivary and lacrimal glands of patients with Sjögren's syndrome [26][27][28], the brains and spinal cords of patients with multiple sclerosis [29][30][START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF][32][33][34][START_REF] Szentistványi | Drainage of interstitial fluid from different regions of rat brain[END_REF], the kidneys of patients with lupus nephritis [START_REF] Weller | Lymphatic drainage of the brain and the pathophysiology of neurological disease[END_REF], the pancreata of patients with diabetes [START_REF] Yamada | Albumin outflow into deep cervical lymph from different regions of rabbit brain[END_REF][START_REF] Clarkson | CCR7 deficient inflammatory dendritic cells are retained in the central nervous system[END_REF][START_REF] Goldmann | T cells traffic from brain to cervical lymph nodes via the cribroid plate and the nasal mucosa[END_REF], and the thymus of patients with myasthenia gravis [START_REF] Hatterer | How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicles of cervical lymph nodes[END_REF][START_REF] Kaminski | Migration of monocytes after intracerebral injection at entorhinal cortex lesion site[END_REF]. In many cases, increased severity of disease is associated with more or larger TLOs in the affected tissue [START_REF] Mohammad | Immune cell trafficking from the brain maintains CNS immune tolerance[END_REF][START_REF] Shechter | Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates[END_REF]. As a result, many investigators conclude that TLOs exacerbate local immune reactivity and contribute to pathogenesis.

Consistent with the idea that TLOs exacerbate local autoimmune-mediated inflammation and tissue damage, TLOs in target tissues often contain B and T cells responding to local autoantigens [START_REF] Silver | Mast cells in the brain: evidence and functional significance[END_REF]. For example, B cells in TLOs from the synovium or lungs of rheumatoid arthritis patients often produce antibodies that bind citrullinated histones [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF][START_REF] Goldmann | Origin, fate and dynamics of macrophages at CNS interfaces[END_REF], which are target antigens Tertiary Lymphoid Structures in the Lung in this disease. Similarly, B cells in TLOs from patients with rheumatoid arthritis [START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF][START_REF] Korin | High-dimensional, single-cell characterization of the brain's immune compartment[END_REF], Sjögren's syndrome [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF], and lupus nephritis [START_REF] Silver | Mast cells in the brain: evidence and functional significance[END_REF] have evidence of clonal expansion and somatic hypermutation, suggesting that they are actively responding to local antigens.

In many cases, the formation of TLOs in the context of autoimmunity is associated with the presence of Th17 cells. For example, patients with lupus nephritis, rheumatoid arthritis, diabetes, and multiple sclerosis commonly have Th17 cells in TLOs that form in target tissues [15,[START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF]32,[START_REF] Radjavi | Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice[END_REF][START_REF] Kipnis | Multifaceted interactions between adaptive immunity and the central nervous system[END_REF][52][53][54]. Unfortunately, it is difficult to dissociate cause from consequence in snapshots of human disease. As a result, it is not entirely clear whether these Th17 cells are produced by TLOs under these conditions, or alternatively whether TLOs are forming in response to the activities of Th17 cells that were produced elsewhere [32, 55,56]. Nevertheless, the role of IL-17 in the formation of TLOs is well documented in mouse studies [15,32,57,58], suggesting that IL-17 or its downstream mediators may be important therapeutic targets for pathogenic immune responses that involve TLOs.

The role of IL-17 in the formation of TLOs is complex. Although Th17 cells are the primary producers of IL-17 in many autoimmune diseases, IL-17 is also made by γδT cells, ILC3 cells, LTi cells, and some Tfh cells, all of which may contribute to TLO formation under various conditions. These same cells make other cytokines, such as IL-22, TNF-α, and LT, which also contribute to TLO formation in both autoimmunity and infection [20,59,60]. Importantly, IL-17 can directly trigger fibroblastic cells to express CXCL13 and CCL19, which attract B cells, T cells, and DCs and are required for the formation of B-cell follicles and T-cell zones, respectively [15,58]. IL-17 also promotes the expression of CXCL8, CXCL9, and CXCL10, inflammatory chemokines that potently recruit neutrophils [61]. In fact, neutrophils are instrumental in the formation of some TLOs [START_REF] Foo | Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation[END_REF], possibly by providing B-cell-activating cytokines, such as APRIL [START_REF] Mohr | Dendritic cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature[END_REF], or because they release serine proteases that damage local tissues and trigger repair processes [START_REF] Solleti | Serpine2 deficiency results in lung lymphocyte accumulation and bronchusassociated lymphoid tissue formation[END_REF].

Although IL-17 is important for initiating TLO formation, the reciprocal interactions between stroma-derived homeostatic cytokines/chemokines and LT-producing lymphocytes and DCs are required to maintain the structure of TLOs. In fact, the blockade of IL-17 has little effect on established TLOs, whereas the blockade of LT causes their dissolution [15]. In the context of autoimmunity or other chronic inflammatory conditions, IL-17-driven inflammation and LT-driven homeostasis most likely occur simultaneously, making therapeutic intervention more difficult.

TLOs and Protective Immunity

Despite the fact that TLOs are often associated with pathogenic immune responses, the mere presence of a TLO in a particular organ does not promote autoimmune or inflammatory responses. For example, the Ruddle lab generated mice that expressed TNF-α or LT under the control of the rat insulin promoter, with the idea that these cytokines would promote inflammation in the pancreas and lead to the development of diabetes. In fact, the transgenic mice did develop inflammatory infiltrates in the pancreas consisting of B cells, CD4 + and CD8 + T cells, as well as ICAM + VCAM + stromal cells, but surprisingly did not develop diabetes [START_REF] Picarella | Transgenic tumor necrosis factor (TNF)-alpha production in pancreatic islets leads to insulitis, not diabetes. Distinct patterns of inflammation in TNF-alpha and TNF-beta transgenic mice[END_REF][START_REF] Picarella | Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas[END_REF][START_REF] Flavell | The contribution of insulitis to diabetes development in tumor necrosis factor transgenic mice[END_REF]. Subsequent studies defined these lymphoid infiltrates as TLOs, complete with B-cell follicles with FDCs and T-cell areas with HEVs [START_REF] Clarkson | CCR7 deficient inflammatory dendritic cells are retained in the central nervous system[END_REF][START_REF] Goldmann | T cells traffic from brain to cervical lymph nodes via the cribroid plate and the nasal mucosa[END_REF]60,68].

In fact, the tissue-specific transgenic expression of a variety of cytokines or chemokines leads to the spontaneous development of TLOs-each revealing a new level of complexity to the mechanisms that promote TLO formation. For example, using the same rat insulin promoter to drive transgene expression, multiple investigators show that LT [START_REF] Mounzer | Lymphotoxin-alpha contributes to lymphangiogenesis[END_REF], TNF-α [START_REF] Zhang | Increased lymphangiogenesis in joints of mice with inflammatory arthritis[END_REF]59], LIGHT [60], CXCL13 [START_REF] Ansel | A chemokine-driven positive feedback loop organizes lymphoid follicles[END_REF], and CCL21, CCL19, and CXCL12 [START_REF] Luther | Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis[END_REF][START_REF] Cyster | Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs[END_REF] independently promote the formation of local TLOs in the pancreas. Moreover, the transgenic expression of these same cytokines or chemokines in other tissues also triggers TLO formation in those tissues [START_REF] Marinkovic | Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid[END_REF][START_REF] Marchesi | CXCL13 expression in the gut promotes accumulation of IL-22-producing lymphoid tissue-inducer cells, and formation of isolated lymphoid follicles[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF][START_REF] Chen | IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology[END_REF]. Each cytokine or chemokine generated TLOs with slightly different features as a consequence of what cell types that particular molecule activated or attracted. Although tissue-specific expression of cytokines and chemokines is associated with TLO development at that site, the systemic expression of some cytokines, such as IL-7, can trigger the formation of TLOs (and even additional SLOs) in multiple tissues due to the expansion and persistence of LTi cells [START_REF] Meier | Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells[END_REF]. Thus, a variety of cytokines impact TLO formation either directly or indirectly without triggering autoimmunity.

Although the examples above demonstrate that TLOs are not always associated with pathogenesis, there are also cases in which TLOs are distinctly protective, particularly in the context of infection. For example, TLOs form in the lungs following infection with a variety of viruses or microbes [57,58,[START_REF] Baluk | Preferential lymphatic growth in bronchusassociated lymphoid tissue in sustained lung inflammation[END_REF][START_REF] Gaur | Bronchus-associated lymphoid tissue lymphoma arising in a patient with bronchiectasis and chronic Mycobacterium avium infection[END_REF][START_REF] Khader | IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung[END_REF][START_REF] Kaushal | Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis[END_REF][START_REF] Kahnert | Mycobacterium tuberculosis triggers formation of lymphoid structure in murine lungs[END_REF][START_REF] Geurtsvankessel | Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice[END_REF][START_REF] Kocks | Chemokine receptor CCR7 contributes to a rapid and efficient clearance of lytic murine gamma-herpes virus 68 from the lung, whereas bronchus-associated lymphoid tissue harbors virus during latency[END_REF]. Interestingly, mice lacking all SLOs, but retaining TLOs in the lung, are actually more resistant to influenza infection than conventional mice [13]. These mice make normal primary B-and T-cell responses, generate long-lived antibody-secreting cells, maintain memory T cells, and are resistant to secondary challenge infections [13]. Similarly, memory CD8 + T cells accumulate in the lung after infection in TLO-like structures termed repair-associated memory depots (RAMDs) [START_REF] Takamura | Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance[END_REF]. These sites are similar to nodular inflammatory Tertiary Lymphoid Structures in the Lung foci (NIFs), which develop in mice infected with murine cytomegalovirus (MCMV) [11]. Myeloid cells accumulate in these sites, differentiate into antigen-presenting DCs, and promote CD8 + T-cell responses against infection. Thus, TLOs can provide effective primary immunity and maintain memory cells, independently of conventional SLOs.

The role of TLOs in controlling pulmonary infection with Mycobacterium tuberculosis (Mtb) is particularly instructive [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF][START_REF] Khader | IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung[END_REF][START_REF] Kahnert | Mycobacterium tuberculosis triggers formation of lymphoid structure in murine lungs[END_REF][START_REF] Nakayama | Vaccination against H9N2 avian influenza virus reduces bronchus-associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection[END_REF]. For example, numerous well-organized TLOs are found in the lungs of primates with well-controlled, latent tuberculosis, whereas fewer and less organized TLOs are observed in the lungs of primates with active disease [START_REF] Slight | CXCR5(+) T helper cells mediate protective immunity against tuberculosis[END_REF]. In fact, TLOs are an important component of the granuloma structure that helps contain Mtb, in part because stromal cells in TLOs recruit CCR7-and CXCR5-expressing T cells [START_REF] Kahnert | Mycobacterium tuberculosis triggers formation of lymphoid structure in murine lungs[END_REF][START_REF] Khader | Th17 cells at the crossroads of innate and adaptive immunity against infectious diseases at the mucosa[END_REF], which are necessary to activate pulmonary macrophages that engulf and kill Mtb.

Similar to the formation of TLOs in autoimmune diseases, the formation of TLOs in response to infection with Mtb requires the IL-23/IL-17 axis as well as the LT signaling pathway [START_REF] Shen | Selective destruction of interleukin 23-induced expansion of a major antigen-specific gammadelta T-cell subset in patients with tuberculosis[END_REF][START_REF] Shen | The crucial roles of Th17-related cytokines/signal pathways in M. tuberculosis infection[END_REF][START_REF] Quesniaux | TNF in host resistance to tuberculosis infection[END_REF][START_REF] Day | Secondary lymphoid organs are dispensable for the development of T-cell-mediated S1P receptor 1[END_REF][94]. Mtb-infected mice lacking IL-17A, IL-22, or IL-23 develop smaller and fewer TLOs and are less able to restrain bacterial growth [START_REF] Khader | IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung[END_REF]. Although IL-17 works by triggering chemokine expression and thereby calling in inflammatory cells, IL-22 seems to be more important for the formation of B-cell follicles [15,20], perhaps due to expansion of CXCL13-expressing FDC networks in TLOs. These data demonstrate that the formation of TLOs, even when triggered by IL-17, can be protective in the context of infection.

Regulatory Functions of TLOs

One might conclude that TLOs simply amplify local immune responses. In the case of autoimmunity or other inflammatory responses, augmented immunity would exacerbate inflammation, whereas in the case of infection augmented immunity would facilitate pathogen clearance and accelerate the healing process. In addition to these two scenarios, some data suggest that TLOs can mediate protective functions via regulatory functions. For example, in the ApoE -/-model of atherosclerosis, TLOs form beneath atherosclerotic plaques [95]. Although atherosclerosis is considered an inflammatory disease, these TLOs appear to temper inflammation by promoting the differentiation of regulatory T cells (Tregs). In the absence of TLOs (eliminated by conditional knockout of the LTβR), ApoE -/-mice developed more and larger plaques [95]. Thus, some aspect of TLO function, perhaps Treg generation, is immunoprotective, even in the context of inflammatory diseases.

What might confer regulatory properties on TLOs? One possibility is the activities of fibroblastic stromal cells that make up the scaffolding of TLOs. In addition to their organizational properties, FRCs in conventional SLOs can present self-antigens and promote tolerance rather than immune activation [96,97]. This activity is achieved in part by promoting Treg differentiation [98,99], and by expressing inducible nitric oxide synthase [100-102], which suppresses T-cell proliferation via the production of nitric oxide [100,103]. Although, TLOs appear to have FDCs and FRCs like conventional SLOs, it is not clear where these cells come from or whether they have additional or different functions that may influence local immune reactions.

The example of Mtb infection highlights another potential role of TLOs-containment. Granulomas not only support the activation of Mtb-killing macrophages, but they also physically contain the infection and prevent dissemination. In a similar way, TLOs may be collecting and sequestering antigens, as well as activated effector T cells, and thereby preventing them from disseminating to target tissues and causing further damage. For example, TLOs in the lungs of asthmatics may collect and sequester Th2 cells and limit their ability to promote airway hyper-responsiveness. The idea seems plausible, as treatment with the S1P agonist, FTY720 (a drug that downregulates the S1PR1 and prevents lymphocyte egress from SLOs [104]), blocks airway hypersensitivity in mice [105,106]. Moreover, FTY720 treatment promotes TLO development [107], in part by preventing the recirculation of lymphocytes. In fact, FTY720 (fingolimod) is now used clinically to treat autoimmune diseases like multiple sclerosis [108], which is characterized by the development of TLOs in the CNS. Thus, the ability of TLOs to sequester antigens and potentially damaging effector cells may by one way in which they attenuate disease progression.

Importantly, the ability of TLOs to sequester antigens or cells is dependent on their ability to collect those cells. Conventional SLOs have well-described methods for antigen collection. For example, lymph nodes collect antigens and cells from regional tissues via lymphatic vessels [109-112], mucosal tissues like Peyer's patches collect antigens across a specialized epithelium [3,5], and fat-associated lymphoid clusters (FALCs) in the mesentery and pericardium and the milky spots of the omentum collect antigens from body cavities [5,[113][114][115]. However, it is less clear how TLOs, which are often embedded in solid tissues, might collect antigens.

In many cases, TLOs form in response to autoantigens or alloantigens that are expressed in the tissues in which TLOs are formed, so there is no need to collect antigen from distal sites. In some cases however, the formation of TLOs is linked with the local development of lymphatic vessels. For example, pulmonary infection promotes the VEGF-dependent growth of lymphatic vessels Tertiary Lymphoid Structures in the Lung surrounding TLOs in the lung [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF][START_REF] Baluk | Preferential lymphatic growth in bronchusassociated lymphoid tissue in sustained lung inflammation[END_REF]. However, it is unclear whether these vessels are afferent lymphatics that bring antigens and cells to the TLOs or are efferent lymphatics that drain to downstream lymph nodes. Understanding how TLOs are connected to the lymphatic vasculature will be important for understanding how TLOs function in many settings.

6 What Controls Whether TLOs Promote or Prevent Immune Activation?

Much like chronic infections, tumors and transplanted organs persistently stimulate the immune system, leading to sustained inflammation. Not surprisingly, TLOs are often observed in these situations [116] and the mechanisms underlying their formation and function are clinically important [117]. For example, in some cases, the formation of TLOs in lung transplants is associated with sustained alloreactivity and chronic graft rejection [118,119]. In fact, the presence of TLOs in allografts is primarily associated with local lymphocyte activation and increased rejection [120], probably via many of the mechanisms discussed above. However, in other cases, the formation of TLOs is associated with acceptance of allografts [121], possibly by priming Tregs that help mediate tolerance [121]. Similarly, the transfer of lymphocytes from accepted transplants into new transplant recipients leads to allograft acceptance [122], due to the expansion of alloantigen-specific Tregs in local TLOs [123]. Thus, it is important to understand the features of TLOs that distinguish whether they promote T-cell activation and rejection or Treg expansion and tolerance.

Although tolerance is a desired outcome in transplantation, the opposite is true in the case of cancer. TLOs are often observed in or near areas of non-small cell lung cancer (NSCLC) [124][125][126]. Not surprisingly, these areas are associated with the expression of homeostatic chemokines linked to TLO formation and T-cell recruitment. Consistent with this idea, tumor-associated TLO areas have PNAd + HEVs that co-localize with CD62L + T cells, suggesting that they can recruit T cells from the blood [START_REF] Fava | Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjogren's syndrome[END_REF]. Although the formation of TLOs in tumors is sometimes prognostic of favorable outcomes to treatment [128], tumor-associated TLOs sometimes exhibit an immunosuppressive phenotype, characterized by a high density of Tregs and absence of NK cells [129,130]. This functional discrepancy may be partly due to their location, as TLOs within pancreatic tumors are correlated with favorable outcomes, whereas TLOs at the periphery of tumors are not [129]. Unfortunately, our understanding of these observations is incomplete and we do not know why tumor-associated TLOs might form in one location or another, whether their location changes the phenotype of responding T cells, or whether they are Aaron Silva-Sanchez et al.

differentially supplying T cells to the tumor or sequestering them. Nevertheless, these results dramatically illustrate the importance of TLOs in both protective immunity and pathogenesis and highlight our gaps in knowledge that need to be filled in order to therapeutically target the activities of TLOs in the context of inflammation, infection, and malignancy.

Conclusion

In summary, we now know that TLOs are formed by a wide variety of stimuli under diverse conditions and, depending on factors that we do not entirely understand, can either exacerbate or restrain inflammation and local immunity. Enhanced immunity is clearly beneficial for clearing infections or tumors, but is also detrimental in the context of autoimmunity and transplantation. As a result, the details of how TLOs form and function under different conditions are critically important, as they will undoubtedly make the difference between transplant rejection or tolerance, tumor killing or progression, and pathogen clearance or chronic infection. In our view, some of the important unanswered questions are these: What are the origins of the fibroblastic stromal cells in TLOs? Are they developmentally or functionally different in TLOs that form in different tissues or in different locations in the same tissue? What controls whether TLOs promote T-cell activation or Treg expansion, thereby leading to opposite clinical outcomes? An understanding of the answers to these questions will almost certainly lead to methods allowing us to control the activity of TLOs and promote desired clinical outcomes, regardless of the type of immune response.

Introduction

The ectopic process that results in the close interaction between leukocytes and stromal cells in the context of inflammation results in the formation of anatomical structures defined as ectopic or tertiary lymphoid structures (TLS). TLS form to satisfy the local requirement for lymphocyte maintenance within an inflamed tissue. As such, these structures develop in organs not previously predisposed to support the independent generation of an immune response, making the process of lympho-neogenesis the progressive acquisition of features able to support basic lymphocyte functions, such as antigen presentation, co-stimulation, survival, affinity maturation, and exit [1]. As such TLS are best defined as the organoid assembly of cells of the adaptive immune system (B and T lymphocytes and by dendritic cells) in nonimmune tissue; they comprise one or more B-cell follicles that may cluster around fibroblastic stromal cells that share features of follicular dendritic cells (FDC) [2]. TLS are also characterized by T cells that, along with interdigitating mature dendritic cells (DCs), are placed around fibroblastic reticular cells (FRCs) [3]. High endothelial venules (HEVs), flat vessels, and lymphatic vessels (LVs) can also be found within TLS, but fail to acquire the complex organization of SLOs [4,5].

Molecular Cues Regulating TLS Assembly, the TNFRS

It is now clear that the process of ectopic TLS formation does not fully recapitulate the embryological development of SLOs and while the same molecules might be expressed at some stage during TLS establishment the sequence of events that regulate SLO formation may not be quite the same in TLS [1].

The tumor necrosis factor superfamily (TNFSF) members TNFα, lymphotoxin (LT) α and β, and their signaling receptors TNFRI/II and LTβR were suspected to promote the formation of TLS when their critical role in SLO development emerged. Seminal work by Dr. Ruddle and her group showed that ectopic expression of TNFα or LTα, but not LTβ, under the control of rat insulin promoter led to the formation of TLS [6,7]. Stronger effects in mesenchyme maturation and leucocyte recruitment were observed when LTα and LTβ were co-expressed, resulting in an invasive leukocyte accumulation of the pancreatic islets and significantly larger TLS than in LTα transgenic mice [7]. Differentiation of specialized vascular structures with high endothelium and expression of peripheral node addressin (PNAd), ligand for L-selectin, were also observed in this model providing the molecular mechanisms for naïve T-cell and B-cell recruitment at these sites [7]. Of the two TNFRs, the TNFRI, the principal mediator of lymphoid tissue organogenesis and germinal center reaction [8], plays the major role in mediating LTα-induced pancreatic TLS [9]. Investigators have more recently demonstrated that LTα expression in tumor cells leads to the formation of intra-tumoral lymphoid tissue able to sustain an efficient immune response [10].

The inclusion of inducible bronchial-associated lymphoid tissue (iBALT), fat-associated lymphoid clusters (FALC), and nasalassociated lymphoid tissue (NALT) in the TLS category is debated [11]. Similarly to classical TLS, iBALT, tear duct-associated lymphoid tissues, and NALTs appear to develop independently of LTαβ and LTβR [12][13][14][15]. However, LT signaling is crucial for the maintenance and organization of these structures; accordingly, iNALT are disrupted in LTα -/-mice [12]. Interestingly, the expan-sion of the lymphatic network in iBALT appears to be directly dependent on LTβR signaling [16].

While an effect of LTα, alone or with LTβ, appears manifest, the role of TNFα is conflicting. In some inflammatory diseases, including those with TLS, TNFα actually exhibits anti-inflammatory activities [17]. For instance, insulitis in NOD mice and lupus in New Zealand lupus-prone mice are improved after injection of TNFα [18,19]. Nevertheless, the activation of both LTβR and TNFRI has been implicated in the formation of aortic TLS (ATLS) that form in association with atherosclerotic lesions within the adventitia of apolipoprotein-deficient (apoE -/-) mice. Here, interruption of the LTβR signaling has been demonstrated to suppress lymphoid chemokine (CXCL13 and CCL21) expression and to reduce HEV formation, thereby disrupting the anatomical structure and maintenance of the TLS [20,21]. In NOD mice, pancreatic TLS show local upregulation of LTαβ and LIGHT, an alternative LTβR ligand [22,23]. Similarly, in a model of TLS formation in the meningeal tissue, LTβ is expressed and important for TLS establishment [24].

LIGHT is an alternative ligand for LTβR and its transgenic overexpression drives TLS formation in animal models of melanoma and fibrosarcoma [10,25]. In the TLS of NOD mice there was a local upregulation of LTαβ and LIGHT [26]. Pancreatic LIGHT overexpression in NOD mice also exacerbates local inflammation and disease development [27].

Cellular Requirements for TLS Establishment, ILC3

SLO development depends on the interaction between hematopoietic lymphoid tissue inducer (LTi) cells and lymphoid tissue organizers (LTo) of mesenchymal origin [28]. Recently, in addition, the importance of LTi cell interaction with RANK-activated lymphatic endothelial cells has been highlighted [29]. LTi cells are the major producer of LTαβ in response to IL-7, TNFα, and RANKL [30] and their attraction into the lymph node (LN) anlagen in sufficient numbers for complete organogenesis is regulated by chemoattractive signaling mediated by CXCL13 and CCL21 [START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF]. Their assembly is secured by the reciprocal interaction of integrins with MAdCAM-1 and VCAM-1 that are upregulated on the vascular endothelium and the mesenchyme of the anlagen [28].

The requirement for innate lymphoid cell type 3 (ILC3) for TLS formation is debated. ILC3 cells have been suggested to support isolated lymphoid follicle (ILF) formation, via IL-22 production. However, the extent to which the structures can be considered TLS is controversial [11]. Indeed, there is evidence from different animal models that ILCs, including LTi/ILC3 cells, are not essential for the formation of ectopic lymphoid aggregates. In a model of thyroid CCL21 overexpression, TLS formation occurs in the absence of transcription factor Id2 that is required for LTi/ILC3 cell maturation [11]. IL-17-mediated iBALT formation also appears to be independent on LTi cells [12].

Proinflammatory Cells and Cytokines

It is now accepted that in inflammatory conditions, the signals required for TLS establishment and maturation can be provided by a variety of proinflammatory immune cells that reprise the role of LTi cells during SLO formation [13]. B cells and T cells can provide an alternative source of LTαβ when appropriately stimulated, and a similar role can be played by dendritic cells, both in terms of TLS formation and maintenance [1,11]. Myeloid CD68 + cells have also been demonstrated to be able to produce lymphoid chemokines such as CXCL13 or CXCL12 [32].

Intriguingly, the signals delivered by these inflammatory cells exceed the classical lymphotoxin/TNFR activation pathway. For instance, the ubiquitous transgenic co-expression of IL-6 and IL-6R leads to perivascular accumulation of lymphocytes with a substantial proportion of B cells and mature plasma B cells [33]. Additionally, overexpression of IL-5 in the respiratory epithelium also results in the development of organized iBALT. However, unlike the models of homeostatic chemokines or TNF-family ligands mentioned above, which do not necessarily develop associated pathology despite TLS formation, the IL-5-dependent induction of iBALT leads to epithelial hypertrophy, goblet cell hyperplasia, accumulation of eosinophils in the airway lumen and peribronchial areas, and focal collagen deposition, which are all signs of severe lung pathology [34].

IL-17 has emerged as an important mediator or iBALT induced by lipopolysaccharides [12]. IL-17 elicits inflammatory and homeostatic chemokine production in the absence of LTα and LTβ, including the aberrant production of CXCL12 [START_REF] Szentistványi | Drainage of interstitial fluid from different regions of rat brain[END_REF]. Using a T-cell transgenic animal model of experimental autoimmune encephalomyelitis (EAE) that mimics multiple sclerosis, Peters and colleagues demonstrated that T cells expressing IL-17 can support the formation of ectopic lymphoid tissues in the central nervous system (CNS) [START_REF] Weller | Lymphatic drainage of the brain and the pathophysiology of neurological disease[END_REF]. BALTs induced by Mycobacterium tuberculosis are also dependent on IL-17 [START_REF] Yamada | Albumin outflow into deep cervical lymph from different regions of rabbit brain[END_REF].

Studies of human SS have detected IL-22 mRNA in the affected salivary glands [START_REF] Clarkson | CCR7 deficient inflammatory dendritic cells are retained in the central nervous system[END_REF] and serum levels of IL-22 have been shown to correlate with clinical manifestations of the disease, including hypergammaglobulinemia and autoantibody production [START_REF] Goldmann | T cells traffic from brain to cervical lymph nodes via the cribroid plate and the nasal mucosa[END_REF]. In a mouse model of viral induced SS, inhibition of IL-22 but not of IL-17 has been shown to strongly reduce TLS size [START_REF] Hatterer | How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicles of cervical lymph nodes[END_REF].

Lymphoid Chemokines and Survival Factors

Ectopic lympho-neogenesis has been typically associated with the aberrant expression of lymphoid chemokines, classically implicated in the compartmentalization of adult SLO. CXCL13 is expressed by fibroblastic stromal cells in SLO. It is required for the embryonic migration of LTi cells in the anlagen and, in postnatal life, for the compartmentalization of the lymphocytes in the follicles. Overexpression of CXCL13 under the rat insulin promoter (RIP) in the pancreas and kidneys [START_REF] Kaminski | Migration of monocytes after intracerebral injection at entorhinal cortex lesion site[END_REF] leads to TLS characterized by segregated B/T-cell zones, presence of conventional DCs, and a dense network of stromal cells and HEV-type blood vessels (Fig. 1) [START_REF] Mohammad | Immune cell trafficking from the brain maintains CNS immune tolerance[END_REF]. Similarly, increased expression of CXCL13 and B-cell infiltration has been observed in the central nervous tissue of mice in experimental autoimmune encephalomyelitis (EAE) [START_REF] Shechter | Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates[END_REF] and, among other chemokines (notably CXCL10), CXCL13 has been found in the spontaneous mouse model of autoimmune gastritis [START_REF] Silver | Mast cells in the brain: evidence and functional significance[END_REF].

The expression of several other chemokines has been described in TLS. For instance, spontaneous TLS formation in the pancreas of diabetic NOD mice is associated with local upregulation of CXCL13, CXCL12, and CCL19 concomitant with FDC formation and B-cell activation [26]. ATLS that form in the aortic adventitia display expression of CXCL13 and CCL21 [20].

CXCL12 (or stromal cell-derived factor 1, SDF1) is critical in bone marrow hematopoiesis and B-cell development, where it is expressed by bone marrow stromal cells [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF]. CXCL12 is displayed by HEVs in SLO and acts as an important B-cell recruiting factor [START_REF] Goldmann | Origin, fate and dynamics of macrophages at CNS interfaces[END_REF]. RIP-CXCL12 transgenic mice present small infiltrates comprising few T cells but enriched in DCs, B cells, and plasma cells [START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF]. Significant upregulation of CXCL12 is observed in TLS associated with lymphoma development in the salivary glands of patients with Sjögren's syndrome [32].

CCL19 and CCL21, expressed by endothelial cells and some stromal cells, are ligands for CCR7 carried by T cells, dendritic cells, and LTi cells. CCL21 is more effective than CCL19 in forming ectopic lymphoid structures [START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF][START_REF] Korin | High-dimensional, single-cell characterization of the brain's immune compartment[END_REF]; however they lack B-cell follicles [START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF]. Ectopic expression of CCL21 in the thyroid gland was sufficient to induce TLS formation that resembled the structures seen in Hashimoto's thyroiditis and Graves' disease [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF] and both chemokines were detected in RA and SS [5,[START_REF] Radjavi | Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice[END_REF].

In human pathology, CXCL13, CCL21, and CXCL12 were found in Sjögren's syndrome tissue [32, [START_REF] Kipnis | Multifaceted interactions between adaptive immunity and the central nervous system[END_REF]52], Hashimoto's thyroiditis [53], rheumatoid arthritis [5], and non-small cell lung cancer (NSCLC) [54]. While different patterns of lymphoid arrangements usually coexist, inflamed tissues harboring highly organized TLS tend to express significantly higher levels of LTα, CXCL13, and CCL21 than those with diffuse lymphoid infiltrates [5,[START_REF] Radjavi | Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice[END_REF][55][56][57]. In fact, the expression levels of CXCL13 and LTβ may be highly predictive of the presence of ectopic germinal centers in synovial biopsies of patients with RA and SS [5,[START_REF] Radjavi | Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice[END_REF][55][56][57][58].

One of the primary functions of TLS is to support the survival of incoming lymphocytes. Accordingly, ectopic expression of Band T-cell survival factors has been found in TLS. B-cell-activating factor (BAFF) regulates B-cell survival and has been found to be overproduced in meninges-associated ectopic GC formation in a mouse model of CNS inflammation [START_REF] Shechter | Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates[END_REF]. Similarly, LTα-dependent IL-7 overexpression leads to ectopic lymphoid structures in different nonlymphoid tissues [59] and has been attributed to increased local survival of lymphocytes and disease persistence.

Non-hematopoietic Compartments of TLS

The organizational program that regulates the maturation of the embryonic LTo into adult lymph nodes is tightly regulated and gives rise, in postembryonic life, to a distinct and well-organized anatomical structure. This organization is maintained by mature fibroblastic reticular cells (FRCs) in the T-cell zone, marginal reticular cells (MRCs) of the marginal zone, and FDCs of the B-cell follicle. Together, these stromal cell populations regulate organ compart-

Mesenchymal Cells

Fig. 1 Four-stage process of TLS formation. Inflammatory cytokines prime endothelium, tissue-resident fibroblasts, or possibly other structural cells (i.e. pericytes, epithelial cells) to express chemokines and adhesion molecules leading to the recruitment of circulatory or tissue-resident hematopoietic cells. Adhesion molecules subsequently anchor the cells to fibroblasts and/or endothelial cells. During the maturation phase, cytokines such as lymphotoxin and TNF-α foster fibroblast differentiation into lymphoid tissue organizers that more specifically and more efficiently cluster B cells and T cells. Innate lymphoid cells (ILCs) are thought to play an important role by the production of lymphotoxin α and β, but possibly also through Th-17. Similar to SLOs, these structures support B-and T-cell activation leading to TLS expansion through cell proliferation and recruitment. These chronic inflammatory lymphoid structures can then cause severe immunopathology mentalization, cell mobility, and distribution of cells and small molecules [60]. The modification of local stromal cells into functional lymphoid tissue fibroblasts together with the mostly segregated expression of lymphoid chemokines (CCL19, CCL21, CXCL12, and CXCL13) and survival factors (IL-7, BAFF, and APRIL) define most TLS [11].

Once fully matured, TLS can display a specialized network of FDCs capable of driving a functional GC response and differentiation of HEVs [61]. FDCs predominantly form in aggregates where B-cell follicles and/or a germinal center are observed [START_REF] Foo | Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation[END_REF]. FRCs, commonly characterized by the expression of gp38 and the production of CCL21, are generally found within the T-cell area of the TLS in primary biliary cirrhosis, RA, and other conditions [3,11,[START_REF] Radjavi | Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice[END_REF][START_REF] Mohr | Dendritic cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature[END_REF][START_REF] Solleti | Serpine2 deficiency results in lung lymphocyte accumulation and bronchusassociated lymphoid tissue formation[END_REF].

Different expression of chemokines and stromal cell markers has been reported in diverse TLS models [START_REF] Szentistványi | Drainage of interstitial fluid from different regions of rat brain[END_REF][START_REF] Hatterer | How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicles of cervical lymph nodes[END_REF][START_REF] Picarella | Transgenic tumor necrosis factor (TNF)-alpha production in pancreatic islets leads to insulitis, not diabetes. Distinct patterns of inflammation in TNF-alpha and TNF-beta transgenic mice[END_REF][START_REF] Picarella | Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas[END_REF][START_REF] Flavell | The contribution of insulitis to diabetes development in tumor necrosis factor transgenic mice[END_REF], suggesting that the production of different chemokines can be elicited in response to diverse stimuli dependent on the locations.

Although epithelial cells can transform into mesenchymal cells, there is so far little evidence of this occurring during TLS formation. In a model of skin inflammation (not characterized by full TLS maturation), stromal cells are derived from local fibroblasts but not from keratinocytes [START_REF] Picarella | Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas[END_REF]. Epithelial cells appear involved in SS, where CXCL12 is expressed by the salivary duct epithelium and CXCL13 in acini and ducts [32,68].

Conceptually, TLS develop when the forces that recruit and retain leukocytes exceed those that expel them from the tissue. This results either through an overactive recruitment via blood endothelial cells or as a consequence of a diminished lymphatic endothelial cell-regulated cell output. Nonetheless, lymphedema is unlikely to be sufficient to create TLS when the retaining force is underdeveloped [61]. Therefore, a coordinated interplay between entry-retention-exit is probably required for TLS formation. Moreover, lymphocyte retention within the tissue rarely achieves the level of organization sufficient to form a TLS, thus suggesting that an active process of recruitment and organization is required for TLS formation [11].

Nonetheless, the activation of the resident vascular structures, that includes upregulation of homing molecules to enable lymphocyte recruitment, appears to be a prerequisite of TLS assembly. Blood endothelial vessels undergo remodeling during inflammation signaling for the entry of blood-derived hematopoietic cells through expression of integrins, addressins, and chemokines in response to LTαβ signals [START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF]. Endothelial cells and fibroblasts express cell adhesion factors such as mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1), vascular addressin cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1). This active expression of adhesion molecules is accom-

Vascular System

Requirements for TLS Formation and Maintenance panied by the loss of vascular integrity, which results in exposure of the sub-endothelium matrix and efflux of plasma from the intravascular space [START_REF] Mounzer | Lymphotoxin-alpha contributes to lymphangiogenesis[END_REF]. This newly formed extravascular matrix supports leukocyte extravasation [START_REF] Zhang | Increased lymphangiogenesis in joints of mice with inflammatory arthritis[END_REF][START_REF] Ansel | A chemokine-driven positive feedback loop organizes lymphoid follicles[END_REF]. The process of angiogenesis in TLS is supported by local release of VEGFα, angiopoietin, PDGF, IL-1, TNFα, and CXCL8, some of which are expressed by fibroblasts under inflammatory conditions [11]. Ectopic expression of proinflammatory chemokines such as CXCL8/IL-8 and CCL2/ MCP-1 support the chemotactic gradient necessary for influx of various inflammatory cells into peripheral tissue [START_REF] Luther | Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis[END_REF]. Blood endothelial cells are also involved in leukocyte activation by expressing MHC class II molecules [START_REF] Cyster | Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs[END_REF] and co-stimulatory molecules including OX40L, ICOSL, and CD25 [START_REF] Marinkovic | Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid[END_REF].

Within TLS, blood endothelial cells undergo a complex process of conversion of flat venular endothelial cells into tall and plump endothelial cells that closely resemble the HEVs present in the T-cell areas of the lymph nodes. TLS-associated HEVs are characterized by expression of the lymph node trafficking code, peripheral node addressin (PNAd) which recognizes and binds L-selectin (also named CD62L), expressed on naïve/central memory T lymphocytes and mature dendritic cells. This is responsible, together with the ectopic expression of CCL21, ligand for CCR7, for the homing of naïve and central memory T cells within those structures [4,5,[START_REF] Marchesi | CXCL13 expression in the gut promotes accumulation of IL-22-producing lymphoid tissue-inducer cells, and formation of isolated lymphoid follicles[END_REF]. HEV differentiation and in particular PNAd expression are regulated by lymphotoxin [START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF][START_REF] Chen | IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology[END_REF][START_REF] Meier | Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells[END_REF].

Under homeostatic conditions lymphatic vessels drain and transport extracellular fluid and macromolecules into the systemic circulation. While blood endothelial vessels mainly deliver cells to inflamed tissues, the lymphatic bed is responsible for the transport of tissuederived cells (i.e. DCs, T cells) and antigens toward the draining lymph node. LVs also regulate interstitial fluid drainage preventing excessive swelling of the inflamed organ. LVs undergo dramatic remodeling during inflammation to support the increased need for tissue exit of cellular material [START_REF] Baluk | Preferential lymphatic growth in bronchusassociated lymphoid tissue in sustained lung inflammation[END_REF]. Lymphangiogenesis in embryonic life is tightly regulated, so that LV development is intimately connected with the process of lympho-neogenesis. LVs sprout out from the cardinal vein, in a process that is orchestrated by the homeobox genes Prox1 and Sox18, the growth factor VEGF-C, and its receptor VEGFR3. However, a non-venous origin of lymphatic vasculature has been also demonstrated [START_REF] Gaur | Bronchus-associated lymphoid tissue lymphoma arising in a patient with bronchiectasis and chronic Mycobacterium avium infection[END_REF]. Recent studies have shown that lymphatic vessel formation also requires interaction between lymphatic vessels and platelet-derived Clec-2 for separation of lymphatic vessels from the blood endothelial vessels [START_REF] Khader | IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung[END_REF][START_REF] Kaushal | Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis[END_REF].

Within immunized lymph nodes, a significant expansion of the lymphatic vessels occurs that coincides with an increased influx of immune cells, thereby maximizing the potential for interaction between different cell types and enabling an effective immune response to occur.

Lymphatic Vessels

This process is very tightly regulated by LTβR signaling and remodels the LN in a manner that allows an effective balance between the input and output of the cells. This in turn helps in the recovery of the LN to homeostatic conditions [START_REF] Kahnert | Mycobacterium tuberculosis triggers formation of lymphoid structure in murine lungs[END_REF]. The expansion of LV endothelial cells not only supports the need for cellular interaction but may also play an important role in immunological function, as lymphatic endothelial cells express peripheral tissue antigens (PTA) under the control of AIRE for presentation to T cells as well as immunoregulatory factors that are able to modulate the local immune responses [START_REF] Geurtsvankessel | Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice[END_REF].

LVs also undergo remodeling in peripheral tissue during inflammatory conditions, whereby the activation of NF-κB pathway can stimulate the expression of Prox1 and increase the transcripts for the VEGFR3 [START_REF] Kocks | Chemokine receptor CCR7 contributes to a rapid and efficient clearance of lytic murine gamma-herpes virus 68 from the lung, whereas bronchus-associated lymphoid tissue harbors virus during latency[END_REF][START_REF] Takamura | Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance[END_REF]. Neo-lymphoangiogenesis in TLS is also supported by the expression of LT, IL-1, and TNFα [START_REF] Nakayama | Vaccination against H9N2 avian influenza virus reduces bronchus-associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection[END_REF][START_REF] Slight | CXCR5(+) T helper cells mediate protective immunity against tuberculosis[END_REF][START_REF] Khader | Th17 cells at the crossroads of innate and adaptive immunity against infectious diseases at the mucosa[END_REF][START_REF] Shen | Selective destruction of interleukin 23-induced expansion of a major antigen-specific gammadelta T-cell subset in patients with tuberculosis[END_REF][START_REF] Shen | The crucial roles of Th17-related cytokines/signal pathways in M. tuberculosis infection[END_REF]. Immune cell migration into the developing LVs has been suggested, alongside proliferation of existing endothelial progenitors [START_REF] Quesniaux | TNF in host resistance to tuberculosis infection[END_REF][START_REF] Day | Secondary lymphoid organs are dispensable for the development of T-cell-mediated S1P receptor 1[END_REF][94].

Interestingly TLS, that lack differentiated marginal reticular cells and a proper capsule, are devoid of a subcapsular sinus, contributing to the anatomical disorganization of the vascular system in TLS. However, LVs are present in TLS and to a certain extent localized mainly in the outer follicular area. While there is no evidence that an organized lymphatic afferent system exists in TLS, the presence of efferent lymphatics draining antigens and activated immune cells to the draining lymph nodes has been demonstrated [95]. Similar to the SLO, LVs in inflamed tissue act in an immuneregulatory role, upregulating the expression of D6/CCBP2, a decoy receptor capable of reducing the inflammatory response by scavenging inflammatory CC chemokines [96] and suppressing dendritic cell maturation [97]. There is no evidence that this function is not maintained in TLS. Lymphangiogenesis in TLS is believed to represent a productive attempt to resolve inflammation; however, it is not clear whether the newly formed LVs are able to establish viable connection with preexisting lymphatic vessels, which would hamper the drainage of activated dendritic cells, macrophages, and locally displayed antigens to the draining LN [98-103] (Bugatti et al., EULAR Communication, 2011). It is also unclear to what extent the newly formed lymphatic vessels contribute to the chemokine gradient established in the TLS and whether or not the ectopic expression of CCL21 on LVs can be considered a positive factor for the functional development of TLS [99, 100]. Of interest is the finding that RANK activation of LVs plays a critical factor in SLO formation [29]. While the underlying mechanisms are still unclear, the finding that lymphatic and blood endothelial cells express cell adhesion molecules upon RANK stimulation [104] suggests that the endothelial cells function as a first retention signal to LTi cells. Indeed, pharmacological blocking lymphocyte exit by acting on lymphatic sphingosine-1-phosphate (S1P) receptors lead to an additional LN in about half of the animals [29].

Requirements for TLS Formation and Maintenance

Nayar et al. demonstrated that expansion of the lymphatic bed within TLS follows a bimodal course, with a first phase of expansion induced by IL-7 and the second phase that coincides with the peak of organization of the TLS, being mediated by LTβR signaling [95]. While we could not identify a direct effect of LTβR in lymphatic cell proliferation, others have recently showed that LT might act as a proliferative factor for LV endothelial cells via NF-κB activation [105]. Other proinflammatory cytokines have been implicated in this process, some with positive and some with detrimental effects on lymphangiogenesis. Interestingly, blocking some of these cytokines has been sought after as a therapeutic intervention in inflammation, leaving open the possibility that counteracting the inflammatory cascade might hamper the same innate attempt to its resolution [95].

Conclusions

TLS assembly is a complex phenomenon and can be regulated at different sites by diverse cytokines and cellular requirements. While the pathogenic versus tolerogenic role of those structures is still debated [106, 107], in chronic autoimmune disease TLS persistence is considered a negative predictive factor for disease progression [2,32]. Recent advances in the understanding of SLO biology and the development of novel tools to dissect leukocytes/stromal cell interaction provided critical insights into TLS assembly and regulation [12,108]. This will translate to the development of compounds able to interfere with TLS structure and persistence in the tissue, thus decreasing local autoimmunity and risks associated with ectopic lymphocytic expansion.

Immune Privilege Concept and Immunity of the Central Nervous System

The central nervous system (CNS) is a compartmentalized organ composed of the parenchyma (brain and spinal cord) surrounded by meninges. The meninges are comprised of the pia mater which covers the parenchyma, the arachnoid mater, and the dura mater that is attached to the skull. The subarachnoid space lies between the arachnoid and the pia mater and contains the cerebrospinal fluid (CSF). Both the CSF and the meninges ensure the protection of the parenchyma from endogenous/exogenous threats and provide buoyancy. The CNS is also protected from the entry of pathogens, circulating immune cells, and blood factors by the blood-brain barrier (BBB), which is formed by a unique set of tight junctions expressed by the endothelial cells of the brain along with a lining of astrocyte end-feet.

In the mid-1940s, Sir Peter Medawar proposed the concept of immune privilege to describe tissues in which the introduction of a foreign tissue does not elicit an immune response. Indeed, implan-tation of tumors or fetal tissues into the brain parenchyma leads to their successful growth and maintenance [1][2][3]. Similarly, transplantation of skin allograft inside of the brain parenchyma only elicited a delayed graft rejection compared to a similar graft in the periphery [4]. These findings suggest that the brain is a tolerogenic organ unable to induce an immune response as efficient as peripheral tissues. Several features of the CNS were proposed to explain this unique feature: (1) the lack of professional antigenpresenting cells in the brain parenchyma, (2) the lack of lymphatic drainage from the brain parenchyma, and (3) the blood-brain barrier. Interestingly, if an immune response against the graft's antigens was induced prior to the transplantation in the parenchyma, the brain's immune response to the allograft is similar to the peripheral organs' response [4]. This suggests that while antigens within the brain fail to prime an efficient immune response, they can maintain and potentiate it. This highlights that immune cells can cross the BBB if activated in the periphery. Furthermore, if the allograft was implanted close to the ventricle or within the subarachnoid spaces, the rejection rate was similar to peripheral graft suggesting that the CSF/meningeal compartments behave differently than the parenchyma and are more in touch with peripheral immunity [5,6]. Overall these pioneer experiments revealed that the different CNS compartments have a different relationship toward the immune system.

One major difference between the brain parenchyma and its surroundings is their drainage capacity. Indeed waste clearance out of the brain relies on the brain interstitial fluid and the CSF [7,8]. Injection of tracers (blue dextran or horseradish peroxidase) directly into the brain parenchyma resulted in their accumulation in the perivascular spaces of cerebral blood vessels [9,10]. These observations were confirmed with a large variety of compounds of vastly different molecular weights [11,12]. Similarly, horseradish peroxidase (HRP), or other compounds injected into the CSF of mammals, appears to use similar perivascular pathways to rapidly enter the brain [12][13][14]. A system was therefore proposed where the CSF and the interstitial fluid (ISF) would intermix at the level of the perivascular space (the degree of mixture of both fluids remains a matter of debate [15][16][17]), allowing the removal of molecular waste from the brain parenchyma into the CSF, and consequently might favor the delivery of beneficial CSF constituents and nutrients to deeper structure of the brain parenchyma. The exact mechanism allowing the efflux of CSF into the brain remains a matter of debate between a bulk fluid flow and a simple diffusion mechanism [9,[17][18][19]. Pioneer work from the laboratory of Maiken Nedergaard has demonstrated that this system is dependent on cerebral arterial pulsation [20], CSF volume, and homeostasis [21]. Sleep states, anesthesia, and postural altera-

Brain Parenchyma Versus Meningeal Compartment: Drainage System

tions have been shown to modulate the perivascular draining system [22,23]. Reports have identified the water-channel AQP4, expressed on the end-feet of astrocyte, as a major regulator of perivascular drainage and driving of the CSF into the brain. This leads to the nomenclature of glial associated lymphatic system or glymphatic system [7,21,24]. More recent reports have challenged the role of AQP4 in that system [19]. While immune cells are unlikely to use that system to exit the brain parenchyma [25], perivascular drainage of molecular waste might play a major, and central, role in neurological disorders [15,17,26]. In aged mice, and in murine transgenic model of Alzheimer's disease, glymphatic CSF influx is reduced and results in impaired β amyloid (Aβ) clearance [7,27,28], suggesting that the glymphatic system might play a major role in the pathology of Alzheimer's disease. Furthermore, glymphatic impairment seems to be a hallmark of multiple neurological disorders, including subarachnoid hemorrhage, multiple microinfarction, and traumatic brain injury [29,30]. Reduced perivascular clearance of solute also appears to be associated with motor and cognitive deficits [START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF]. Overall, perivascular drainage represents the system for the brain to remove its waste into the CSF.

Injection of tracer into the CSF leads to its accumulation into the brain parenchyma via perivascular drainage (as described earlier), but also can be seen accumulating in the periphery into the lymph nodes of the neck [32][33][34][START_REF] Szentistványi | Drainage of interstitial fluid from different regions of rat brain[END_REF][START_REF] Weller | Lymphatic drainage of the brain and the pathophysiology of neurological disease[END_REF][START_REF] Yamada | Albumin outflow into deep cervical lymph from different regions of rabbit brain[END_REF]. Similarly, immune cells with migrating properties, mainly T cells and dendritic cells, when injected into the CSF have been shown to migrate into the cervical lymph nodes [START_REF] Clarkson | CCR7 deficient inflammatory dendritic cells are retained in the central nervous system[END_REF][START_REF] Goldmann | T cells traffic from brain to cervical lymph nodes via the cribroid plate and the nasal mucosa[END_REF][START_REF] Hatterer | How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicles of cervical lymph nodes[END_REF][START_REF] Kaminski | Migration of monocytes after intracerebral injection at entorhinal cortex lesion site[END_REF][START_REF] Mohammad | Immune cell trafficking from the brain maintains CNS immune tolerance[END_REF]. These observations suggest that, contrary to the brain parenchyma, the meningeal spaces have a direct access to the periphery. The ability of CSF constituents to drain out of the CNS, using both classical and nonclassical paths, which will be described in detail in the next section of this chapter, demonstrates that the CNS is neither excluded nor ignored by the immune system.

Another major discrepancy between the brain and the meningeal compartment is the presence of immune cells. Meningeal endothelial cells present different features than parenchymal and lack astrocytic end-feet, resulting in a more permissive barrier compared to the BBB [START_REF] Shechter | Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates[END_REF]. While the brain parenchyma is virtually devoid of immune cells (with the exception of the brain-resident macrophages, that is, microglia, but also potentially mast cells [START_REF] Silver | Mast cells in the brain: evidence and functional significance[END_REF]), the meningeal compartment is populated by numerous immune cells, even under physiological conditions [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF][START_REF] Goldmann | Origin, fate and dynamics of macrophages at CNS interfaces[END_REF][START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF][START_REF] Korin | High-dimensional, single-cell characterization of the brain's immune compartment[END_REF][START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF]. The meningeal immune compartment appears to be a dynamic compartment [START_REF] Radjavi | Dynamics of the meningeal CD4(+) T-cell repertoire are defined by the cervical lymph nodes and facilitate cognitive task performance in mice[END_REF], participating in brain immune surveillance [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF][START_REF] Kivisäkk | Human cerebrospinal fluid central memory CD4+ T cells: evidence for trafficking through choroid plexus and meninges via P-selectin[END_REF][START_REF] Kipnis | Multifaceted interactions between adaptive immunity and the central nervous system[END_REF]52] and ensuring brain function [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF]53]. Indeed, meningeal T cells have notably been implicated in the regulation of cognitive behavior

Brain Parenchyma Versus Meningeal Compartment:

Immune Cells [START_REF] Derecki | Regulation of learning and memory by meningeal immunity: a key role for IL-4[END_REF][53][54][55][56][57], social behavior [54], and adult neurogenesis [56,57] by way of cytokine secretions. These works illustrate how the immune compartment, residing in the surrounding of the brain, can have a direct impact on its function. All cell types in the brain parenchyma express receptor for cytokines [54,[58][59][60][61], including neurons, and can therefore have their function modulated by cytokines released in the CSF [54]. Under pathological conditions, the meningeal immune compartment is also gaining momentum. Longitudinal clinical studies have revealed that in multiple sclerosis patients, inflammatory events can be observed prior to being detected in the brain parenchyma [START_REF] Foo | Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation[END_REF][START_REF] Mohr | Dendritic cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature[END_REF]. In experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, encephalitogenic T cells have been shown to enter the CNS through the meningeal blood vasculature where they will encounter local macrophages [START_REF] Solleti | Serpine2 deficiency results in lung lymphocyte accumulation and bronchusassociated lymphoid tissue formation[END_REF]. It appears that the meningeal compartment serves as a key checkpoint for encephalitogenic T cells, where they could supposedly acquire the phenotype that allows them to infiltrate the brain parenchyma. Recently, locally residing NKp46 + innate lymphoid cells (ILCs) have been shown to coordinate meningeal inflammation during EAE and facilitate the invasion of the brain parenchyma by encephalitogenic T cells [START_REF] Picarella | Transgenic tumor necrosis factor (TNF)-alpha production in pancreatic islets leads to insulitis, not diabetes. Distinct patterns of inflammation in TNF-alpha and TNF-beta transgenic mice[END_REF]. Moreover, meningeal resident mast cells have also been proposed as a sentinel, controlling the activation and invasion of the brain parenchyma by encephalitogenic T cells [START_REF] Picarella | Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas[END_REF]. The meningeal immune compartment has started being studied in other pathological conditions. In a stroke model, IL-17-producing T cells have been reported to migrate into the meningeal compartment and influence ischemic neuroinflammation [START_REF] Flavell | The contribution of insulitis to diabetes development in tumor necrosis factor transgenic mice[END_REF]. In animal models of Alzheimer's disease, targeting the meningeal immune system through blockade of PD-1 resulted in an amelioration of disease pathology and clinical symptoms [68], suggesting that the meningeal immune compartment represents a key therapeutic target for neurological disorders.

While the brain parenchyma and the meningeal spaces have different features, they are not isolated from one another and can communicate. Indeed, molecular waste from the brain is removed via perivascular drainage and accumulates in the CSF where it would then drain into the cervical lymph nodes via lymphatic drainage. Molecules, including cytokines produced by the meningeal immune compartment, can directly affect (via perivascular drainage) brain function.

Meningeal Lymphatic Vasculature and Regulation Of Brain Immunity

As described earlier, injection of tracers or immune cells into the CSF leads to their drainage into the cervical lymph nodes, mostly the deep cervical lymph nodes, but also the superficial cervical

Lymphatic Vasculature in the CNS

lymph nodes and, in some instances, the lumbar lymph nodes [15,17] (Fig. 1). In almost all peripheral tissues, drainage of extracellular fluids and immune cells is ensured by the lymphatic system [START_REF] Mounzer | Lymphotoxin-alpha contributes to lymphangiogenesis[END_REF]. The lymphatic vasculature is a unique blind-ended, unidirectional absorptive and transport system [START_REF] Zhang | Increased lymphangiogenesis in joints of mice with inflammatory arthritis[END_REF]. Unlike blood vessels, the lymphatic network does not form a closed circulatory network. The presence of lymphatic vessels in the dura mater has been suggested over the years [START_REF] Ansel | A chemokine-driven positive feedback loop organizes lymphoid follicles[END_REF][START_REF] Luther | Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis[END_REF][START_REF] Cyster | Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs[END_REF][START_REF] Marinkovic | Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid[END_REF][START_REF] Marchesi | CXCL13 expression in the gut promotes accumulation of IL-22-producing lymphoid tissue-inducer cells, and formation of isolated lymphoid follicles[END_REF] and has been recently molecularly and functionally characterized in both rodents [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF] and humans [START_REF] Chen | IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology[END_REF]. The meningeal lymphatic vasculature, expressing all the hallmark markers of lymphatic endothelial cells [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF], runs along the major veins of the meninges, but also along the middle meningeal artery [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF]. Furthermore, lymphatic vessels can be seen in the vicinity of the cribriform plate, and even crossing the cribriform plate [START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF], a route previously described as a major route for CSF, immune cells, and macromolecules to drain into the peripheral lymph nodes [34,[START_REF] Goldmann | T cells traffic from brain to cervical lymph nodes via the cribroid plate and the nasal mucosa[END_REF][START_REF] Kaminski | Migration of monocytes after intracerebral injection at entorhinal cortex lesion site[END_REF][START_REF] Meier | Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells[END_REF][START_REF] Baluk | Preferential lymphatic growth in bronchusassociated lymphoid tissue in sustained lung inflammation[END_REF]. While most work has focused on the meningeal lymphatic of the brain region, a similar network of lymphatic appears to be present in the meninges covering the spinal cord, enabling drainage into lower back lymph nodes such as the lumbar lymph nodes [START_REF] Weller | Lymphatic drainage of the brain and the pathophysiology of neurological disease[END_REF][START_REF] Gaur | Bronchus-associated lymphoid tissue lymphoma arising in a patient with bronchiectasis and chronic Mycobacterium avium infection[END_REF][START_REF] Khader | IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung[END_REF][START_REF] Kaushal | Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis[END_REF].

Over the years, several routes have been proposed for macromolecules and immune cells to drain into the cervical lymph nodes: (1) clearance along perineural sheaths of cranial and spinal nerves, (2) meningeal lymphatic vasculature, and (3) arachnoid granulations. Arachnoid granulations are protrusions of the arachnoid mater into the sinuses of the dura and allow the drainage of water content of the CSF directly into the venous system via channels of the arachnoid cap of the granulation. Their roles in the maintenance of CSF homeostasis in human have been extensively described [START_REF] Kahnert | Mycobacterium tuberculosis triggers formation of lymphoid structure in murine lungs[END_REF][START_REF] Geurtsvankessel | Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice[END_REF][START_REF] Kocks | Chemokine receptor CCR7 contributes to a rapid and efficient clearance of lytic murine gamma-herpes virus 68 from the lung, whereas bronchus-associated lymphoid tissue harbors virus during latency[END_REF][START_REF] Takamura | Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance[END_REF][START_REF] Nakayama | Vaccination against H9N2 avian influenza virus reduces bronchus-associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection[END_REF] but appear to have a more limited role in sheep and rodents [START_REF] Slight | CXCR5(+) T helper cells mediate protective immunity against tuberculosis[END_REF]. Accordingly, a recent study suggested that CSF drainage out of the CNS is carried predominantly by the lymphatic vasculature in mice [START_REF] Khader | Th17 cells at the crossroads of innate and adaptive immunity against infectious diseases at the mucosa[END_REF]. Furthermore, this system appears unlikely to allow the excretion of large macromolecules or immune cells [START_REF] Shen | Selective destruction of interleukin 23-induced expansion of a major antigen-specific gammadelta T-cell subset in patients with tuberculosis[END_REF]. Tracers, when injected into the CSF, have been observed into the nasal mucous membrane and its associated lymphatics [34, [START_REF] Meier | Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells[END_REF][START_REF] Baluk | Preferential lymphatic growth in bronchusassociated lymphoid tissue in sustained lung inflammation[END_REF] suggesting a direct drainage of the CSF and its constituents through the cribriform plate into the nasal mucosa prior to reaching the cervical lymph nodes. Immune cells have been suggested to use a similar route when injected into the subarachnoid space or into the brain parenchyma [START_REF] Goldmann | T cells traffic from brain to cervical lymph nodes via the cribroid plate and the nasal mucosa[END_REF][START_REF] Kaminski | Migration of monocytes after intracerebral injection at entorhinal cortex lesion site[END_REF][START_REF] Mohammad | Immune cell trafficking from the brain maintains CNS immune tolerance[END_REF] (Fig. 1). Similar injections of tracers and immune cells into the CSF, or into the brain parenchyma, lead to their accumulation into the meningeal lymphatic vasculature prior to being observed in the cervical lymph nodes [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF]. This suggests that the meningeal vasculature could represent a major route for drainage of CSF constituents (Fig. 1). The exact mechanism is however unknown. Indeed the meningeal lymphatic system is localized in the dura [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF][START_REF] Ansel | A chemokine-driven positive feedback loop organizes lymphoid follicles[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF][START_REF] Chen | IL-23 activates innate lymphoid cells to promote neonatal intestinal pathology[END_REF], therefore separated from the CSF by the impermeable arachnoid mater. Further works are therefore necessary to understand how the CSF and its constituents can reach the meningeal lymphatic and drain into the cervical lymph nodes.

The contribution of the cribriform plate route versus the meningeal lymphatic route in CNS homeostasis and immune surveillance will require more work and the development of a targeted approach. Those routes however seem to play different roles in terms of CSF maintenance. Surgical blockade of the cribriform plate route results in an instantaneous and sustained increase of intracranial pressure in sheep, suggesting that the cribriform plate

Routes of Drainage and Contributions

route is a major regulator of CSF homeostasis [START_REF] Shen | The crucial roles of Th17-related cytokines/signal pathways in M. tuberculosis infection[END_REF]. However, mice born without meningeal lymphatic vessels do not appear to have any defect in intracranial pressure nor brain water content [START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF]. Because the lack of meningeal lymphatic came from birth, it is possible that compensatory or adaptive mechanisms took place in this model. However, substantiated by the low diameter of the meningeal lymphatic network compared to peripheral tissues, it is plausible that the meningeal lymphatic vasculature is not a major contributor in the regulation of intracranial pressure.

Immune cells, notably T cells and dendritic cells, have been shown to be naturally present in the meningeal lymphatic network [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF], suggesting that this system could play a major role in the regulation of the meningeal immune system and participate in the generation and/or maintenance of the immune response in the CNS. We are only starting to explore the contribution of the meningeal lymphatic network under normal and pathological conditions. The meningeal lymphatics are efferent vessels allowing exit of immune cells into the periphery. The potential function of these vessels as afferent vessels participating in the entry of immune cells into the CNS under pathological conditions has however never been addressed.

Tertiary Lymphoid Organ Formation in CNS Disorders

Tertiary lymphoid organs (TLOs) also named tertiary lymphoid structures (TLS) are ectopic lymphoid formations developing in inflamed, infected, injured, or tumoral tissues [START_REF] Quesniaux | TNF in host resistance to tuberculosis infection[END_REF]. They are characterized by the accumulations of lymphoid cells in a structured manner reminiscent of lymph nodes with the presence of high endothelial venules and lymphatic vessels [START_REF] Quesniaux | TNF in host resistance to tuberculosis infection[END_REF][START_REF] Day | Secondary lymphoid organs are dispensable for the development of T-cell-mediated S1P receptor 1[END_REF][94][95]. Structurally, TLS have T-and B-cell compartmentalization with intercalated dendritic cells, follicular dendritic cells, and stromal cells. TLS can be found in many tissues under conditions of chronic inflammation, and while their contribution in pathologies is still a matter of debate, correlation studies are strongly suggesting that their formation is a good prognosis in cancer, while detrimental in transplantation and autoimmune diseases [START_REF] Quesniaux | TNF in host resistance to tuberculosis infection[END_REF]94]. The formation of TLS in the CNS has been described, notably in the context of multiple sclerosis (MS) and its animal model [96], but little is known about their formation and the role of the meningeal immune compartment/lymphatic network in the formation and maintenance of such structure. In this section, we discuss the existing literature about TLS formation in the CNS and hypothesize the potential role of TLS in neurological disorders.

Multiple sclerosis is a chronic inflammatory disease of the CNS, which results in permanent neuronal damage and substantial motor

TLS in Multiple Sclerosis

Meninges and Tertiary Lymphoid Structure disability in patients [97,98]. While autoreactive T cells are a major driver of the disease, B cells have been proven essential for disease pathogenesis [99][100][101]. Morphological studies have demonstrated, in a fraction of patients with MS [102-104], a large degree of meningeal inflammation associated with aggregates of B cells and T cells with various degrees of organization [105]. Some aggregates even presented a reticular network of follicular dendritic cells [104], suggesting the formation of TLS in the meninges of MS patients. Contrary to TLS described in peripheral tissues, meningeal TLS do not appear to harbor high endothelial venules, suggesting particular features for the meningeal TLS (Fig. 2). Interestingly, these aggregates were observed in patients at different stages of the disease but were not systematically observed, suggesting that these structures might be only transiently formed in MS patients [103,104], or that only a fraction of patients develop such structures for a reason that remains to be determined. The incidence of meningeal TLS in MS pathology remains unclear. However, the presence of such structures correlates with a more severe disease course. In MS, B-cell repertoire analysis revealed a high degree of overlap between the CSF and the brain lesions in patients, suggesting that local hypermutation and affinity maturation of immunoglobulins might occur locally [106,107]. Meningeal TLS would represent a perfect structure enabling such an event. This hypothesis was recently demonstrated in a mouse model of MS [108]. Meningeal TLS have been shown to form in some mouse models of MS, especially the ones that present dependency on B cells [108][109][110][111][112][113][114][115]. Similarly to patients, meningeal TLS particularly developed on the brain meninges lining the brainstem and ventricles [96]. Again, most meningeal TLS did not present high endothelial venule structures but are formed within the highly vascularized meninges suggesting that meningeal TLS might function using their niche environment. The mechanism for the induction of meningeal TLS during EAE remains unclear but reports have identified lymphotoxins as a driver of B cells attracting CXCL13 in the CNS, a known chemokine for the formation of TLS [110,113]. Moreover, infiltrating Th17 cells have been described to be capable of functioning similarly to lymphoid tissue inducer (LTi) cells and induce remodeling of meningeal fibroblast into follicular dendritic cells to promote TLS formation [114,115].

The presence of lymphatic vessels, normally observed in peripheral TLS [95], in meningeal TLS has not been described yet (Fig. 2). Because of their preferential formation in the brain meninges, they could be inducing the invasion of the meningeal lymphatic vessels directly into the TLS. It is also possible that if localized in close vicinity to the meningeal lymphatics, TLS might use the endogenous network without inducing de novo formation of lymphatic vessels, similarly to what is suggested for high endothelial venules.

Meningeal Lymphatics and CNS TLS

CCL21 has been shown as a key chemokine for the formation of TLS [116]. CCL21 is highly expressed by the meningeal lymphatic endothelial cells [START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF][START_REF] Furtado | TNFalpha-dependent development of lymphoid tissue in the absence of RORgammat(+) lymphoid tissue inducer cells[END_REF] and could therefore represent a source of chemokines capable of initiating the formation of TLS in the CNS. Furthermore, the communication of the TLS with the periphery in the context of MS/EAE remains understudied, but B-cell repertoire studies do suggest a degree of communication of the TLS/meningeal compartment with the cervical lymph nodes [105, 106].

As of now, the presence of TLS has only been described in the context of MS/EAE. The meningeal compartment is representing a major, yet understudied, site and further studies are necessary to analyze the immune activity of this compartment, most notably the formation of TLS in many more neurological disorders such as injury and brain tumors. We are only starting to decipher the regulation of meningeal immunity under physiological conditions: How are immune cells maintained in the meningeal compartment? How are they responding to changes in homeostasis? How do those changes affect the brain both structurally and functionally? It is possible that dysregulation of the meningeal immune compartment contributes to the incidence of neurological disorders. Similarly, we do not understand the mechanisms of regulation of

TLS in Other Neurological Disorders

Lymphatic cell

Lymphatic cell? meningeal immunity during CNS inflammation, including the contribution of naturally resident immune cells in the formation of TLS. Therefore, understanding the mechanism of meningeal TLS formation might uncover a new therapeutic approach in context where the immune system fails to mount a sufficient immune response in the brain, notably in the context of primary brain tumors.

Conclusion

In conclusion, while initial experiments conducted by Peter Medawar established the idea that the CNS was an immuneprivileged organ by way of being ignored by the immune system, studies over the past 50 years or so have clearly demonstrated immune activity in the brain under both physiological and pathological conditions. Indeed, the homeostatic meningeal spaces are dynamically populated by numerous cells and can communicate with the periphery through the meningeal lymphatic network. Moreover, tertiary lymphoid structures can form in those spaces and participate in the local inflammatory response. However, Medawar's initial observations remain valid, suggesting that the CNS is immunologically unique, and understanding the dynamic of the immune response in the CNS will uncover new therapeutic avenue to impact neurological disease.

Introduction

Under pathological conditions, the infiltration of tissues by immune cells, the density of this immune infiltrate, and its potential organization in tertiary lymphoid structures (TLS) often mirror the development of an active immune response. TLS are transient ectopic lymphoid aggregates displaying the same organization and functionality as canonical secondary lymphoid organs (SLOs), with a T-cell-rich area characterized by DC-LAMP + mature dendritic cells (mDCs) forming clusters with T cells, and a B-cell-rich area characterized by CD20 + B cells [1,2]. Like in conventional SLOs, B-cell follicles are composed of a mantle of IgD + naive B cells, surrounding a germinal center (GC) identified by highly proliferating Ki67 + B cells, a network of CD21 + follicular dendritic cells (FDCs), presence of CD3 + CD4 + CXCL13 + PD-1 high follicular helper T cells (T FH ), and expression of activationinduced cytidine deaminase (AID) and Bcl6 (B-cell lymphoma 6), both associated with class-switch recombination (CSR) and somatic hypermutation (SHM) activities [2][3][4][5]. An increasing number of studies over the past decades have provided evidence that TLS represent sites for the local initiation and expansion of antigen-specific T-and B-cell responses [2,6,7], subverting the dogma that adaptive immune responses only take place in SLOs.

So far, these TLS have been observed in many situations of chronic inflammation, including lung inflammatory diseases (fibrosis, pneumonia, hypersensitivity pneumonitis, diffuse pan-Christophe Klein et al.

bronchiolitis, tobacco-induced inflammation, …), infectious diseases, graft rejection, autoimmune disorders (rheumatoid arthritis (RA), multiple sclerosis …), allergy, atherosclerosis, and cancer [8][9][10]. While in autoimmune diseases and transplantation the presence of TLS was associated with disease exacerbation and poor outcome [11][12][13][14][15], T-and B-cell responses initiated within TLS during infections were shown to be associated with pathogen clearance and reduced morbidity [16]. Similarly, in cancer, many studies demonstrated that the presence of TLS in the tumor microenvironment was associated with anti-tumor immune responses and prolonged patient survival, such as in non-small cell lung cancer (NSCLC), oral squamous cell carcinoma, metastatic colorectal cancer, and breast cancer [1,2,4,6,10,[17][18][19][20].

Most of these studies based their analysis on the quantification by immunohistochemistry (IHC) of CD20 + B-cell aggregates in the tumor tissue or of DC-LAMP + mDCs, as a hallmark of TLS [21]. However, proper TLS visualization and quantification within tissues may be challenging, and the complete characterization of fully active TLS may require the use of additional markers and thus the development of multiplexed immunolabeling. A way to achieve multiplexing is to perform sequential IHC stainings on the same tissue section, with tyramide-based signal amplification and antibody striping between each staining. However, the analysis of the sequential stainings obtained requires the use of sophisticated methods of image acquisition, as well as image processing by a spectral unmixing algorithm, especially when cell-to-cell interactions have to be studied at high resolution. In the case when the desired information are the absolute quantification and/or the potential co-localization of different immune cell types within a same structure, at a relatively lower resolution, virtual multiplexing of different IHC stainings performed on serial tissue sections can be developed more easily. The first step is the digitalization of the immunostained tissue sections as whole-slide images (WSI), which can be performed using more accessible slide scanners. These WSI can then be analyzed with an image analysis software, and highthroughput quantitative studies can then be achieved by counting a large amount of cells on a large number of tissue sections. Another advantage of WSI is that vast areas of tissue can be imaged at high magnification, giving the opportunity to detect and quantify rare isolated cells within the entire tissue section. Finally, the spatial relationship between different cell types can also be investigated starting from different WSI, provided that a step of image registration, extensively described in this chapter, has been performed before the analysis.

Here we propose three standardized IHC protocols for the visualization and/or subsequent quantification of TLS-associated CD20 + B-cell follicles, TLS-associated DC-LAMP + mDCs, and TLS-associated PD-1 high T FH , on formalin-fixed, paraffin-embedded (FFPE) human tissue sections, using NSCLC tumor samples as an example. The computer-assisted semiquantitative and quantitative analysis of IHC stainings proposed here represents a solution superior to the manual method in terms of reproducibility and applicability in the clinic, on large cohorts of patients. We also describe a virtual multiplexing method based on image registration, using an open-source software broadly used in scientific imaging and microscopy, ImageJ (IJ), to visualize the co-localization of several immune cell populations within a same structure, starting from different IHC stainings performed on serial tissue sections.

Materials

FFPE NSCLC tumor tissue sections, 5 μm thick. All specific antibodies used in the three following protocols are additionally listed in Table 1.

1. 1× Antigen retrieval solutions:

(a) For double IHC anti-human CD20/anti-human CD21 and anti-human CD20/anti-human PD-1: Target retrieval solution (TRS) pH6 (Dako, 10×; 1 volume + 9 volumes of distilled water, pH adjusted at 6).

(b) For double IHC anti-human DC-LAMP/anti-human CD3: Citrate buffer pH6 (4.5 mL of 0.1 M citric acid + 20.5 mL of 0.1 M sodium citrate in 225 mL of distilled water; pH adjusted at 6).

2. 3% H 2 O 2 .

3. 5% HS: 5% Decomplemented human serum (HS) in PBS.

4. Antibody diluent (Dako REAL ™ , Dako).

Samples

Buffers

Reagents

Christophe Klein et al.

Primary antibodies:

(a) For double IHC anti-human CD20/anti-human CD21: Anti-human CD20 (mouse IgG2a, clone L26, Dako, working dilution 1/250e) and anti-human CD21 (mouse IgG1, clone 1F8, Dako, working dilution 1/30e).

(b) For double IHC anti-human DC-LAMP/anti-human CD3: Anti-human DC-LAMP (CD208, rat IgG2b, clone 1010E1.01, Dendritics/Clinisciences, working dilution 1/80e, see Note 1) and anti-human CD3 (rabbit polyclonal IgG antibodies, Dako, working dilution 1/80e).

(c) For double IHC anti-human CD20/anti-human PD-1: Anti-human CD20 (mouse IgG2a, clone L26, Dako, working dilution 1/250e) and anti-human PD-1 (mouse 12. ImageJ software.

Specialist Equipment

Christophe Klein et al.

Methods

1. Dry the tissue sections a minimum of 30 min (or overnight) in a drying oven at 37 °C.

2. Deparaffinize the tissue sections: Under a laboratory fume hood, immerse the slides in three successive baths of 100% xylene (or Clearene) for 5 min each, then in one bath of absolute ethanol for 5 min, one bath of 90% ethanol for 5 min, one bath of 70% ethanol for 5 min, one bath of 50% ethanol for 5 min, and one bath of distilled water for 5 min (use glass containers for slides).

3. Retrieve antigen: Immerse the slides in a bath of pre-warmed antigen retrieval solution (TRS, pH6) for 30 min at 97 °C (in a water bath; use a plastic container for slides).

4. Take out the plastic container from the water bath and let it cool for 30 min at room temperature, on the bench.

5. Wash the slides in TBS for 5 min under agitation (use a glass container for slides) (see Note 2).

6. Remove the excess of buffer with absorbent papers (see Notes 3 and 4).

7. Place the slides in a humidified chamber.

8. Cover the tissue with 200 μL of 3% H 2 O 2 for 10 min at room temperature (for saturation of the endogenous peroxidases).

9. Wash the slides in 1× TBS for 1 min.

10. Remove the excess of buffer with absorbent papers.

11. Cover the tissue with 200 μL of 5% HS, for 30 min at room temperature (for saturation of the receptors for the Fc portion of immunoglobulins).

12. Simply remove the excess of 5% HS with absorbent papers (do not wash).

13. Cover the tissue with 200 μL of primary antibody solution (anti-human CD20 1/250e + anti-human CD21 1/30e in antibody diluent Dako REAL ™ ), for 1 h at room temperature.

14. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

15. Remove the excess of buffer with absorbent papers.

16. Cover the tissue with 200 μL of secondary antibody solution (anti-mouse IgG2a-biotin 1/100e + anti-mouse IgG1 1/30e in 1× TBS), for 30 min at room temperature. 17. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

18. Remove the excess of buffer with absorbent papers.

Double Immunohistochemistry Anti-human CD20/Anti-human CD21 for TLS-Associated B-Cell Follicle Quantification

IHC-based TLS Visualization and Quantification

19. Cover the tissue with 200 μL of tertiary reagents (streptavidin-HRP 1/300e + APAAP 1/30e in 1× TBS), for 30 min at room temperature.

20. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

21. Wash the slides in 1× TBS for 5 min, under gentle agitation.

22. Remove the excess of buffer with absorbent papers.

23. CD20 immunostaining revelation with AEC: Cover the tissue with 200 μL of AEC solution (prepared according to the manufacturer's instructions, in distilled water), for up to 30 min at room temperature (CD20-immunostained cells appear in red (Fig. 1a), check under the microscope) (see Note 5).

24.

Stop the reaction in a bath of 1× TBS, for 5 min, under gentle agitation.

25. Remove the excess of buffer with absorbent papers.

26. CD21 immunostaining revelation with SAP: Cover the tissue with 200 μL of SAP solution (prepared according to the manufacturer's instructions, in 0.1 M Tris-HCl pH 8.2, and supplemented with Levamisole, to inhibit endogenous alkaline phosphatases, 1 drop per mL of SAP solution), for 10-30 min maximum at room temperature (CD21-immunostained cells appear in blue (Fig. 1a), check under the microscope) (see Note 6).

27. Stop the reaction in a bath of 1× TBS, for 5 min, under gentle agitation.

28. Wash the slides in distilled water, for 5 min, under gentle agitation.

29. Remove the excess of buffer with absorbent papers.

30. Mount the slides with glass coverslips using glycergel (liquefied by heating at 70 °C).

31. Let the slides dry overnight at room temperature before storing.

1. Dry the tissue sections a minimum of 30 min (or overnight) in a drying oven at 37 °C.

2. Deparaffinize the tissue sections: Under a laboratory fume hood, immerse the slides in three successive baths of 100% xylene (or Clearene) for 5 min each, then in one bath of absolute ethanol for 5 min, one bath of 90% ethanol for 5 min, one bath of 70% ethanol for 5 min, one bath of 50% ethanol for 5 min, and one bath of distilled water for 5 min (use glass containers for slides).

Double Immunohistochemistry Anti-human DC-LAMP/Anti-human CD3 for TLS-Associated mDC Quantification

Christophe Klein et al. 3. Retrieve antigen: Immerse the slides in a bath of pre-warmed antigen retrieval solution (citrate, pH6) for 30 min at 97 °C (in a water bath; use a plastic container for slides).

4. Take out the plastic container from the water bath and let it cool for 30 min at room temperature, on the bench.

5. Wash the slides in TBS for 5 min under agitation (use a glass container for slides) (see Note 2).

6. Remove the excess of buffer with absorbent papers (see Notes 3 and 4).

7. Place the slides in a humidified chamber.

8. Cover the tissue with 200 μL of 3% H 2 O 2 , for 10 min, at room temperature (for saturation of the endogenous peroxidases).

9. Wash the slides in 1× TBS for 1 min.

10. Remove the excess of buffer with absorbent papers.

11. Cover the tissue with 200 μL of 5% HS, for 30 min, at room temperature (for saturation of the receptors for the Fc portion of immunoglobulins).

12. Simply remove the excess of 5% HS with absorbent papers (do not wash).

13. Cover the tissue with 200 μL of primary antibody solution (anti-human DC-LAMP 1/80e + anti-human CD3 1/80e in antibody diluent Dako REAL ™ ), for 1 h, at room temperature.

14. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

15. Remove the excess of buffer with absorbent papers.

16. Cover the tissue with 200 μL of secondary antibody solution (anti-rat IgG-biotin 1/500e + anti-rabbit IgG-AP 1/300e in 1× TBS), for 30 min, at room temperature. 17. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

18. Remove the excess of buffer with absorbent papers.

19. Cover the tissue with 200 μL of tertiary reagent (streptavidin-HRP 1/300e in 1× TBS), for 30 min, at room temperature.

20. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

21. Wash the slides in 1× TBS for 5 min, under gentle agitation.

22. Remove the excess of buffer with absorbent papers.

23. DC-LAMP immunostaining revelation with AEC: Cover the tissue with 200 μL of AEC solution (prepared according to the manufacturer's instructions, in distilled water), for up to 30 min, at room temperature (DC-LAMP-immunostained cells appear in red (Fig. 1c), check under the microscope) (see Note 5).

24.

Stop the reaction in a bath of 1× TBS, for 5 min, under gentle agitation.

25. Remove the excess of buffer with absorbent papers.

26. CD3 immunostaining revelation with SAP: Cover the tissue with 200 μL of SAP solution (prepared according to the manufacturer's instructions, in 0.1 M Tris-HCl pH 8.2, and supplemented with Levamisole, to inhibit endogenous alkaline phosphatases, 1 drop per mL of SAP solution), for 10-30 min maximum, at room temperature (CD3-immunostained cells appear in blue (Fig. 1c), check under the microscope).

27. Stop the reaction in a bath of 1× TBS, for 5 min, under gentle agitation.

28. Wash the slides in distilled water, for 5 min, under gentle agitation.

29. Remove the excess of buffer with absorbent papers.

30. Mount the slides with glass coverslips using glycergel (liquefied by heating at 70 °C).

31. Let the slides dry overnight at room temperature before storing.

1. Dry the tissue sections for a minimum of 30 min (or overnight) in a drying oven at 37 °C.

2. Deparaffinize the tissue sections: Under a laboratory fume hood, immerse the slides in three successive baths of 100% xylene (or Clearene) for 5 min each, then in one bath of absolute ethanol for 5 min, one bath of 90% ethanol for 5 min, one bath of 70% ethanol for 5 min, one bath of 50% ethanol for 5 min, and one bath of distilled water for 5 min (use glass containers for slides).

3. Retrieve antigen: Immerse the slides in a bath of pre-warmed antigen retrieval solution (TRS, pH6) for 30 min at 97 °C (in a water bath; use a plastic container for slides).

4. Take out the plastic container from the water bath and let it cool for 30 min at room temperature, on the bench.

5. Wash the slides in TBS for 5 min under agitation (use a glass container for slides) (see Note 2).

6. Remove the excess of buffer with absorbent papers (see Notes 3 and 4).

7. Place the slides in a humidified chamber.

8. Cover the tissue with 200 μL of 3% H 2 O 2 , for 10 min, at room temperature (for saturation of the endogenous peroxidases).

Double Immunohistochemistry Anti-human CD20/Anti-human CD21 for TLS-Associated T FH Cell Visualization

IHC-based TLS Visualization and Quantification 9. Wash the slides in 1× TBS for 1 min.

10. Remove the excess of buffer with absorbent papers.

11. Cover the tissue with 200 μL of 5% HS, for 30 min, at room temperature (for saturation of the receptors for the Fc portion of immunoglobulins).

12. Simply remove the excess of 5% HS with absorbent papers (do not wash).

13. Cover the tissue with 200 μL of primary antibody solution (anti-human PD-1 pre-diluted + anti-human CD20 1/250e, in anti-human PD-1 pre-diluted solution), for 1 h and 30 min, at room temperature.

14. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

15. Remove the excess of buffer with absorbent papers.

16. Cover the tissue with 200 μL of secondary antibody solution (anti-mouse IgG2a-biotin 1/100e + anti-mouse IgG1 1/30e in 1× TBS), for 30 min, at room temperature. 17. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

18. Remove the excess of buffer with absorbent papers.

19. Cover the tissue with 200 μL of tertiary reagent (streptavidin-HRP 1/300e + APAAP 1/30e in 1× TBS), for 30 min, at room temperature.

20. Wash the slides in 1× TBS-T for 5 min, under gentle agitation, twice.

21. Wash the slides in 1× TBS for 5 min, under gentle agitation.

22. Remove the excess of buffer with absorbent papers.

23. CD20 immunostaining revelation with AEC: Cover the tissue with 200 μL of AEC solution (prepared according to the manufacturer's instructions, in distilled water), for up to 30 min, at room temperature (CD20-immunostained cells appear in red (Fig. 1f), check under the microscope) (see Note 5).

24.

Stop the reaction in a bath of 1× TBS, for 5 min, under gentle agitation.

25. Remove the excess of buffer with absorbent papers.

26. PD-1 immunostaining revelation with SAP: Cover the tissue with 200 μL of SAP solution (prepared according to the manufacturer's instructions, in 0.1 M Tris-HCl pH 8.2, and supplemented with Levamisole, to inhibit endogenous alkaline phosphatases, 1 drop per mL of SAP solution), for 10-30 min maximum, at room temperature (PD-1-immunostained cells appear in blue (Fig. 1f), check under the microscope).

Christophe Klein et al.

27.

Stop the reaction in a bath of 1× TBS, for 5 min, under gentle agitation.

28. Wash the slides in distilled water, for 5 min, under gentle agitation.

29. Remove the excess of buffer with absorbent papers.

30. Mount the slides with glass coverslips using glycergel (liquefied by heating at 70 °C).

31. Let the slides dry overnight at room temperature before storing.

Image Analysis and TLS Quantification

TLS-associated B-cell follicle quantification can be performed automatically, using an image analysis software (Calopix software), after scanning of the whole slide using a slide scanner (NanoZoomer slide scanner).

1. Import the image generated after scanning of the slide into the Calopix database.

2. Delineate the "region of analysis" (ROA): In the case of NSCLC tumor tissue, the region of analysis corresponds to the tumor area, including the invasive margin; necrotic areas are excluded.

3. Use the "Ilastik" tool to educate the software: Create a protocol with five different classes, for (1) red CD20 + B cells, (2) blue CD21 + FDCs, (3) black anthracosis, (4) white empty spaces, and (5) the rest of the tissue (i.e., the tumor nests, tumor stroma, and remodeled lung tissue at the invasive margin); save the protocol for subsequent application to the complete tissue.

4. Launch a "Tissue recognition for surface quantification" analysis on Calopix: This protocol determines the ratio (expressed as a percentage) between a numerator corresponding to the surface (in mm 2 ) of the area of interest and a denominator corresponding to the surface (in mm 2 ) of the total tissue. In this case, the numerator is the total surface of all TLS-associated B-cell follicles, identified by red CD20 + B cells (class 1) and/ or blue CD21 + FDCs (class 2) (Fig. 1a), and the denominator is the total surface of the region of analysis, after exclusion of white empty spaces (class 4):

TLS-Associated B-Cell Follicle Quantification

IHC-based TLS Visualization and Quantification

During the analysis, a size criterion for TLS-associated B-cell follicle surface is used: only TLS-associated B-cell follicles with a minimum surface of 7000 μm 2 are considered and included in the numerator (Fig. 1b) (see Note 7).

When immunostained with anti-DC-LAMP antibody, mDCs within TLS are visualized as very small red dots (like nuclear circular structures), scattered among blue CD3 + T cells (Fig. 1c,e). Sometimes, very thin, free dendrites are visible. However, in lungs including NSCLC, immunostaining with DC-LAMP marker stains not only mDCs but also type II pneumocytes (Fig. 1e). The presence of two different types of red-colored cells in a same area limits the full-automated counting of mDCs using the aforementioned Calopix software. However, TLS-associated mDC quantification can be performed semiautomatically, using Calopix for determining total tissue surface area, but with the manual counting of the red DC-LAMP + mDCs within blue CD3 + T cell areas by two independent observers.

1. Import the image generated after scanning of the slide (on NanoZoomer slide scanner) into the Calopix database.

2. Delineate the "region of analysis" (ROA): In the case of NSCLC tumor tissue, the region of analysis corresponds to the tumor area, including the invasive margin; necrotic areas are excluded.

3. Use the "Ilastik" tool to educate the software: Create a protocol with three different classes, for (1) tissue, (2) black anthracosis, and (3) white empty spaces; save the protocol for subsequent application to the complete tissue.

4. Launch a "Tissue recognition for surface quantification" analysis on Calopix: Exclude class 3 while launching the protocol. This protocol determines the surface (in mm 2 ) of the total tissue within the ROA, that is, tumor nests, tumor stroma, and invasive margin.

5. For counting manually DC-LAMP + mDCs, first screen the entire slide to pre-visualize the mDCs within the T-cell areas of TLS.

6. In the window "Annotations" of Calopix, use the tool "counter" to start the manual counting. On the slide, left click on each DC-LAMP + mDC within the predetermined ROA: each click appears as a small number "1" on the slide (Fig. 1d), and is incremented in the "Annotations" window of Calopix.

7. The final count can be exported from the "Annotations" window into a separate Excel file.

8. Divide this final count by the surface area (in mm 2 ) previously determined to get the density of DC-LAMP + mDCs in the tissue section (expressed as a number of cells per mm 2 ).

TLS-Associated mDC Quantification

Christophe Klein et al.

The complete characterization of fully active TLS within a (tumoral) inflamed tissue may require the simultaneous identification, localization, and quantification of several immune cell populations and subpopulations on the same tissue section. By IHC, it is theoretically possible to distinguish up to three different immuno-markers, as images are usually recorded with RGB cameras. However, in practice, it is difficult to distinguish more than two chromogens on the same slide. In this regard, immunofluorescence (IF) presents some advantages, as signals from different fluorochromes can be acquired independently. In conventional fluorescence microscopy, up to four biomarkers (or three biomarkers plus counterstained nuclei) can be easily recorded with standard equipment. For higher degrees of multiplexing, several problems may arise, including potential antibodies' cross-reaction but also difficulties in secondary fluorescence signal detection and separation. Indeed, when more than four biomarkers are used simultaneously, in order to distinguish the different fluorochromes, image acquisition has to be done with a spectral imager, either a spectral confocal laser scanning microscope or a conventional optical microscope equipped with a spectral detector. The multispectral data acquired have then to be processed with a spectral unmixing software. This method allows the simultaneous detection of up to six fluorescent biomarkers associated with nuclear counterstaining, but still suffers from some drawbacks, including a time-consuming process of labeling, pricey reagents, and expensive equipment for image acquisition. In addition, those equipment are usually not able to acquire very large image mosaics (WSI), or only at low speed as compared to conventional slide scanners. Finally, some errors in spectral unmixing, which is an image processing step, can arise. Virtual multiplexing methods have also been proposed and represent affordable alternatives. One strategy is to perform on the same tissue section sequential standard immuno-peroxidase labelings, with imaging and then erasing of the staining obtained between each labeling ("SIMPLE" method). It has been shown that up to six consecutive staining/erasing steps can be achieved [22]. This method has the advantage to allow the use of standard reagents and imaging equipment but is still time consuming. A more simple approach consists of performing IHC (and/or IF) labelings on serial tissue sections [23]. This technique has to be performed on maximum 3-5 μm thick consecutive sections to obtain good results, and has for main limitation that antigen coexpression at the cell level can only be checked on one unique tissue section. However, this method can be performed with standard procedures and equipment, and enables to determine the colocalization and thus the potential interactions of several different cell types in specific tissue areas.

A crucial point for these two methods is the image registration process, which allows the comparison of different labelings from

Full TLS Visualization After Image Registration of Three Serial Tissue Sections: CD20/CD21 (B-Cell Follicles and FDCs), DC-LAMP/CD3 (mDCs and T-Cell Areas), and CD20/PD-1 (B-Cell Follicles and T FH )

IHC-based TLS Visualization and Quantification consecutive (different) tissue sections in the same tissue area. Image registration consists of computing a geometric transform that maps points from one image, the moving image, to homologous points in a fixed reference image. This transform is called rigid when the spatial transform is limited to translation, rotation, scaling, shearing, or a combination of those operations, and is called deformable or elastic when local displacements are also allowed. Usually, B splines are used to keep the track of those local deformations. An optimizer routine then tests different geometric transforms by evaluating the similarity between the moving image and the reference image until an optimal transform is found. The similarity is quantified at each step of the optimization by some metrics, for example the sum of squared differences between the pixel intensity of the two images.

Image registration methods have been well developed for many years in different domains such as satellite imagery, radiology imagery, and even histology, for example for the reconstruction of monomodal (single stained) 3D serial sections. Indeed, registration of histological images is a well-studied field of research, and issues such as nonlinear deformation, steering, shearing, folding, or shrinking due to tissue processing have been addressed and have received some solutions. Multimodal image series (images of different labelings from different tissue sections) registration presents additional challenges. As pixel values can vary a lot for different labelings in the same area of tissue, the metrics used to quantify the similarity between the modalities has to be chosen carefully. Usually, the metrics successfully used in monomodal image series registration fail when applied to the registration of multimodal image series. Alternatively, landmark points positioned on remarkable features of the images can be used by the registration algorithm so that their distance in both images is minimized at the end of the process. The landmark points can be selected manually by the user or determined automatically by an algorithm. The use of landmark points is generally a good strategy for the registration of multimodal images; however the selection of landmark point positions can be imprecise, hence limiting the accuracy of the registration.

Finally, an additional challenge is the usually huge size of the WSI created by the slide scanners. The most straightforward strategy for WSI registration is to perform at first a registration at low magnification, followed by a registration at high magnification on a small field of view based on the results of the low-magnification registration.

ImageJ (IJ) is a well-known image processing and analysis opensource software [24]. It is used in many domains of scientific imaging and especially in microscopy. The FIJI version of ImageJ [25] is bundled with many plug-ins dedicated to microscopy image processing and analysis, including image registration tools. However,

Registration with ImageJ Software

Christophe Klein et al.

the main drawback of IJ is its limitation regarding the size of the images it can manage. Indeed, IJ is only able to deal with images smaller than 2 31 pixels (about 46,000 × 46,000 pixels), even if enough memory is allocated to the software. This restrains the use of this tool to small or low-resolution virtual sections. Another limitation of IJ is the slide scanner's file formats it can open. Nearly each slide scanner vendor uses its own file format. To open those different file formats in FIJI, the "LOCI's Bio-Formats importer" plug-in [26] may be useful. However, it can only deal with some of them. Fortunately, regarding the file format generated by the NanoZoomer slide scanner (Hamamatsu), an IJ plug-in, "NDPItools," has been developed recently [27], making those files convenient for IJ.

Among the different tools for image registration available in FIJI we used two plug-ins based on their performances and ease of use: the "Linear stack alignment with SIFT" written by Dr. Stephan Saalfeld, based on the SIFT algorithm [28], and the "Stackreg" plug-in [29], which is based on the minimization of the sum of squared differences between the pixel intensity of the two images. The "Scale Invariant Feature Transform" (SIFT) algorithm finds automatically distinctive features in both images, and identifies those that are matched. Then, a geometric transform is applied to the moving image so that the distance between matching points is minimized. This tool gave good results in our hands. In contrast, because of the metrics it uses, "Stackreg" is not well adapted to the registration of multimodal images and is more suited to the registration of monomodal images. However, "Stackreg" is very fast and easy to use and we found that it gives satisfactory results when the labelings present in the different images to register are roughly localized in the same tissue areas.

In the case when significant physical deformations of the tissue sections appeared during sample preparation, elastic registration may be required. In this situation "BUnwarpJ" [30] proved its usefulness. This plug-in can run fully automatically, or with userdefined landmarks. It also accepts landmarks automatically defined by the SIFT algorithm which is also present in FIJI, as a standalone plug-in (plugin>feature extraction>Extract SIFT correspondences). Unfortunately, "BUnwarpJ" will function only on small images as it requires a large amount of memory, and thus can only be used for the registration of WSI at very low resolution.

Download and install the FIJI version corresponding to your

computer's operating system from this web page: https://fiji. sc/#download.

Check that enough memory is allocated to IJ:

Edit>Options>Memory & threads. Usually about 75% of the computer RAM can be used by IJ.

3. Download and install "NDPItools" plug-in from this web page: http://www.imnc.in2p3.fr/pagesperso/deroulers/software/ndpitools/.

4. Download NDPItools.jar and copy this file in the /Fiji.app/ plugins folder or alternatively drag and drop the NDPItools.jar file on the FIJI menu bar when FIJI is started.

1. Extract the different layers of the NPDI pyramidal file using Plugins>NDPI tools>Extract to tiff. The plug-in will try to load the highest resolution layer. This usually fails because of the size of the image. As a result, a message error box will pop up. Close it and go on for the next step.

2. Load the required layer using NDPI tools>Open Tiff (not higher than 5× because 20× images are usually too big to be loaded).

3. Repeat the process (steps 4 and 5) for the different WSI to register (see Note 8).

4. Put the different images in one image stack using Image>Stacks>image to stack (copy center).

5. Plugins>Registration>Linear Stack Alignment with SIFT.

6. Save the registered image stack (File>Save as TIFF format).

1. Load low-resolution preview of the WSI using NDPItools>PreviewNDPI.

2. Draw a region of interest (ROI) with the rectangle selection tool of the IJ menu bar (see Note 9).

3. Extract this region using NDPItools>Extract to TIFF format.

4. Repeat step 1 with the next WSI to register.

5.

Copy the ROI of interest on this preview image: Edit>Selection>restore Selection. Move the position of the ROI to the desired area (drag and drop the selection).

6. Repeat steps 4 and 5 if other WSI have to be registered.

7. Close all the preview images.

8. Put the different images in one image stack using Image>Stacks>image to stack (copy center).

9. Plugins>Registration>linear Stack Alignment with SIFT.

10. Save the registered image stack (File>Save as TIFF format) (Fig. 2a-c). 2. Crop the stack using Image>Crop.

3. Split the images from the stack using Image>Stacks>Stack to Images (Fig. 2d-f).

4. For each image, separate the stainings, using Image>Color>Color Deconvolution.

5. Keep only the useful images containing one labeling. Invert the contrast using Edit>Invert.

6. Apply the desired color map on each image: Image>Look Up Tables. Choose among Gray, Red, Green, Blue, Cyan, Magenta, and Yellow (Fig. 2g-k).

7. Create the overlay using Image Color>Merge Channels (Fig. 2l).

Of course, the usefulness of the registration of WSI at low resolution or of small areas at full resolution is limited, except for demonstrative purpose. For analysis purpose, it is highly desirable to register full-resolution WSI. In this regard, open-source solutions have been proposed using "Elastix," a free software based on the ITK library [START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF]32]. More recently, a plug-in was developed [33] for the open-source software "ICY" [34] as well as a stand-alone software named "Hyper-Stain inspector" [START_REF] Szentistványi | Drainage of interstitial fluid from different regions of rat brain[END_REF]. However those solutions are still experimental and not well suited for the analysis of large-scale datasets containing several tenths of WSI images. At this time, the best solution is to use commercially available software such as the "Visiopharm Visiomorph" software and its WSI registration module "TISSUEALIGN." This module allows the registration of several WSI either automatically or thanks to landmarks manually defined by the user. This task is achieved with a good accuracy and speed. It is also possible to register IF images together with bright-field (IHC) images. Once registered, a stack of WSI is obtained. It is then possible to define ROIs on a reference slide, for example on a hematoxylin and eosin-stained tissue sections, and to report those ROIs on the other registered images for analysis in the same areas at full resolution, if required.

Notes

1. Anti-human DC-LAMP clone 1010E1.01 also displays a crossreactivity with canine, feline, murine, and sheep DC-LAMP.

2. In each protocol, after antigen retrieval, it is possible to let the slides in a bath of 1× TBS overnight at room temperature before continuing the experiment (step 5 in Subheadings 3.1, 3.2, and 3.3).

3. When removing the excess of buffer with absorbent papers, pay attention to the tissue.

Overlay of the Registered Images

Registration of WSI at Full Resolution

Christophe Klein et al.

4

. Process the slides one by one: Do not let the slides dry for longer than 1 min before adding the following reagent, to avoid tissue alteration and staining background.

5. During the revelation steps (steps 23-26 in Subheadings 3.1, 3.2, and 3.3), the substrate for HRP (AEC) is commonly added first. However, it is also possible to add first the substrate for AP (SAP).

6. For double IHC anti-human CD20/anti-human CD21, carefully check under the microscope CD21 staining. CD21 in blue must not be too dark for subsequent analysis on Calopix software (otherwise it can be confounded with black anthracosis in the lung).

7. During TLS-associated B-cell follicle quantification, red CD20 + B-cell aggregates without the presence of blue CD21 + FDCs are also considered as TLS-associated B-cell follicles (quantified) (Fig. 1a,b), provided that they display a minimum surface of 7000 μm 2 (the visualization or not of FDCs within B-cell follicles may strongly depend on the tissue section).

8. Instead of different IHC stainings performed on serial tissue sections, the different WSI to register may also correspond to sequential IHC stainings performed on the same tissue section, with steps of imaging and then erasing of the staining obtained between each labeling (multiplexed IHC). In that case, image registration is much less challenging. 9. For image registration, during the "registration of selected areas at high magnification," the selected areas have to be large enough to contain a sufficient amount of unambiguous features.

Funding

This

Introduction

Lymphoid neogenesis resulting in the development of tertiary lymphoid structures (TLS) occurs under chronic inflammatory conditions and involves different cell types including perivascular stromal cells, B cells, high endothelial venules (HEVs), T cells, macrophages, and dendritic cells (DCs) (reviewed in [1,2]). We recently demonstrated that TLS development in the microenvironment of lung and colorectal cancer follows sequential maturation stages [3,4]. The process begins with the accumulation of lymphocytes in response to chemokine cues. Next, follicular dendritic cells (FDCs) differentiate to generate TLS that resemble primary follicles of the secondary lymphoid organs. Finally, a germinal center (GC) reaction develops resulting in TLS that resemble secondary follicles. The presence of TLS in human tumor microenvironment is often associated with improved patient outcome [2,5,6]. We showed that the generation of GCs was crucial for the prognostic power of TLS in untreated lung and colorectal cancer patients [4]. This suggests that TLS promote antitumor immunity. However, despite the frequent observation of GC-containing TLS in various human tissues, the molecular and cellular mechanisms that initiate TLS development and drive TLS maturation in different inflammatory contexts are incompletely understood.

The dissection of the interplay between different cell types and signaling molecules leading to TLS formation within the histological context requires the detection of multiple parameters on tissue sections. We focus here on immunofluorescence because it enables analysis of more markers simultaneously than bright-field imaging. Several intrinsic limitations must be considered when using immunofluorescent staining of multiple parameters on the same tissue section. First, the signal of tissue immunofluorescence has lower intensity when compared to flow cytometry, which often precludes the use of directly labeled primary antibodies. In such cases, signal amplification using labeled secondary antibodies is necessary. Second, the limited number of species in which most primary antibodies are produced thus restricts the number of markers one can analyze within the same tissue section. This obstacle can, however, be overcome by using tyramide signal amplification (TSA): Dyeconjugated tyramide molecules are activated by horseradish peroxidase (HRP), which initiates a radical chain reaction leading to the covalent binding of dye-tyramide residues to the antibodybound protein [7]. Because of this permanent labeling, the detection antibodies can be removed by heating without losing the fluorescent signal of the target antigen. A second antigen can now be detected using primary antibodies raised in the same species as for the first antigen, followed by HRP-conjugated secondary antibody and a tyramide conjugated to a different dye. This detection/ removal process can be repeated several times. TSA considerably increases the detection sensitivity of tissue immunostaining [7], thus allowing the detection of molecules that are present in low concentrations such as secreted cytokines.

Multispectral imaging (MSI) is a recent technological advancement that allows increasing the number of fluorescent colors that can be simultaneously detected by a conventional fluorescence microscope [8]. MSI also provides the possibility to separate tissue autofluorescence from the specific fluorophore signals. This involves the use of a multispectral camera for the acquisition of spectral profiles of each individual fluorophore as well as the spec-Karīna Siliņa et al.

tral profile of tissue autofluorescence using unstained tissue. Further, using a dedicated image-processing software, the spectral profiles present in each pixel can be assigned to specific fluorophores and/or autofluorescence through a process called spectral unmixing. Thus, the main factor limiting the number of parameters that can be detected simultaneously is the spectral overlap of fluorophores, which cannot be properly unmixed if their emission spectra overlap too much. The development of fluorophores with narrower emission spectra in combination with the multispectral imaging and TSA currently allows for robust seven-color immunofluorescence protocols. This number may still increase by development of new dyes.

Here, we describe a quantitative pathology approach for the analysis of TLS in different maturation stages and in combination with other relevant TLS-associated cell types including HEVs and conventional DCs. This method can be further adapted to other tissues or scientific questions. To distinguish the different TLS maturation stages, a minimal combination of CD20, CD21, and CD23 expression needs to be analyzed. The remaining three markers can be used to assess chemokine expression, vasculature, tissuespecific niche cells, or other parameters.

The method we developed and describe here consists of (i) immunostaining of formalin-fixed paraffin-embedded (FFPE) human tissues for the detection of six markers plus a nuclear staining, (ii) multispectral imaging using the Vectra 3.0 ™ automated microscopy system (PerkinElmer), and (iii) image data analysis using Inform ™ (PerkinElmer) and RStudio software. We provide details on tissue slide preparation, staining procedure using tyramide-conjugated Opal ™ dyes (PerkinElmer), multispectral image acquisition, and analysis of quantitative imaging data using tissue and cell segmentation algorithms of Inform ™ software (PerkinElmer).

Materials

1. Freshly cut FFPE tissue sections (2-5 μm) on routine pathology glass slides (see Note 1) including sections from secondary lymphoid organs.

2. 55 °C Incubator.

3. Pressure cooker (routine kitchen cooker, 5 L) and stove for antigen retrieval by autoclaving (see Note 2).

4.

Metal forceps, at least 20 cm long.

5. Pretreatment and antigen retrieval buffer: 1× Trilogy ™ solution (Cell Marque) (see Note 3).

Preparation of FFPE Tissue Slides

Analysis of TLS by Quantitative Pathology 5. HRP-conjugated secondary antibodies (Table 1): Donkey anti-rabbit IgG (H+L), donkey anti-mouse IgG (H+L), donkey anti-rat IgG (H+L), goat anti-rat IgM (μ chain specific) All HRP-conjugated secondary antibodies were purchased from Jacksons ImmunoResearch. The optimal working dilutions were 1:500 for anti-mouse IgG, anti-rat IgM, and IgG, and 1:1000 for anti-rabbit IgG with minimal cross-reactivity to immunoglobulins from other species (see Note 6).

Immunostaining of Human FFPE Tissues

6. Tyramide-fluorophore conjugates: Opal™ 7-color Manual IHC Kit (PerkinElmer) (Table 2). This kit also contains the Amplification Diluent buffer necessary for generating the working dilutions of the tyramide-conjugated dyes. The kit contains an optimized combination of six modified fluorescent dyes with narrow emission spectra and the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). The use of such dyes is crucial for multispectral image processing to enable unmixing of the spectra from dyes that are detected with the same emission filter.

7. 95 °C Water bath (see Note 7).

8. Heat-resistant slide trays (glass jars with glass slide holders or plastic 50 mL tubes, depending on the number of slides being processed simultaneously) for antibody removal. 9. Regular slide trays for washing steps.

10. Antibody removal buffer (ARB): 10 mM Citric acid, pH 6.0.

11. WB, rocking platform, and wet chamber, as in Subheading 2.1.

12. Antifade mounting medium without DAPI like ProLong ™ Diamond (Thermo Fisher) (see Note 8).

13. Glass coverslips.

1. Vectra 3.0 ™ multispectral imaging system (PerkinElmer).

Mounted tissue slides:

(a) A fully unstained tissue section that has been subjected to the same processing protocol as the stained slides (antigen retrieval, blocking, washing, heating) but omitting all fluorescent dyes. This slide is necessary to determine the spectral profile of autofluorescence of the tissue of interest (see Note 9).

Multispectral Imaging

(b) Tissue sections that have been stained with only one fluorescent dye, including a separate slide for the DAPI staining (see Note 10). These slides are necessary to determine the spectral profile of each dye used in the multiplex protocol.

(c) Slides stained with the seven-color protocol.

1. Phenochart ™ software (PerkinElmer).

2. Inform ™ software (PerkinElmer).

3. RStudio software (see Note 11).

Methods

The protocol described here has been optimized for the manual processing of up to 20 slides in parallel with the least possible number of heating steps for antibody removal to reduce the risk of tissue and epitope damage. If not indicated otherwise, all steps are performed at room temperature (RT). During all incubation steps the sections should be protected from light (i.e., washing containers and wet chambers covered with aluminum foil). Apart from the slides of interest, ten additional slides must be included (see above under step 2 in Subheading 2.3): one slide that is not stained with any of the fluorophores, seven slides that are stained with each individual fluorophore only, one negative control slide where all primary antibodies are omitted, and one positive control slide (any of the secondary lymphoid organs). The single-color and unstained slides need to be included only once if the same combination of fluorescent dyes is used and the same tissue is analyzed. The singlestained slides are necessary for the generation of the spectral profile for each dye and the unstained slide is necessary to establish the autofluorescence profile of the tissue of interest. These spectral profiles are used for the unmixing of fluorophore signals in the multiplex-stained tissue sections using the Inform ™ software. Of note, positive and negative control slides need to be included in every experiment.

1. Incubate FFPE tissue slides at 55 °C for 2-16 h.

2. Prepare two equal heat-resistant glass trays containing 1× Trilogy ™ solution. Place slides in a glass slide holder and transfer it into one of the trays so that all tissues are fully immersed in the buffer without filling the tray to the brim. Leave the second tray with an equal amount of Trilogy ™ solution without slides.

Image and Data Processing

Immunostaining

Slide Pre-processing

Karīna Siliņa et al.

3.

Fill the pressure cooker with 2 L of ddW, and place a heatresistant platform (e.g., an autoclavable tip box) to prevent submersion of the slide trays inside the pressure cooker.

4. Cover both trays with loose lids and place them in the pressure cooker on a stove. Close the lid of the pressure cooker, turn the stove on, and wait until the pressure has reached its maximum, which is indicated by the fully lifted pressure nob on the pressure cooker's lid.

5. Autoclave the slides for 15 min, then remove the cooker from the heat, and release the pressure by keeping the pressure nob lifted using long forceps or other heat-resistant tool (see Note 12).

6. Immediately after opening the pressure cooker, carefully transfer the slides to the second tray containing the hot Trilogy ™ solution. Do not shake any of the trays or the slides to avoid boiling retardation, which will damage the tissue.

7. Incubate slides in the second tray for 5 min to remove remaining paraffin residues.

8. Transfer the slides to another tray containing ddW and place it on a rocking platform for 5 min (see Note 13).

9. One by one, dry the glass area surrounding the tissue with a paper napkin and encircle the tissue with the PAP pen. Do not allow the tissue to dry at any time throughout the duration of the protocol. 3. Decant the primary antibody mixture and wash in WB for 3 × 5 min on a rocking platform. 21. Decant the DAPI solution and wash slides in WB for 5 min on a rocking platform.

22. One by one, mount the coverslips using an antifade mounting medium that is suitable for the detection of fluorophores emitting also in the far-red spectrum (see Note 8).

23

. Store slides at 4 °C until imaging as well as for long-term storage.

The process described below corresponds to the standard operating procedure of the Vectra 3.0 ™ multispectral imaging system with automated slide loading. This microscope is equipped with a LED white and UV light source, 1 bright-field (position 1) and 5 fluorescence excitation/emission filters (positions 2-6), and the Nuance ™ multispectral camera (PerkinElmer). The excitation/ emission data and the respective detection filters for each fluorophore/maker are depicted in Table 2.

For clarity, in the description of the use of the dedicated software, we have highlighted buttons and tabs in bold italics and the options of choice in italics.

1. Using the Edit Protocol tab of Vectra ™ software, indicate the use of 10× objective for the whole-slide overview scan and 20× objective for the multispectral high-power field (HPF) acquisition. Determine the optimal exposure times for each of the five fluorescent channels for each of the objectives using slides that have been stained with only a single fluorophore. Exposure times between 50 and 100 ms are recommended. Set DAPI as the reference signal for finding the focal plane and choose the Saturation Protection option. Save the scanning protocol in the respective study folder.

2. Using the single-color-stained slides and the determined exposure times, find an optimal region of interest with the highest marker expression and acquire a multispectral HPF snapshot

Multispectral Imaging

Establishment of Imaging Protocol

Analysis of TLS by Quantitative Pathology for each individual fluorophore. Save these snapshots under the respective fluorophore name.

3. Using the unstained slide and the determined exposure times, acquire a multispectral HPF snapshot of a tissue area containing blood vessels, fibers, or other autofluorescent structures. Save this snapshot under the Autofluorescence name.

1. Using the Scan Slides tab of Vectra ™ software, generate the task list containing the slide names with the respective positions in the slide-loading cassette, indicate the name of the generated scanning protocol, and choose the Prescan task, which will generate whole-slide overview scans.

2. Perform whole-slide scans of all slides of interest stained with the seven-color protocol.

3. Open whole-slide scan images using Phenochart ™ software and select the regions of interest for multispectral HPF acquisition for all slides of interest, optimally containing one TLS per HPF.

4. In the Scan Slides tab of Vectra ™ software, modify the task list by selecting the Acquire MSI Fields task, which will automatically find the position of the selected HPFs from the position annotations generated by Phenochart ™ software for each slide.

5. Perform multispectral HPF acquisition.

1. Generate the spectral library of the used fluorophores by choosing the Build Library tab in Inform ™ software. Open the multispectral HPF snapshots of each individual fluorophore, extract, and save the respective spectral profiles (Fig. 1, top left).

2. Open 3-5 multispectral HPF images from each slide of interest and the Autofluorescence image in Inform software ™ . To perform spectral unmixing, select the seven fluorophores from the generated spectral library. Using the Autofluorescence Selection tool draw a line over the regions with most autofluorescent signal in the Autofluorescence image. By clicking on the Prepare Slides button, the software will perform unmixing of all indicated fluorophore and autofluorescence profiles, which can now be viewed as individual channels or in any combination assigning colors as necessary (Fig. 1, top right). Figure 2a depicts the detection of each individual marker and the marker combination in human tonsil. Figure 2b (left) depicts a GCcontaining TLS in a human lung tumor section. Next, save the established image view settings as an Inform ™ project file.

Slide Scanning

Image Analysis

Spectral Unmixing

Karīna Siliņa et al.

1. Configure the Inform ™ project by activating the Trainable Tissue Segmentation, Cell Segmentation, and Phenotyping options. Because FDCs and HEVs are cells with highly irregular shape, we recommend using tissue segmentation rather than cell segmentation for quantification of these cell types.

The cell segmentation and phenotyping algorithms can be reliably trained to recognize T cells, DCs, and B cells. 2. In the Review and Merge tab of Inform ™ software, select the folder containing the exported data. This opens a review window where exported images can be verified and images with mistakes in segmentation or artifacts can be excluded from the final data file. After reviewing all images, click on the Merge button to generate a merged text file containing data from all approved images. This also creates a second file containing data from the rejected images (Fig. 1, bottom right).

In the

3. Using RStudio, create an Excel file by arranging the data from the merged text file according to tissue categories for each patient, and generate a summary data table containing the information about the size of analyzed tissue categories and the number of each cell phenotype in the different tissue categories for each patient (Fig. 1, bottom right). The data can now be subjected to various statistical analyses. Specific R scripts are available upon request.

Notes

1. Cut FFPE tissue sections can be stored at 4 °C for up to 3 months without apparent decrease in signal for the described antigens.

2. A microwave, which allows for the regulation of power intensity, can be used instead of a pressure cooker (see the Opal ™ 7-color Manual IHC Kit description for the requirements of the microwave).

Image Data Processing

3. We have compared the Trilogy ™ solution pretreatment with the conventional pretreatment protocol (xylene deparaffinization, graded ethanol rehydration, and antigen retrieval by autoclaving in 10 mM citric acid pH 6.0, or Tris-EDTA buffer pH 9.0) and found that the immunostaining signal intensity was superior for all antibodies using the Trilogy ™ solution.

4. We have tested several PAP pens and found that the ImmEdge pen from Vector Laboratories stays on the slides after multiple washing and heating rounds without the necessity to redraw the border, which saves a considerable amount of time especially when processing many slides in parallel.

5. The optimal dilution of the primary antibodies should be determined for your experimental setting and antibody manufacturer. As a rule of thumb, the use of TSA provides strong immunostaining signals using 5-10 times lower primary antibody concentrations than in conventional immunostaining protocols using fluorophore-labeled secondary antibodies.

6. It is important to use secondary antibodies with the least possible cross-reactivity due to the high sensitivity of the tyramide signal amplification approach. The dilutions will depend on the manufacturer.

7. A microwave (see Note 2) can be used instead of the water bath.

8. Not all antifade mountants are suitable for the detection of far-red dyes. Make sure to use one that is. In our experience, Prolong ™ Diamond mounting medium provides reliable detection of all above-described fluorophores for up to 3 months. 9. Use a separate washing tray for the unstained slide to avoid any fluorescent dye transfer especially DAPI from the stained slides.

10. Use separate washing trays for the single Opal ™ color-stained slides to avoid any fluorescent dye transfer especially DAPI from other stained slides. This is crucial for the identification of the pure spectral profiles for each dye, which is necessary for the spectral unmixing step by the Inform ™ software.

11. Matlab, Excel, or other suitable software can be used instead of RStudio.

12. Protecting your hand with a towel keep the pressure nob lifted. Take care not to release the pressure too fast as this might cause boiling of the solution inside the slide trays causing tissue damage.

13. Using ddW instead of WB for this step makes it easier to dry the glass surface surrounding the tissue in order to draw the barrier.

14. Slides can be kept in the WB at 4 °C for up to 3 days.

Karīna Siliņa et al.

15. The incubation time for all antibodies except anti-PNAd could be reduced to 2 h at RT. 16. The manufacturer recommends diluting the Opal ™ tyramidedyes 1:50. In our experience, 1:100 dilution is detected equally well, with the exception of Opal-690. Because Opal-650 and Opal-960 are detected through the same microscope filter (see Table 2), it is crucial to establish such antibody and tyramidedye dilutions that are detected with the same exposure time. A recommended exposure time for all filters is 50-100 ms. In our experience, Opal-650 needs to be diluted 1:150 and Opal-690 1:50 to achieve the same exposure time and to obtain signals that can be reliably unmixed by Inform ™ software.

17. Here, we have combined three primary antibodies raised in different species in the same incubation step. This allows to use TSA by sequential incubation of HRP-conjugated secondary antibodies (with minimal cross-reactivity) in combination with H 2 O 2 treatment after each tyramide reaction. This quenches the HRP of the previous antibody before applying the next antibody followed by the next tyramide-dye conjugate. This way no heat-induced antibody removal is necessary, which decreases the chance of tissue and epitope destruction and considerably shortens the duration of the manual protocol. Antibody removal by heating is necessary only after all three secondary antibodies have been used in order to proceed with the next round of primary antibodies. If, however, most primary antibodies were raised in the same species, heatinduced antibody removal is necessary after each tyramide reaction. In this case, it is important to determine which epitopes are most sensitive to multiple heating rounds in order to establish the optimal staining order.

Introduction

The immune response to cancer is fundamentally complex, with a variety of cell types and numerous proteins present in various regions of the tumor microenvironment. In the field of immunooncology, the relationships between such cells expressing one or another protein is a particular area for which our understanding seems limited. Tertiary lymphoid structures (TLS) exemplify a microcosm of this overall spatial complexity, and are important in that their presence indicates the existence of an active immune response to a number of cancers [1,2]. Multiplex immunohistochemistry (IHC) or immunofluorescence (IF) to label multiple proteins in tissue sections is a useful means to get increased amounts of information out of limited available tissues or to demonstrate relationships among several key cell types expressing one or another protein. A number of multiplex IHC and IF methods have been reported, each with advantages and disadvantages [3][4][5][6][7][8]. As a practical solution suitable for use by most research histopathology laboratories currently, we find the use of multiplex IHC to provide substantial value in understanding the complexity of TLS in terms of the different cell types present and the key immune proteins they express. Further value can be gained when this approach is combined with image analysis.

Here, we demonstrate a single assay that combines two individual chromogenic IHC segments performed on two serial sections of formalin-fixed paraffin-embedded (FFPE) human lung cancer to demonstrate cells that express PD1, PD-L1, CD3, and CD68 in the context of cytokeratins. The complexity of performing this procedure should be obvious, as illustrated by the number of individual reagent steps involved (not counting wash steps). The use of automated immunostaining instruments is therefore almost a requirement to achieve quality results. Based on this combination of multiplex IHC, we illustrate the kinds of information evident in the tumor microenvironment and TLS. And to that, we add digital transformation of classified images to illustrate a "virtual IF"-like single image. This combination takes advantage of the relative simplicity of chromogenic IHC and bright-field digital scanning. It further takes advantage of image analysis to combine these markers to better understand the localization of individual cell types as well as the relationships among the different marker-positive cells within the TLS and other parts of the complex tumor microenvironment. We use this approach to also illustrate how additional data parameters can be generated from images processed in this way.

Materials

Methods

Prepare all bulk instrument reagent solutions according to the manufacturer's instructions, unless otherwise specified below. Follow all safety, hazardous material handling, and hazardous waste disposal procedures according to applicable regulations and institutional requirements. Follow all manufacturer's instructions for use of individual automated IHC instruments, unless otherwise specified below. Wash steps, omitted below, are also to be completed according to the manufacturer's instructions. 1. Unless otherwise noted, seemingly similar reagents (e.g., Tris-EDTA buffer) are not used interchangeably between automated IHC stainers. We typically utilize the appropriate reagent supplied by each manufacturer for use on their instrument.

2. This combined "5-plex" assay requires two segments of 2 markers + 3 markers because the numbers of quality chromogens available commercially are limited and different chromogens may not have suitable spectral characteristics to combine for a given set of markers on a single tissue section. Thus, our assay represents a compromise approach that requires two tissue sections, to each of which is applied a single combination of markers. As explained subsequently (see Note 12), it is possible to combine all five markers visually or through the use of digital software that co-registers paired immunostained sections in order to form a single "virtual multiplex" of the combined markers.

3. The specific positive control tissues to be included are at the discretion of the user. Human tonsil contains cells expressing all five markers included in this assay, and is affordable and readily available as FFPE blocks. Human tonsil is therefore recommended. For positive control tissue, pairs of tonsil sections should be run in parallel. One section receives all primary antibodies, in proper sequence, while the second section receives isotype-matched (rabbit and mouse) immunoglobulins in place of the primary antibodies, also in sequence. Duplicate sections of test samples may be included for application of isotype antibodies. We typically do not include them in the assay described.

Having optimized and tested these assays in a variety of tumor and normal tissues, we find their inclusion to rarely provide helpful information, that is, unexpected staining patterns (see also Note 7).

4. The application of these reagents combined with high heat serves as a denaturation step to strip the preceding primary antibody and polymer-labeled enzymes, but not the preceding chromogen. This prevents interference between these molecules and the subsequently applied reagents.

5. The HRP-reactive purple chromogen can currently only be applied on the Ventana autostaining instruments. We find the use of this particular purple chromogen to be very useful for combining with DAB and yellow chromogens. Thus, we feel the effort to transfer slides between instruments is of sufficient value to enable the performance of this and similar multiplex IHC stains done in our lab.

6. This step removes the "liquid coverslip" that is part of the Discovery autostaining instrument prior to dehydration and coverslipping.

7. Microscopic analysis of the positive control tissues is the primary quality control step for each IHC "run." FFPE sections of human tonsil should have all markers included in this multiplex assay present. The exact numbers and locations of cells expressing the five included markers may vary somewhat, however, from one tonsil specimen to another. PD1 + lymphocytes are typically intensely labeled within germinal centers and should also be evident in other locations. PD-L1 + intensely labeled epithelial cells should be evident in tonsillar crypts and other cells (e.g., macrophages in germinal centers) should also be labeled. CD68 + macrophages should be evident in germinal centers and elsewhere. CD3 + lymphocytes should be evident in T-lymphocyte-dependent regions such as the parafollicular zone, and elsewhere. Cytokeratin labeling should be evident in epithelial cells of the tonsil per se, or any adjacent epithelial tissue (e.g., salivary gland) that may also be present. It is important to utilize the same positive control tissue in the case that multiple "runs" of that assay are performed. And then, confirm that the staining qualities of immunostaining for each marker are consistent among the different runs. This quality control aspect is necessary to ensure that both the sensitivity and specificity of staining are appropriate for all runs from which data is generated. In addition, tonsil sections incubated with isotype control immunoglobulins (negative control) should be examined to assess appropriate (i.e., minimal) levels of nonspecific staining. Additional QC of the test slides is also required for general aspects of immunostaining because inappropriate staining of individual slides can occasionally happen, even for those stained on automated instruments.

8. Digital scanning at high magnification is slower and generates image files of greater size than at lower magnification. However, it also provides greater resolution of histological information for either visual inspection or potentially relevant to the quality of image analysis data. We typically scan using a 20× objective lens, but occasionally 40× scans are required. Each user should take these factors into account to determine the image resolution that meets the needs of their study. 9. We frequently use Aperio I ImageScope ™ to view images scanned on the Aperio scanner. Other image visualization software may equally serve the needs of other users without access to ImageScope. We also use the proprietary Definiens VeriTrova ™ software (Definiens AG, Munich, Germany) for image viewing and to perform other processes related to computational image analysis.

10. The initial value of the multiplexing approach described here is evident in the two separate images shown in Fig. 1 viewed adjacent to each other. Accordingly, PD1 + lymphocytes (Fig. 1a, brown) are conspicuous in the germinal centers (GCs) of TLS of the lung cancer specimen shown. They are present as well in T-zone regions, where they co-localize with CD3 + lymphocytes (Fig. 1b, purple), and also in the tumor microenvironment. Note the intensity of PD1 labeling in the GCs in particular versus other PD1 + cells. CD68-positive macrophages (Fig. 1b, brown) are present mainly in GC (i.e., tingible-body macrophages), T-zone regions, and infiltrating tumor islands.

A few PD-L1 + cells (Fig. 1a, purple) are present in the GC; however most PD-L1 + cells are neoplastic cells and macrophages of the tumor region (Tu), as well as small rafts of intensely positive epithelioid cells in TLS (arrowheads) that also express cytokeratins (Fig. 1b, yellow). The co-localization with cytokeratins identifies these rafts of PD-L1 + cells as epithelial; however their origin is otherwise uncertain.

11. We use Definiens Developer XD ™ software to perform image analysis, though other commercial and publicly available software can be used. We frequently apply regional tissue annotations manually to scanned images prior to image analysis. This serves to partition the subsequent data according to the specific tumor regions in which particular marker-positive cells reside. Manual annotations are done using Aperio ImageScope or Definiens VeriTrova software, and drawn either with a computer mouse or using an interactive pen tool and a touch screen display (e.g., Wacom Cintiq ™ ). Examples of annotations appropriate to analysis of TLS are shown in Fig. 2, representing a larger field of view of the lung tumor shown in Fig. 1. Several TLS are annotated (blue). The tumor invasive margin is annotated separately (green) to mark a band of tissue approximately 500 μm wide and centered on the outermost neoplastic cells.

The tumor center represents a third annotation (red) to include tumor inside the invasive margin. The use of suitable image analysis software to classify cells expressing one or another of the five markers included in this multiplex assay can therefore separate data according to these tumor regions.

12. The classification of marker-positive cells by image analysis software (Developer XD ™ ) for individual tumor sections can be digitally overlaid to produce a pseudo-colorized virtual multiplex (see Fig. 3). Co-registration of the CD3/CD68/cytokeratins and PD1/PD-L1 images was performed, as previously described [9]. In this image, CD3 + lymphocytes are colored green, PD1 + lymphocytes yellow, CD68 + macrophages blue, and PD-L1 + cells red. Relationships among these various cells can therefore be understood visually in a more direct way. For example, the prevalence of PD1 + cells in the T-lymphocyte-rich zone of the TLS, that is co-localized with CD3 + lymphocytes, becomes more easily seen than in Fig. 1. A limitation of this approach, however, is that detection of co-expression of PD1 and CD3 is not possible, mainly because these two markers were performed on separate tissue sections. Another observation evident here, and one specifically relevant to immune oncology, is the spatial relationship between PD1 and PD-L1 in various regions of the tumor microenvironment. In this single image, a number of PD1 + lymphocytes are nearby to PD-L1 + cells, both in the GC and elsewhere. Such a proximity relationship between this important immuno-inhibitory receptorligand pair is relevant and has even previously been reported to correlate with therapeutic response to pembrolizumab in melanoma patients [10]. This permits more direct identification of the spatial relationships between markers, for example, PD1 + lymphocytes (yellow) versus PD-L1 + cells (red, e.g., macrophages) in the germinal center (GC) and CD3 + lymphocytes (green) and CD68 + macrophages (blue) in multiple regions. A limitation of this approach is the determination of dual-positive cells when markers are applied to separate tissue sections (cytokeratin was intentionally left out of this virtual multiplex to permit visualization of PD-L1 + cells in tumor islands (Tu) and TLS (*) also depicted in Fig. 1). Original magnification: 200×

Introduction

High endothelial venules (HEVs) are structurally and antigenically distinct blood vessels especially adapted for lymphocyte trafficking [1]. HEVs develop in every secondary lymphoid organ except the spleen (i.e., lymph nodes (LN), tonsils, and Peyer's patches) during embryonic and neonatal life, and are fully integrated into the blood vasculature supplying these organs. The endothelial cells (EC) lining HEV in neonatal mice are immature and express the vascular addressin, mucosal addressin cell adhesion molecule (MAdCAM)-1. During the first weeks of neonatal life, HEVs mature into peripheral node addressin (PNAd) expressing HEV [2]. PNAd expression is induced by gut-derived dendritic cells in neonates [3] and maintained by lymphotoxin-β receptor (LTβR) signaling in HEV EC stimulated by LTαβ-expressing dendritic cells and lymphocytes [4,5]. HEVs play a critical role in delivering naïve and memory T and B lymphocytes, regardless of antigen receptor specificity, from the bloodstream into lymphoid organs under homeostatic conditions. Here, lymphocytes scan dendritic cells and stromal networks for activating, tolerogenic, and homeostatic stimuli. HEVs, therefore, regulate the outcome of an immune response be it activation, tolerance, or homeostatic proliferation of lymphocytes. PNAd-expressing blood vessels that resemble structurally distinct HEV in LN develop postnatally in nonlymphoid organs during chronic inflammation driven by autoimmunity, infection, and allografts [6]. These extranodal HEVs are characteristically surrounded by dense lymphocytic infiltrates organized into lymph node-like structures with discrete T-and B-cell-rich areas and germinal centers and are called tertiary lymphoid structures (TLS). Extranodal HEVs facilitate entry of blood-borne lymphocytes and are therefore critical to the development and immune function of TLS. HEV-containing TLS also develop in solid, vascularized cancers and are receiving increasing attention because of their potential role in regulating immunity at the tumor site [7,8].

The presence of TLS in resected solid cancers has been correlated with prolonged patient outcome in some cancers such as breast cancer [9,10], melanoma [11,12], and lung cancer [13]. In other cancers, TLS have been shown to either promote carcinogenesis such as virus-associated hepatocellular carcinoma [14] or accumulate with disease progression such as in primary breast carcinoma [15]. In colorectal cancer, TLS often containing germinal centers (Crohn's-like aggregates) develop in the surrounding peritumoral stroma as well as inside cancerous tissue. The number of TLS in colorectal cancer has been reported to correlate either with improved patient outcome or with disease progression depending on the stage of the disease [16][17][18][19].

In breast cancer and melanoma, the density of HEV correlated with improved patient outcome highlighting the important role that HEVs play in orchestrating anticancer immunity [20,21]. HEV neogenesis correlates with regression of established tumors in preclinical mouse tumor immunotherapy models, such as depletion of Foxp3 + regulatory T cells [22,23] or combined checkpoint blockade inhibition and angiogenesis therapy [24]. HEV neogenesis and tumor regression are also seen when the TNF superfamily member LIGHT (TNSF14) (which signals via LTβR, an important driver of lymphoid organ development) is expressed by tumor cells or targeted to tumors or tumor blood vessels [25][26][27]. It is Emma Jones et al.

thought that the antitumor effects are due to the generation of cancer cell-destroying lymphocytes from naïve T cells which have been recruited from the bloodstream into the cancer by newly formed HEV.

PNAd-expressing blood vessels lined by flat endothelial cells, rather than the cuboidal or plump endothelial morphology typical of mature HEV, are found inside cancer-induced ectopic lymphoid aggregates that are not organized into distinct T/B-cell areas [28]. These could represent immature HEV-containing structures in the process of forming TLS, or dedifferentiating HEV as found in reactive LN or following disruption of the LN microenvironment [5,29]. Interestingly, PNAd-expressing blood vessels that form following depletion of Foxp3 + regulatory T cells from methylcholanthrene-induced tumor-bearing mice are not generally associated with histologically distinct, TLS [22] which suggests that HEV neogenesis may occur independently of lymphoid neoorganogenesis. The development of HEV in the absence of fullblown TLS correlates with T-cell infiltration and cancer regression in this experimental model, highlighting the important role of HEV in controlling antitumoral immunity.

PNAd-expressing blood vessels have been reported in preclinical mouse tumor models particularly following immunotherapy. In tumor cell transplant models, PNAd expression is induced in tumor blood vessels by infiltrating CD8 + T cells and NK cells [30]. In marked contrast to HEV development in LN, PNAd expression is not dependent on LTβR signaling but is stimulated instead by lymphotoxin α3 (LTα3). Although comprising <10% of the tumor vascular network and lined by flat endothelial cells PNAd-expressing tumor blood vessels are functional in that they recruit naïve, L-selectin-expressing T cells from the bloodstream into the tumor where they are activated to kill tumor tissue [30]. In methylcholanthrene-induced fibrosarcomas in mice, HEV neogenesis is also not dependent on LTβR but is driven mainly by T-cellderived TNF-α and LTα3 [23]. However, HEV neogenesis during antiangiogenic/anti-PD-L1 therapy is dependent on LTβR signaling and in poorly infiltrated tumors where HEV neogenesis is low, such as glioblastomas, the combination of agonistic antibodies to LTβR alongside antiangiogenic/anti-PD-L1 therapy boosts HEV neogenesis [22]. Together, these findings suggest that TNFR and/ or LTβR signaling in endothelial cells stimulates the development of PNAd-expressing HEV blood vessels that promote antitumor immunity by recruiting naïve T cells into cancerous tissues.

We are studying the role of homeostatic T-cell trafficking via HEV and the mechanisms underpinning HEV neogenesis in controlling local immunity inside mouse tumors and human cancers. We use immunohistochemistry to identify HEV in clinical and murine tissues by detection of PNAd, a specific marker of HEV (Fig. 1). We measure L-selectin-dependent recruitment of naïve T HEV and TLS in Cancer lymphocytes from the bloodstream into tissues, via mature, PNAdexpressing HEV, in mice using short-term homing assays and analyze tissue-infiltrating T cells by flow cytometry (Fig. 2) [START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF][32][33] and immunohistochemistry to colocate infiltrating T cells and HEV (Fig. 3) [START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF]34].

Materials

All reagents which are not commercially available should be prepared with deionized, sterile water. All reagents are stored at +4 °C unless otherwise specified by the manufacturer. Tissues should be The following reagents are used in all methods: calcium-and magnesium-free phosphate-buffered saline (PBS), complete RPMI 1640 growth media supplemented with penicillin and streptomycin (RPMI), heat-inactivated (30 min at 56 °C) fetal calf serum (FCS), and 70% alcohol for sterilization. The antibodies used for immunofluorescence staining of frozen or paraffin-embedded tissue sections and flow cytometry of T cells are listed in Table 1.

1. Tissue-Tek plastic base molds (Thermo Fisher Scientific).

2. Optimum cutting temperature (OCT) compound.

3. Dry ice with or without ethanol for snap freezing murine tissue in OCT.

4. Cryostat capable of cutting cryosections. 6. Humidified chamber containing wet paper towel to prevent evaporation of staining reagents. be revealed using antibodies to T cells (e.g., rabbit anti-human CD3, which also detects mouse CD3, DAKO A0452, used at 2 μg/mL), B cells (biotinylated rat anti-mouse/human CD45R/B220, clone RA3-6B2, rat IgG2a, used at a working concentration of 2 μg/mL, Biolegend), and FDC (rat antimouse FDC, clone FDC-M1, rat IgG2c, used at a working concentration of 2 μg/mL, BD Biosciences).

Immunolabeling HEV and TLS in Murine Tumor Cryosections

13. For every antibody used, an isotype control antibody should be used at the same working concentration, in parallel on another section to check the staining is specific. A rat IgM isotype control antibody is used to check MECA-79 staining is specific. Biotinylated rat IgG2a is used to check pan-EC antigen (clone MECA-32), MAdCAM-1 (clone MECA-367), and B cell (clone RA3-6B2) staining is specific (Fig. 1). Rat IgG2c is used to check FDC (clone FDC-M1) staining is specific and rabbit IgG polyclonal isotype control is used to check CD3 (DAKO A0452) staining is specific. 17. Glass coverslips (thickness #1.5 coverslips should be used in fluorescence microscopy to obtain the brightest images with minimal spherical aberration; Thermo Fisher Scientific).

1. Tissue-Tek plastic base molds (Thermo Fisher Scientific).

2. Neutral buffered formal saline (NBFS).

3. Paraffin wax (Shandon Histoplast, Thermo Fisher Scientific).

4. Microtome capable of cutting paraffin sections (e.g., Thermo Fisher Scientific).

5. Blades (MX35, Thermo Fisher Scientific).

6. Hydrophobic pen for creating a hydrophobic barrier around tissue section (e.g., Pap pen, Vector).

Immunolabeling HEV and TLS in Paraffin-Embedded Murine Tumor Sections

7. Humidified chamber containing wet paper towel to prevent evaporation of staining reagents.

8. 100% Xylene.

9. Graded alcohols: 100, 95, 90, 80, and 70% ethanol.

10. PBS for washing steps.

11. Peroxidase blocking solution: Either 1% hydrogen peroxide/ methanol or a commercial blocking reagent (e.g., Bloxall; Vector) (see Note 7).

12. Serum blocking solution to block nonspecific binding of antibodies: Often 2.5-5% serum of the species in which the secondary antibody is made is used. Other sera for example horse or mouse serum may also be used.

13. Primary antibody diluted in 1% BSA in PBS: Rat anti-mouse PNAd antibody (MECA-79, rat IgM, used at a working concentration of 2.5 μg/mL) can be used to detect HEV. 4. Labeled tubes containing 1 mL ice-cold PBS for collection of spleens from T-cell donor mice.

5. Labeled tubes containing 1 mL ice-cold PBS for collection of cancerous tissues, lymph nodes, and spleens from experimen-

L-Selectin-Dependent Homing of Naïve T Lymphocytes in Mice

Emma Jones et al.

tal mice after injection of donor T cells (labeled with mouse number (e.g., 1-5) and organ (e.g., cancer, spleen, LN)).

6. Red blood cell lysis buffer.

7. Naïve T-cell isolation kits using negative selection.

8. Cell tracker dye: Carboxyfluorescein diacetate-succinimidyl ester (CFSE, Molecular Probes).

9. Fluorescently conjugated antibodies to TCR (clone H57-597, Armenian hamster IgG), L-selectin/CD62L (clone MEL-14, rat IgG2a), and CD44 (clone IM7, rat IgG2b). Low endotoxin, azide-free antibodies to L-selectin (MEL-14, rat IgG2a) and rat IgG2a isotype control for T cell homing studies in mice.

10. Rat-anti mouse PNAd (clone MECA 79, rat IgM), fluorescently conjugated or biotinylated anti-rat IgM to detect MECA79 in tissue sections.

11. Fixative: 2% Formaldehyde/5% sucrose in PBS, 20% sucrose in PBS.

12. Freezing and storing tissues: Liquid nitrogen.

Methods

1. Collect tumor tissue and place immediately in optimal cutting temperature compound (OCT) in a plastic mold. Add sufficient OCT to completely cover the tissue.

2. Snap-freeze in dry ice and store blocks in small plastic ziplock bags at -80 °C until needed.

3. Cut 5-10 μm sections on a cryostat and mount sections on glass slides. Typically, we mount two sections per slide.

4. Allow slides to dry for approximately 1 h at room temperature (RT) and store in a slide box at -80 °C until needed.

5. Remove slides from freezer, allow them to warm up to RT, and wipe away any residual moisture, taking care not to touch the tissue section.

6. Fix sections in ice-cold 100% acetone, 4% formaldehyde, or PLP fixative for 10 min (see Note 1). Wash slides three times in PBS over 5 min at RT.

7. Drain slides and carefully wipe off excess liquid before next step. Be careful not to let the section dry out.

8. Draw a circle around each section with a hydrophobic marker.

9.

Optional: If fixed in 4% formaldehyde or PLP quench free aldehydes with 0.3 M glycine in PBS for 10 min at RT and then wash slides three times in PBS over 5 min at RT.

Immunolabeling HEV and TLS in Murine Tumor Cryosections

HEV and TLS in Cancer 10. Optional: If using a biotinylated antibody block endogenous biotin with avidin/biotin block and then wash slides three times in PBS over 5 min at RT.

11. Block nonspecific protein binding with serum-blocking solution for 30 min at RT.

12. Drain slides (no need to wash the slides).

Incubate with MECA-79 antibody or rat IgM isotype control in 1% BSA/PBS overnight at 4 °C or for 1 h at RT in a humidified chamber. Optional: Include other antibodies such as MECA-367 to identify immature HEV, MECA-32, to confirm that MECA-79 is detected on EC (see Fig. 1) or T-cell, B-cell, and FDC antibodies (Table 1) to determine if TLS are present.

13. Wash slides three times in PBS over 5 min at RT.

Immunolabeling HEV and TLS in Paraffin-Embedded Murine Tumor Sections

Emma Jones et al.

4.

Precool tissue blocks on ice or on a cold plate (Leica).

5. Cut 5-10 μm sections on a microtome and mount on glass slides.

6. Place slides in a slide rack at 60 °C for 1 h to overnight to melt off wax.

7. Hydrate sections in a slide chamber with 3× 100% xylene washes, 5 min each, and then descending alcohols (100, 100, 90, and 70%) for 3 min each. Wash in running water for 5 min and then rinse in dH 2 0.

8. Use either Tris/EDTA (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0) or sodium citrate (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) antigen retrieval buffer for heat-induced epitope retrieval (see Note 6).

9. Wash slides three times in PBS over 5 min.

10. Drain slides and carefully wipe off excess water before next step. Be careful not to let the section dry out.

11. Draw a circle around each section with a hydrophobic marker.

12. Neutralize endogenous peroxidase activity with 1% hydrogen peroxide/methanol for 10 min at RT or alternatively use a commercial blocking reagent (e.g., Bloxall, Vector; see Note 7).

13. Wash slides three times in PBS over 5 min. 2. Place cell strainer in the neck of a labeled 50 mL polypropylene tube in a tube rack and pour spleen and PBS through cell strainer.

3. Split open paper covering of 2 mL plastic syringe, and remove plunger using paper covering to maintain sterility of flat end (not rubber seal end). Mash spleen using flat end of plunger. Wash strainer using 5 mL of PBS; repeat if necessary to completely disaggregate tissue; discard strainer.

4.

Seal tubes and collect splenocytes by centrifugation at 250 g for 5 min at +4 °C. Aspirate or remove PBS by pouring.

5. Tap the bottom of the tube to dislodge cell pellet, carefully resuspend in 2 mL red cell lysis buffer using 10 mL strippette, make up to 5 mL with red cell lysis buffer, and incubate for 5 min on ice. Add 10 mL PBS, collect cells by centrifugation, and repeat PBS wash once.

6. Isolate naïve T cells by negative selection using commercially available kits according to the manufacturer's instructions (see Note 12).

7. Resuspend T cells to 5 × 10 7 /mL in FCS-free PBS for labeling with CFSE (see Note 13) and remove 1 × 10 5 cells for staining to check naïve T-cell purity (see Note 14).

8. Label T cells in 2 μM CFSE in FCS-free PBS in the dark for 15 min at 37 °C, wash twice in 5-10 mL PBS containing 1% FCS, resuspend to 2.5 × 10 7 cells/mL in saline, and split into two equal aliquots. Collect cells by centrifugation and preincubate one aliquot with 100 μg/mL rat anti-mouse L-selectin (clone MEL-14, rat IgG2a) and the second aliquot with 12. Lyse red blood cells in spleen, tumor, and peripheral blood, as described in step 5.

L-Selectin-Dependent Homing of Naïve T Lymphocytes in Mice

13. Analyze CFSE-labeled lymphocytes on a flow cytometer analyzer using the fluorescein isothiocyanate filter. Data on 2 × 10 5 viable cells in spleen and blood, and 5-10 × 10 5 viable cells for LN, is acquired and the data analyzed using FlowJo software. The percentage of CFSE-labeled cells recovered in the spleen, blood, and each group of LN is determined and compared between control and treatment groups. If L-selectindeficient T cells are available label using CMTMR, mix them 1:1 with CFSE-labeled L-selectin-sufficient T cells, and inject a total of 20 × 10 6 cells into a single group of experimental mice. In tissues with mature HEV, L-selectin-sufficient T cells are highly enriched over L-selectin-deficient T cells (Fig. 2).

14. Cryostat sections of 8 μm are cut from fixed, frozen tissues (see step 11), HEV stained using MECA-79 and red fluorescenceconjugated secondary antibody, as described in Subheading 3.1, step 12, but without additional fixation (step 6). The presence of TLS in fixed tumor tissue may be revealed using antibodies that detect T and B cells in formalin-fixed paraffinembedded tissues (Table 1). The position of CFSE-labeled lymphocytes (green) in relation to HEVs (red) is determined

HEV and TLS in Cancer by fluorescence microscopy using a confocal laser scanning microscope. For each tissue, 10-15 images containing complete cross sections through 30-55 HEVs (average total HEV area 2500 μm 2 ) are collected and analyzed using Image J-NIH software. The total cross-sectional area of HEVs and the remaining area within each image are calculated. CFSE-labeled cells "inside HEVs" are those attached to the luminal surface of the vessel wall and within the HEV wall (Fig. 3a; arrows). The remaining CFSE-labeled cells are scored as "outside HEVs" (Fig. 3a

Notes

1. Fixation is required to maintain tissue morphology. The choice of fixative depends on the antigen and antibody used to detect it. Formaldehyde is a cross-linking fixative and maintains good tissue architecture; however, some antigens cannot easily be detected after formaldehyde fixation (e.g., mouse CD4). Acetone removes lipids, dehydrates cells, and precipitates proteins so cell structure is poorly preserved but may allow better detection of some antigens (e.g., mouse CD4). PLP can be prepared with varying concentrations of formaldehyde and was designed to primarily cross-link carbohydrates to maintain antigenic sites on proteins. Since membranes are rich in glycoproteins and glycolipids, PLP provides good preservation of membrane antigens of the immune system. HEVs are detected well with all of these fixation methods; however if other cells of the immune system are also immunolabeled then different fixation protocols should be tested.

2. MECA-79 is a rat IgM and can precipitate over time resulting in speckled staining at which point the antibody needs to be discarded.

3. MECA-79 can detect HEV in both mouse and human cryosections or paraffin sections. However other antibodies, for example, clones MECA-32 and MECA-367, are mouse specific so alternative human-specific antibodies would be needed.

4. When two rat primary antibodies are used simultaneously (e.g., clones MECA-79 and MECA-367 or MECA-32) fluorescently or biotin-conjugated primary antibodies or isotype-specific secondary antibodies should be used so that anti-rat secondaries are avoided.

5. Since MECA-79 is a rat IgM antibody, it can be detected with an anti-rat IgM secondary antibody. However this secondary antibody will cross-react to endogenous mouse IgM, which can be detected in plasma cells in mouse tissue. Plasma cells can be identified by co-staining with anti-mouse CD138 or alternatively MECA79 staining of HEV can be positively identified by morphology or co-staining with a pan-EC marker.

6. Either Tris/EDTA or sodium citrate antigen retrieval methods are both suitable for detecting HEV in paraffin tissue sections. The choice of antigen retrieval is determined by other antibodies used to sequentially stain tissue sections.

7. Hydrogen peroxide/methanol will quench endogenous peroxidase that would otherwise react with the HRP substrate (e.g., DAB) resulting in undesirable background. Some proteins may be sensitive to methanol or hydrogen peroxide treatment and may reduce staining of the antigen; in this case quenching with hydrogen peroxide/methanol may be performed after the primary antibody incubation step or alternatively a commercial blocking reagent such as Bloxall may used.

8. It is difficult to distinguish by eye two colocalized stains on a single structure or cell by immunohistochemistry, for example, MECA-79 and an EC marker. If this is required immunofluorescence is recommended. However, spatially separated antigens can be detected by immunohistochemistry.

9. Light staining with hematoxylin for 10-20 s is usually sufficient. However, if sections are stained sequentially with multiple antibodies, it may be unnecessary to counterstain with hematoxylin since the antibody staining might be obscured.

10. Each mouse receives 5-10 × 10 6 labeled donor T cells and two groups of 5 mice are required per experiment, one group receiving naïve T cells pretreated with control antibody and the second pretreated with anti-L-selectin antibody to inhibit binding of naïve T cells to HEV. The total number of naïve T cells per experiment is therefore 50-100 × 10 6 T cells. The yield of T cells is 30-50 × 10 6 cells per spleen so 2-3 donor mice will provide sufficient T cells for each experiment. If donor mice are limiting, T cells can be isolated from peripheral (axillary, brachial, and inguinal) and mesenteric LN and pooled with splenic T cells.

11. Dissecting instruments are sterilized by autoclaving in sealed bags or by submerging in 70% alcohol.

12. We have used kits for isolating T cells and CD8 + T cells from Miltenyi Biotec and Stem Cell Technologies. In tissues with functional HEV, L-selectin-sufficient T cells are highly enriched over L-selectin-deficient T cells (Fig. 2). Cell tracker dyes need to be reversed to check for effects of cell tracker dyes on T-cell recruitment from the bloodstream via HEV in wild-type mice (Fig. 3b).

16. To avoid using cell tracker dyes, we have injected unlabeled T cells isolated from CD90.2 (Thy.1.2) mice and injected in CD90.1 (Thy.1.1) cancer-bearing mice and used antibodies to CD90.2 to detect donor T cells by flow cytometry and immunohistochemistry. 17. To measure recruitment directly from the bloodstream into tissues time points of 1-3 h are preferred since this is before T cells exit third-party organs such as the spleen or LN. However, the level of T-cell recruitment into cancerous tissues may be low and longer time points of 24-48 h are required [30,[START_REF] Szentistványi | Drainage of interstitial fluid from different regions of rat brain[END_REF].

18. Collect peripheral (axillary, brachial, inguinal) LN as positive controls for L-selectin/CD62L-dependent T cell homing.

Organs and tissue compartments that do not shown L-selectin/ CD62L dependent T cell homing include mediastinal LN, mesenteric LN, spleen, and peripheral blood (Fig. 2).

19. Tissue disaggregation using enzymes may increase the yield of infiltrating T cells from fibrotic tissues and cancers over mechanical disaggregation; this needs to be determined empirically for each type of tumor.

Introduction

Tertiary lymphoid structures (TLS) have been described in mammalians at various anatomical sites and in many inflammatory contexts, comprising cancer, autoimmune diseases, graft rejection, idiopathic diseases, and viral and bacterial infections [1][2][3][4][5]. TLS share most of the structural characteristics found within canonical secondary lymphoid organs (SLO) such as lymph nodes, including a T-cell zone infiltrated with mature dendritic cells [6][7][8], and a germinal center with a network of follicular dendritic cells and proliferating B cells [9][10][11][12], surrounded by lymphatic vessels and high endothelial venules that express peripheral node addressin (PNAd) [11,13].

The immunological function of TLS is the center of intense investigations as these structures have been recently appreciated as major drivers of the local adaptive immune reaction within various types of inflamed tissues [1,3,5]. For example, in autoimmunity or graft rejection, the presence of TLS has been associated with the aggravation of the disease and thus a deleterious effect [3,4]. Conversely, TLS presence in tumors or at sites of infection has been mainly associated with a better control of the disease and, most of the time, a favorable prognosis [1,7,14,15]. Thus, TLS are a newly appreciated driver of the local adaptive immune reaction that can have harmful or beneficial impact depending on the pathological context.

A number of methods that are described in this issue, like immunohistochemistry, flow cytometry, cell sorting, and deep gene analyses, are powerful approaches to deeply investigate the organization, composition, and immune function of TLS in various anatomical sites and pathological contexts. Nevertheless, interrogating the role of TLS in situ and deciphering their cellular and molecular interactions within the local microenvironment remain a major challenge, especially in humans, as these nonencapsulated structures spontaneously emerge within and/or adjacent to nonlymphoid tissue under inflammatory condition. To comprehensively interrogate the in vivo interactions between TLS and their environmental tissue, specific approaches allowing the molecular characterization of TLS in situ, that is, within the context of their intrinsic tissue microecology, are needed.

Laser capture microdissection (LCM) offers a relatively rapid and accurate method to isolate and routinely analyze tissue regions of interest from their native tissue microenvironment under direct microscopic visualization. LCM can be used to isolate precise anatomical regions of tissue, single cells, or in the present case a concentrated and organized population of lymphocytes from frozen or embedded tissue sections on a microscope slide for various downstream applications like RNA, DNA, protein, or metabolite analyses [16][17][18]. As a testimony of the effectiveness of this method and proof of applications for TLS investigations, we previously applied LCM to successfully isolate TLS from a cohort of primary lung cancer patients [11]. Using RNA transcript profiling, we demonstrated that lung tumor-associated TLS exhibit a specific chemoattractant molecular signature within the tumor microenvironment, including a network of chemoattractant molecules (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL-16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1), and integrins (αL, α4, and αD) [11]. This specific molecular signature was confirmed at proteic level and is associated with the selective presence of high endothelial venules and T-cell infiltration within TLS [11]. Thus, LCM combined with RNA transcript profiling allowed to identify the molecular network mediating the migration of tumor-infiltrating T cells into tumor-associated TLS [11]. LCM is a powerful tool to decipher TLS functions in various tissues and inflammatory contexts, and has been recently applied to inform the role of TLS in lung and skin tumors [12,19], colitis [20], pulmonary hypertension [21], experimental autoimmune encephalomyelitis [22], aortic aneurysm [23], atherosclerosis [24,25], and uveitis [26].

A variety of downstream applications from LCM exist, including DNA genotyping, RNA transcript or signal pathway profiling, cDNA library generation, proteomics or biomarker discovery, and metabolomics analyses. With the emergence of next-generation sequencing technologies [27], LCM now represents a practical and very efficient method to conduct deep molecular analyses of TLS in various pathological contexts. In addition to TLS, LCM can also be applied to simultaneously isolate distinct components of the tumor microenvironment, like tumor nests, stroma, blood, or lymphatic vessels, to further conduct molecular comparisons. In this chapter, we describe a detailed and straightforward method for the isolation of TLS and RNA extraction by LCM from human or murine frozen tissues to perform a comprehensive downstream RNA analysis.

Materials

1. Solid frozen tissue sample pre-screened for a high density of TLS and a good RNA quality (RIN ≥8).

Cryostat microtome (e.g., Leica or other manufacturers) with

glass anti-roll preset to -20 °C.

3. Dry ice.

4. Dry ice container with lid.

5. RNaseZap ® (Applied Biosystems) for RNase removal.

6. Clean tweezers and brushes for cryostat cleaning.

7. Cryostat O-ring chucks.

8. Disposable microtome blades HP35n non-coated (Thermo Fisher Scientific). 9. Leica LMD slides (e.g., 0.2 μm PEN membrane).

10. Cryomatrix optimal cutting temperature (OCT) embedding medium (Thermo Fisher Scientific).

11. 1000 and 100 μL pipets dedicated for RNA work.

12. Filter pipet tips (for 1000 and 100 μL pipets).

13. Nuclease-free water.

14. 100% Ethanol. 1. RNeasy Micro Kit (Qiagen).

Preparation of Frozen Tissue Sections for Laser Capture Microdissection

2. DNase I (1500 Kunitz units, Qiagen).

3. Microcentrifuge (≥13,000 × g).

4. 80% Ethanol prepared with ethanol (96-100%) diluted with nuclease-free distilled water.

5. RNase-free 50 mL Falcon tube.

6. Ice.

1. Agilent RNA 6000 Pico kit (Agilent Technologies).

2. Agilent 2100 Bioanalyzer with Chip priming station and IKA vortex mixer (Agilent Technologies).

3. Heating block.

4. Microcentrifuge (≥13,000 × g).

5. RNaseZap ® (Applied Biosystems).

6. Nuclease-free water.

7. 0.5 mL RNase-free microcentrifuge tubes.

8. Pipet 1000 and 10 μL dedicated for RNA work. 9. Ice.

Laser

Methods

This section describes the successive steps that should be conducted for TLS isolation by LCM, RNA extraction, and gene expression analyses from frozen tissue samples. The protocol provided is a general method for LCM that has been successfully applied for TLS RNA profiling on human lung tumor tissues [11].

The experiments are described in the following order: frozen tissue preparation, LCM isolation, RNA extraction, RNA integrity assessment, reverse transcription cDNA generation, quantitative realtime (qRT) PCR, and analysis. This method is intended to be a starting point for extensive LCM and molecular analyses of TLS on various types of mammalian tissues. Thus, conditions should be tested and optimized by the users for the application on other types of human or murine tissues before proceeding.

The frozen tissue sample has been snap-frozen by immersion into liquid nitrogen immediately after surgery or organ sample to preserve RNA integrity and was stored in liquid nitrogen or at -80 °C before the experiment (see Note 1). Frozen tissues must be transported in an appropriate container containing dry ice for further cryosection (see Note 2). When possible, it is highly recommended to first screen the frozen tissue of interest to confirm preservation of the histological architecture, the presence of TLS, and a good RNA quality (RIN ≥8) before proceeding to the LCM.

1. Set the temperature of the cryostat to -20 °C. Clean the workstation, the cryostat chamber, and the knife holder (not the knife blade) and treat the tweezers and brushes that will be used to handle the tissue sections with RNaseZap (see Note 3).

Reverse Transcription for cDNA Generation 2.6 Quantitative Real-Time Polymerase Chain Reaction Assays

Preparation of Frozen Tissue Sections for Laser Capture Microdissection

TLS Laser Capture Microdissection 2. Place the frozen tissue sample into the cryostat, and let it equilibrate for at least 10 min at -20 °C (see Note 4).

3. Add one drop of Cryomatrix OCT embedding medium on the surface of a cold chuck serving as the "glue" at the interface (see Note 5), and quickly attach the frozen tissue with the help of the tweezers before the OCT hardens. Apply just the minimal amount of OCT needed to finely cover the surface of the chuck and securely mount the tissue (see Note 6).

4. Wait for 10 min for the OCT to solidify at -20 °C (the OCT will turn from translucent to white when frozen). Assure that the tissue is solidly stuck and mount the chuck in the specimen holder orienting the tissue to expose the region of interest.

5. Gently bring the tissue section near to the blade. Using a fresh disposable blade, shave OCT (set the cutting thickness from 5 to 10 μm) from the tissue block until the tissue becomes visible. If necessary, start with successive cuttings of 30 μm until a decent amount of tissue has been sectioned giving access to a large region of interest. At the end of this step, the investigator should obtain a clear and well-oriented plan section of the tissue.

6. Set the thickness of the tissue section at 5-14 μm (depending on the nature and the hardness of the tissue to be microdissected) and start cutting gently. Manipulate the newly cut tissue section onto the glass anti-roll plate and use a small brush to straighten out the section (see Note 7).

7. Collect the tissue section on a PEN membrane LMD glass slide (that investigator has labeled using the pencil) by carefully placing the slide (with the membrane side facing down) against the tissue section and pressing quickly and gently. The tissue section should stick immediately on the membrane. During this step, the tissue section must be handed with extra careful precautions to preserve the native architecture of the tissue (see Note 8). This will be critical to facilitate the detection and the successful microdissection of TLS during the LCM step.

8. To maximize resources per slide and the potential number of TLS to be microdissected, proceed to cut several tissue sections (as described in steps 6-7) and collect them sequentially on the same PEN membrane LMD glass slide to load a total of 4-10 samples (depending on the nature and the size of the tissue) per slide (see Notes 9 and 10).

9. Remove the PEN membrane LMD glass slide from the cryostat and immediately apply a 70% ethanol fixative solution for 1 min on the tissue sections (see Note 11). The fixation and staining steps should then be performed under a hood dedicated for RNA work.

10. Drain the 70% ethanol and gently rinse with nuclease-free water for 10 s.

Drain the water and start the coloration by gently applying

RNase-free 0.01% toluidine blue (≈100 μL) onto the slide to completely cover the tissue sections and let the slide stain for 90 s (see Note 12).

12. Gently wash the slide with 70% ethanol for 30 s and then with 100% ethanol for 30 s. Additionally, the slide may also be rinsed in xylene for a maximum of 5 min to enhance dehydration before proceeding with LCM (see Note 13). Blot the slide on an absorbent paper in between the different solution washes, to prevent carryover from the previous solution (see Note 14).

13. Allow the slide to air-dry for 5 min before loading on the microscope platform. When the tissue section is dry, the slide is ready to proceed to LCM.

1. Turn on power for the microscope and laser control box. Let the laser warm up during approximately 10 min (see Note 15).

2. Use a cover-slipped toluidine blue-stained section of the tissue to pre-analyze the tissue and estimate the number of TLS before starting the LCM. This step will help the investigator to estimate the number of LCM sessions required to yield sufficient RNA quantity for downstream analyses. This step will also avoid losing time analyzing the tissue and risking potential RNA degradation when performing the LCM on the PEN membrane glass slide.

3. Pipet 35 μL of lysis buffer (RLT + 1% ß-mercaptoethanol) for OCT-embedded tissues into the cap of a clean 0.2 mL RNasefree collection tube with flat cap. Load the tube in the collection for tube caps on the holder apparatus of the LCM system under the microscope platform.

4. Place the PEN membrane glass slide on the microscope stage with the membrane and the stained tissue sections facing down.

5. Calibrate the laser (see Note 16) and adjust the laser parameters (power, aperture, speed, frequency, and focus) appropriately according to the type of tissue to be microdissected (see Note 17).

6. Make sure that the cap is aligned with the objective on the microscope (it should be visible through the aperture in the stage) before starting the LCM. Focus the microscope with the 20× objective to get a clear view of the tissue. Locate all the TLS-enriched regions of interest by moving the tracking beam.

7. Using the drawing tool, draw an area that surrounds the TLS, proceed to fire the laser (if a specific number of TLS is desired

Laser Capture Microdissection of TLS

to collect, then investigator can optionally locate and assign all the areas where TLS are present and then proceed to the LCM), and drop the microdissected TLS in the RLT buffer. A clear and confident detection of TLS is critical for this step; thus TLS microdissection should always be performed by an investigator who is well trained to identify and confidently distinguish TLS among the tissue complexity. We define TLS as an aggregate of more than 50 cells with lymphocyte morphology and demonstrating nodular formation with close cell-tocell contact within the structure (Fig. 1).

8. After completing the LCM, unload the collection tube, carefully close the cap, and briefly spin down the dissectates (see Note 18).

9. Gently open the lid and rinse the cap surface with another 40 μL of lysis buffer (total final volume = 75 μL). Carefully close the cap to avoid any loss of material and mix gently by inverting the tube so that the lysis buffer rinses the cap (see Note 19).

Keep the collection tube at -20 °C inside the cryostat until the end of the LCM session (see Note 20).

We recommend using the RNeasy Micro kit (Qiagen) for RNA extraction that is adapted for RNA purification from small amounts of tissues or cells (maximum 45 μg of RNA per sample). Investigators should work quickly at room temperature (15-25 °C) and in an RNA work-dedicated area during all the RNA extraction process.

1. Vortex the tubes for 30 s and add 1 volume of 70% ethanol to the homogenized lysate. Mix well by pipetting up and down and

RNA Extraction of Microdissected TLS

Fig. 1 Laser capture microdissection of TLS. TLS from a frozen human lung tumor before (a) and after (b) the laser capture microdissection. The microdissected area is designated by the green line. Tissue section was fixed and stained with 0.01% toluidine blue before performing the LCM immediately transfer the lysate (including any precipitate that might have formed after ethanol addition) to the RNeasy MinElute spin column placed in an RNase-free 2 mL collection tube.

2. Gently close the lid and centrifuge for 1 min at 100 × g and then centrifuge for 1 additional minute at 10,000 × g.

3.

Discard the effluent present in the collection tube (see Note 21) and briefly wipe the tube on an absorbent paper before replacing the filter.

4. Add 350 μL of RW1 buffer inside the RNeasy MinElute spin column.

5. Gently close the lid and centrifuge for 1 min at 10,000 × g. Discard the effluent present in the collection tube and briefly wipe the tube on an absorbent paper before replacing the filter. 8. Add 350 μL of RW1 buffer inside the RNeasy MinElute spin column and centrifuge for 1 min at 10,000 × g. Discard the effluent and the collection tube and transfer the column to a new RNase-free 2 mL collection tube. 9. Add 500 μL of RPE buffer (see Note 24) and centrifuge for 1 min at 10,000 × g. Discard the effluent present in the collection tube and briefly wipe the tube on an absorbent paper before replacing the filter.

10. Add 500 μL of 80% ethanol to the RNeasy MinElute spin column. Close the lid gently, and centrifuge for 2 min at 10,000 × g to wash the spin column membrane. Discard the effluent present in the collection tube and briefly wipe the tube on an absorbent paper before replacing the filter.

11. Centrifuge for 1 min at 10,000 × g with the lid open to allow the silica membrane to dry.

12. Place the RNeasy MinElute spin column in a new and labeled RNase-free 1.5 mL tube, and add 14 μL of nuclease-free water into the middle of the column membrane.

13. Centrifuge for 1 min at 1000 × g and then centrifuge for 1 min a 10,000 × g to collect total RNA. Discard the column. Vortex well for 10 s and centrifuge for 10 min at 13,000 × g. The gel-dye mix must then be used within 1 day and one tube can be used to run two chips.

Start the 2100 Expert Software and turn on the Bioanalyzer.

To wash the electrodes of the Bioanalyzer, place an electrode cleaner containing 350 mL of nuclease-free water in the instrument, close the lid, and let it wash for 5 min. In the meantime, select "Assays," "Electrophoresis," "RNA," and finally "Eukaryotic Total RNA Pico Series" on the instrument menu.

Then define the number of samples (from 1 to 11 per chip) to be assayed and enter the corresponding sample information. After cleaning, remove the electrode cleaner and allow the remaining water to evaporate for 30 s before closing the lid of the Bioanalyzer (see Note 27).

Prepare the chip priming station and insert a new RNA Pico

Chip within the station. Pipet 9 μL of the gel-dye mix into the well marked with an encircled "G" (see Note 28). Make sure that the syringe is positioned back to 1 mL and then close the chip priming station until you hear a click confirming that the priming station is closed properly. Press the plunger down until it is secured beneath the syringe clip. Wait for exactly 30 s and then release the plunger with the clip-release mechanism. Wait for 5 s and then slowly pull back the syringe to the 1 mL position.

7. Open the chip station and pipet 9 μL of the gel-dye mix into the two remaining wells marked "G." Then, add 9 μL of the RNA 6000 Pico Conditioning Solution (white tube) to the well marked "CS."

Assessing RNA Integrity

8. Pipet 5 μL of the RNA 6000 Pico Marker (green tube) into the ladder well and in all wells containing RNA samples. Add 6 μL of Pico RNA Marker to any empty sample wells. 9. Add 1 μL of aliquoted and heat-denatured RNA 6000 Pico Ladder to the ladder well and 1 μL of RNA sample to the appropriate sample wells. After loading all wells, vortex the chip using the manufacturer-supplied IKA vortexer for 1 min at 2400 rpm. Within the next 5 min, place the Pico Chip on the Agilent 2100 Bioanalyzer and start the run (see Note 29).

If investigators are working on single cells isolated within TLS and/or for specific applications, like RNA-seq, requiring an important quantity of material, it may be necessary to first amplify the RNA [28] before proceeding to downstream applications.

The RNA yielded from LCM will be reverse transcribed using the High-Capacity cDNA Reverse 3. Load the plate or tubes into the thermal cycler and let incubate using the following program: 10 min at 25 °C, 2 h at 37 °C, 5 min at 85 °C, and then hold at 4 °C.

Reverse Transcription for cDNA Generation

TLS Laser Capture Microdissection 4. When the reverse transcription reaction is complete, add nuclease-free water to adjust the concentration of the samples to 5 ng/μL.

5. Mix the diluted samples well and centrifuge briefly. Place on ice for proceeding qRT-PCR or store at -20 °C for further applications.

A variety of techniques for deep molecular analyses exist, including next-generation sequencing or microarrays [27]. Each of them comes with its own advantages and limitations in terms of signal sensitivity, specificity, and stability. Quantitative RT-PCR quantification of mRNA molecules remains a reliable and routine method, even in combination with few microdissected cells for sensitive gene expression analyses or target gene confirmation. For the following protocol description, we describe a qRT-PCR method based on TaqMan Gene Expression Assays (see Note 30). To generate accurate and reproducible results from qRT-PCR, be careful and precise when performing each step in the protocol, especially for pipetting.

1. Turn the TaqMan Real-Time PCR instrument on and allow it to warm up for at least 20 min. We used TaqMan2900HD (Applied Biosystems) for this step, but note that TaqMan assays can also be run on a variety of real-time PCR instruments from other constructors. In the meantime, prepare your experiment settings and load the appropriate target genes and fluorophores (FAM ™ , VIC ® dye-labeled MGB probe, or SYBR Green) in your experiment file and save the file. 4. Seal the plate with the appropriate cover adhesive film and apply a compression pad all over the plate to confirm the sealing. Centrifuge the plate briefly.

Quantitative Real-Time Polymerase Chain Reaction Assays

Claudia Gutierrez-Chavez et al.

5.

Load the reaction plate into the real-time PCR system. In the instrument settings, set the volume of reaction to 20 μL and set the following cycles: Calculate the expression level of each specific target (gene) between the different samples by using the relative standard curve or the comparative Ct methods [29].

Notes

1. Frozen sections are the best choice for RNA analyses. Prompt freezing of the sample limits RNA and protein degradation due to nuclease and protease activity. For a long-term storage (i.e., more than 6 months), it is recommended to conserve the samples in liquid nitrogen as it offers the best preservation of proteins and RNA. Conservation at -80 °C is adequate for short-term storage (i.e., less than 6 months). Alternatively, formalin-fixed paraffin-embedded (FFPE) tissue can also be used, as it has been referenced in the literature [17,18]. Nevertheless, it is more challenging obtaining acceptable RNA quality from FFPE tissues after LCM. For these reasons, we recommend to always favor LCM on frozen tissue when possible. Frozen tissue should be tested for RNA quality before proceeding to LCM (we recommend only using tissue with a RIN ≥8). precautions for RNA manipulation must be applied during all these steps. A specific clean area for RNA work should have been designated in the lab. All equipment and laboratory benches should have been cleaned with RNaseZap or 100% ethanol before starting the procedure (carefully wash all the surfaces, supports, and tools in the cryostat). RNase-free material must be used at all stages of work with RNA. Gloves should be worn at all times and changed frequently, especially after contact with liquids or surfaces that may be contaminated with RNases. Filter pipet tips should be used and changed each time you pipet to avoid potential cross-contamination between samples and to prevent RNase contamination. For most procedures, it is advisable to use nuclease-free, hydrophobic, nonstick tubes to minimize loss of sample that may otherwise adhere to the tube walls.

The investigators

4. Make sure that the tissue sample has equilibrated at cryostat temperature before starting cutting. This will avoid undesirable folds in the section and sticking to the membrane slide during the cut collection.

5. Cover the chuck's flat surface with a piece of wet towel paper and let it dry in the cryostat at -20 °C before pasting the tissue section with OCT medium on the chuck. This will facilitate the disassembly of the tissue section from the chuck at the end of the cryosection. Investigator will simply need to remove the towel paper to detach the tissue from the chuck.

6. Be quick when pasting the frozen tissue with OCT on the chuck. The OCT medium generally takes less than 1 min to solidify. As an indication, the OCT will turn white in color when completely frozen and solid. Investigator should use a tissue less than 1.5 cm size to facilitate cutting. It is recommended adjusting the angle of the tissue block before the medium hardens so that the region of interest of the tissue is facing up and aligned in parallel with the chuck surface. This will then facilitate getting a proper orientation of the tissue section with the microtome blade after loading the chuck in the specimen holder of the cryostat.

7. It is critical to avoid tissue carryover during cutting procedure. Carryover contamination of one specimen from another will results in false or non-reproducible results. Thus, the blade used to cut sections should be kept clean during the experiment, and every excess tissue fragments should be systematically wiped from the area with the brush. A fresh microtome blade should be used for each new specimen, and disposable blades used if possible. If LCM is performed for downstream RNA analyses, it is recommended cleaning the stage of the microtome, which contacts the sectioned tissue, with the brush and RNaseZap between each specimen.

8. If investigator encounters difficulty to maintain original structure of the tissue, due to friability of the section or difficulty in cutting, the tissue may be too cold. Therefore, the temperature of the cryostat should be adjusted until finding the optimal temperature point for the cutting. Optimal temperature point will depend on the nature and the thickness of the tissue.

9. Keep the membrane PEN slides at room temperature before applying a tissue section. Tissues don't adhere well on cold membrane slides. Work gently and as quickly as possible. If the investigator wants to include multiple tissue sections on a slide to maximize resources, keep the slide in the cryostat and warm the area of the slide where the tissue will be deposited by pressing your thumb to the reverse side.

10. PEN membrane slide loaded with tissue sections can be conserved 1 month at -80 °C for further microdissection. Nevertheless, please note that the duration for RNA preservation can vary depending on the nature of the tissue. When possible, we always recommend performing the LCM immediately after the tissue cryosection.

11. It is mandatory to prevent the slide from drying or thawing before the contact with 70% ethanol to avoid loss of tissue adherence to the PEN membrane of the glass slide and/or to prevent RNA degradation.

12. Since tissue section on PEN membrane slide is not coverslipped, the microscopic visualization of the tissue is often darker than first expected when starting the LCM. Optimizing the time of staining and diluting the colorant with nuclease-free water will produce lighter staining and will help finding the best staining conditions for each specific type of tissue.

13. A complete dehydration of the tissue is critical to minimize the adhesive forces between the tissue section and the slide and optimize the LCM efficiency. Increasing the incubation time up to 2 min for the 100% ethanol and up to 5 min for xylene will enhance dehydration and increase the efficiency of the microdissection. These steps can be repeated if the investigator encounters issue with the tissue transfer during LCM. Optionally, after fixation, staining, and dehydrating steps, the slides can be gently heated on a heating block for a maximum of 5 min to ensure full dehydration.

14. There are many alternatives to toluidine blue coloration, like hematoxylin and eosin or methylene blue, that can be tested and preferred depending on the type of tissue to be microdissected [17]. Further, immunohistochemistry procedure for TLS Laser Capture Microdissection staining of specific cell subtypes within TLS (like dendritic cells, B cells, or T cells) can also be applied and combined with LCM [18]. This procedure should be set up and optimized by the user for each specific antibody combination.

15. Various methods of LCM exist that can be divided into contact and noncontact methods in reference to the process used to isolate and extract the targeted cells or structures from the native tissue [18]. For this protocol, we are referencing to a Leica LMD system using a noncontact gravity-assisted microdissection method.

16. Move the microscope objective until finding an empty "white area" (without tissue section) on the slide when calibrating the laser parameters for testing its accuracy.

17. The setting up of the laser parameters (power, aperture, speed, frequency, and focus) is critical for the success of the experiment. These parameters are strongly dependent on the nature and the thickness of the tissue as well as the size of the structures to be microdissected. Thus, laser parameters must systematically be optimized depending on the tissue and the experiment. It is also strongly recommended testing the settings of the laser on a control tissue before starting a LCM session on a precious tissue sample. 27. For periodic washing or when you suspect a RNA contamination on the Bioanalyzer electrodes, remove the pin set on the Bioanalyzer and proceed to wash the electrodes with RNaseZap.

Then rinse thoroughly with nuclease-free water.

28. Never use the blowout function of the pipet when loading the Pico chip. This may generate air bubbles within the chip that will interfere with the RNA analysis during the chip run.

29. RNA extracted from TLS should have a concentration of ≥7 ng/μL and a RNA integrity number (RIN) ≥7 for posterior gene expression analysis. A RIN number <5 may raise major concerns about nonspecific amplification during the reverse transcription and the real-time PCR steps, and thus should not be used for downstream analyses. A low RIN number can be due to incorrect snap-freezing of the native tissue or due to a too long session (>60 min) of LCM that has resulted in RNA degradation, or can alternatively come from problems occurred during RNA extraction. If the investigator has confirmed that the initial frozen tissue has a good RNA quality (RIN ≥8), it is recommended repeating the LCM and RNA isolation steps.

30. In addition to TaqMan, there are several detection methods available that can be used for performing qRT-PCR from microdissected tissue, including SYBR Green.

31. In our experience, 10 ng of cDNA material usually allows good gene detection. Nevertheless, when working with lowabundance transcripts gene targets, the starting amount of cDNA needs to be increased to achieve an appropriate range of detection. In this case, the volume of water in the master mix will need to be adjusted, accordingly.

32. TaqMan assay materials are light sensitive and must be kept in the freezer, away from light, until you are ready to use them.

Excessive exposure to light may degrade the fluorescent probes and impact the results.

Introduction

Secondary lymphoid organs (SLOs), including lymph nodes, spleen, Peyer's patches, and mucosal associated lymphoid tissues (MALT; the largest being tonsils), are highly sophisticated structures designed to optimize antigen capture and presentation so that rare antigen-specific circulating T and B cells encounter their cognate antigens and initiate an adaptive immune response [1]. SLOs are essential for systemic protection of our body from viral infection and pathogen invasion. Areas of SLOs are divided into distinct T-cell zones and B-cell follicles to separate priming and activation of naïve T and B cells, respectively. Naïve T cells (CD4 + and smaller numbers of CD8 + ) interact with dendritic cells (DCs) and other professional antigen-presenting cells (APCs) in the T-cell zone. Germinal center (GC) formation, induced in the B-cell follicle during a primary immune response, provides as a controlled microenvironment for activated B cells to undergo clonal expansion and affinity maturation. This process is helped by their interactions with follicular helper T (T FH ) cells and follicular dendritic cells (FDCs), which lead to selection of antigen-specific highaffinity B cells for memory B cell or antibody-secreting plasma cell differentiation and maturation. These processes are a crucial element of humoral immune response in adaptive immunity. Tertiary lymphoid structures (TLS) develop postnatally and occur in response to chronic inflammation at nonlymphoid anatomical locations. These SLO-like entities are generally small in size and situated in or near the affected tissues in diseases including chronic infection, autoimmunity, and, more recently, cancer. Similar to SLOs, T-and B-cell-predominant areas are observed in well-formed TLS, with mature DCs found in the T-cell zone and GCs apparent in the B-cell area that contains proliferating B cells, T FH cells, and a dense FDC network [2,3]. High endothelial venules (HEVs), blood vessels adapted for lymphocyte trafficking into SLOs, also form in the T-cell zones of many TLS [4], providing efficient movement of peripheral lymphocytes (including naïve cells) to and from the tissue [5].

In autoimmunity, TLS are often associated with the more aggressive forms of disease [6]. Recent studies have also correlated their presence with improved survival in several human solid tumors although some investigations found the inverse [7]. TLS are thought to provide a site that facilitates immune cell interactions leading to local antigen-specific T-and B-cell priming, activation, and differentiation into memory and/or effector cells. These activities produce adaptive immune responses that either exacerbate (autoimmunity) or help combat (infection and cancer) the disease.

Accurate TLS detection normally requires examination of specific markers expressed on key cellular components using immunohistochemistry (IHC) [2,8]. These markers can however change because TLS are dynamic sites that can arise or dissipate longitudinally during disease progression. Their level of organization extends from lymphoid aggregates to mature TLS with GCs. Several studies propose that these different structures represent developmental stages of TLS. The least frequent but highest level of organization is considered the GC-containing TLS, which can coexist with suboptimally organized smaller T-and B-cell aggregates [9]. Alternatively, the aggregates could be part of a fully formed TLS detectable on a different tissue plane [10]. Whether the more abundant, sub-organized structures evolve to GC during tumor progression in patients is not currently known but suggested to be possibly true by our recent study [11].

Chunyan Gu-Trantien et al.

Alternative methods for detecting TLS, such as gene expression analysis, likely capture the molecular basis underlying TLS activities and/or formation. Specific genes have been proposed as markers for these morphologically distinct structures, which are commonly associated with them in human autoimmune diseases and cancer. A set of 12 chemokine genes were correlated with a TLS presence in colorectal cancer and subsequently confirmed in melanoma [12,13]. Lung cancer TLS differentially express many chemokines and adhesion molecules compared to the tumor nests (usually lacking TLS) [14]. Consistent with this is that important factors involved in T-and B-cell recruitment to SLOs, CCL19/CCL21 and CXCL13 (T-and B-cell chemoattractants, respectively), are the most specific TLS genes found to date. These signals together with FDC-specific marker genes (e.g., CD21L) and early mediators of SLO development [lymphotoxins α and β (LTA and LTB)] are specifically associated with a GC presence in rheumatoid synovitis and melanoma metastases [15,16]. Our studies have identified PD-1 hi CD200 hi ICOS int CXCR5 -T FH X13 cells as the major CXCL13 producers in human breast cancer (BC) with these specialized helper cells appearing to promote local TLS formation at the tumor site [3,11].

Formalin-fixed paraffin-embedded (FFPE) blocks are the most frequent source of human tumor material that is available from tumor banks and pathology departments today. These samples are used for routine diagnostic analyses including IHC. RNA extracted from these samples frequently exhibits high levels of degradation. Recent advances have improved the quality of gene expression quantification from FFPE tumor tissues and this has increased the material available for molecular analysis in human cancer. The following protocol details the steps for using FFPE breast tumor tissues to quantify TLS-related gene expression, using CXCL13 and LTB as examples, and preparing dual CD3/CD20 IHC for parallel TLS scoring by experienced pathologists.

Materials

1. 1.5 or 2 mL RNase-free microcentrifuge tubes.

2. Microtome and new blade.

RNase decontamination solution (e.g., RNaseZap RNase

Decontamination Solution, Ambion). 2. Microtome and blade.

Reverse Transcription of RNA to cDNA 2.4 Pre-amplification and Real-Time PCR Quantification of Gene Expression

Immunohistochemical Staining of FFPE Tumor Tissues

Chunyan Gu-Trantien et al.

Oven (for 37 °C incubation).

4. Superfrost Plus microscope slides.

5. Rabbit anti-human CD3 polyclonal antibody (Dako).

6. Mouse anti-human CD20 monoclonal antibody (clone L26). 12. Mild liquid dishwashing soap.

13. 96-100% Ethanol.

14. 100% Isopropanol.

15. 100% Xylene.

16. Film coverslipper (e.g., Tissue-Tek Prisma & Coverslipper HQ Plus, Sakura). 17. Coverslipping films (e.g., Tissue-Tek Coverslipping Film, Sakura).

1. SDS software (Applied Biosystems) or equivalent.

2. Bright-field microscope.

3. GraphPad Prism software or equivalent.

Methods

Always work with clean gloves for RNA and avoid touching the inside of any containers (see Note 1).

Correlation Between TLS-Related Gene Expression and IHC-Scored TLS

Preparation of Tissue Sections from FFPE Tumor Blocks

TLS-Related Gene Detection in FFPE Human Samples 1. Before tissue sectioning, chill FFPE blocks on ice for 20 min (see Note 2).

2. Fix the FFPE block to the microtome and ensure, as much as possible, that the tissue surface is vertical to the blade (see Note 3).

3. Set the section thickness at 5 μm (see Note 4).

4.

Use a new blade to avoid any possibility of contamination by RNase.

5. Adjust the distance of the block surface to progressively reach the blade.

6. Start cutting and verify that the sections contain the entire tissue area.

7. Throw out the two first sections that might be oxidized (see Note 5).

8. Continue to cut three sections of 5 μm each (see Note 6).

9. Use a clean forceps (see Note 7) to put the sections immediately into a clean, RNase-free 1.5 mL microcentrifuge tube.

10. Close the lid and keep the tissue sections at 4 °C until RNA extraction, or proceed immediately to RNA extraction (see Note 8).

The following RNA extraction protocol largely follows the manufacturer's instructions for the RNeasy FFPE kit (Qiagen) with some adaptations.

RNA Extraction from FFPE Tumor Tissues

Chunyan Gu-Trantien et al. Currently, several commercial cDNA synthesis kits are of high efficiency. The High-Capacity RNA-to-cDNA kit is composed of two easy-to-use fractions: one tube containing a 2× RT Buffer Mix and another tube containing a 20× RT Enzyme Mix.

cDNA Preparation

TLS-Related Gene Detection in FFPE Human Samples 1. Thaw the TaqMan Gene Expression Assays and the cDNA in the refrigerator (4 °C). Move them to ice once they are completely thawed.

2. In a 1.5 mL microcentrifuge tube, combine equal volumes of each 20× TaqMan Gene Expression Assay (up to a total of 100 assays depending upon the study design) (see Note 19).

Pre-amplification and Real-Time PCR Quantification of Gene Expression

3. Dilute the pooled TaqMan assays using RNase-free water (see Note 20) so that each assay is at a concentration of 0.2× [the volume of the diluted pooled assays (typically 12.5 μL/reaction) accounts for 1/4 of the final volume of the preamplification reaction (typically 50 μL)] (see Note 19).

4. For 50 μL reactions, mix 25 μL of TaqMan PreAmp Master Mix (2×), 12.5 μL of the diluted pooled TaqMan assays, and 12.5 μL of cDNA in 0.2 mL PCR tubes. We use an amount of cDNA that corresponds to 100 ng of initial total RNA (see Note 21).

5. Cap the microcentrifuge tubes.

6. Mix the reactions by gently inverting the tubes, and then centrifuge briefly. 11. For each gene, prepare a mixture of the latter three components for the total number of reactions foreseen (see Note 16).

12. Fill the 96-well plates with 5 μL of cDNA and 15 μL of the mixture, in triplicate (see Note 25).

13. Seal the plates with MicroAmp Optical Adhesive Film.

14. Centrifuge the plates for 2 min at 200 × g (see Note 26).

15. Run the plate on a 7900HT Fast Real-Time PCR System or equivalent using the following program: 50 °C for 2 min, 95 °C for 10 min, then 40 cycles at 95 °C for 15 sec, and 60 °C for 1 min.

IHC is a commonly used technique to monitor protein expression and localization in the context of tissue morphology. IHC uses antibodies to detect and analyze protein expression while maintaining the composition, cellular characteristics, and structure of the native tissue. Chemical fixation secures cellular and molecular interactions in complex tissues. Tissue samples can be embedded in paraffin before being cut into thin sections that are mounted on slides for staining and analysis. To visualize TLS in BC, we devel-

TLS Scoring on IHC-Stained Tumor Sections

TLS-Related Gene Detection in FFPE Human Samples oped a dual-CD3/CD20 IHC staining that clearly reveals dense aggregates of B cells adjacent to a T-cell zone [8] (Fig. 1a,b). Dual-CD3/CD20 IHC is performed by an automated IHC slide staining system (Ventana Benchmark XT; steps 11-22) at our institute that delivers consistent and high-quality results. Dehydration and slide mounting (steps 25 and 26) are performed by the Tissue-Tek Prisma & Coverslipper HQ Plus.

1. Chill FFPE blocks on ice before sectioning, as in Subheading 3.1, step 1 (see Note 2).

2. Fill a water bath with ultrapure water and heat to 40 °C (see Note 27).

3. Place the blade in the holder and insert the paraffin block.

4. Carefully, wind the block to approach the blade and cut a few thin sections to ensure that the positioning is correct.

5. Trim the block to expose the tissue surface at a thickness of 10-15 μm.

6. Cut sections for staining at a thickness of about 4-5 μm.

7. Using a brush, pick up the ribbon sections and float them on the surface of the water in the water bath so they flatten out.

8. Use a dissecting needle to separate the sections.

9. Use microscope slides to pick the sections out of the water bath and store upright in a slide rack.

10. Dry slides overnight at 37 °C in an oven (see Note 28).

11. De-paraffinize and rehydrate the section (see Note 29).

12. Unmask antigen by following antibody-specific recommendations (see Note 30).

13. Apply ultraView Universal DAB Inhibitor to the slide and incubate for 4 min at room temperature. 22. Counterstain sections with hematoxylin and a bluing reagent, which stains the cell nuclei blue to better visualize the tissue morphology.

23. Wash slides in a mild liquid dishwashing soap. 2. The C T values of the real-time qPCR results are calculated using the SDS software with the option "Manual C T " and a threshold of 0.2 (see Note 32).

3. To compare gene expression across samples, we consider ΔC T values that correspond to the difference between the C T values of each target gene and the mean C T values of all the housekeeping genes (ΔC T = C T(target gene) -C T(mean of all housekeeping genes) ). ΔC T values are inversely proportional to the quantity of the specific mRNA in a given sample (see Note 33).

4. The -ΔC T values (positively proportional to gene expression) of the CXCL13 and LTB genes were then correlated with the TLS score (no. of TLS/mm 2 breast tumor surface scored on CD3/CD20 IHC-stained tissue sections) for each individual patient (Fig. 2a,b).

Notes

1. Human body (including hands) is a rich source of RNase.

2. The small amount of moisture that penetrates the paraffin from the melting ice will make the tissue easier to cut.

3. The goal is to quickly get the first section that covers the entire tissue area to avoid wasting material.

Correlation Between TLS-Related Gene Expression and IHC-Scored TLS

Chunyan Gu-Trantien et al. 4. The maximum allowable thickness is 20 μm for the RNeasy FFPE kit (Qiagen). We prefer to use 5 μm sections to ensure complete de-paraffinization.

5. Alternatively one of the first sections could be counterstained with hematoxylin to have an idea about the cellular composition (e.g., lymphocytes, tumor cells) of the tissue sections extracted.

6. The RNeasy FFPE kit (Qiagen) can handle up to four 10 μm sections with a surface area up to 250 mm 2 in a single extraction by using the larger reagent volumes proposed in the standard protocol (cf. Qiagen RNeasy FFPE Handbook). FFPE tumor blocks prepared in routine pathology laboratories contain variable surface sizes depending on the size of the tumor. Breast tumors are usually small and the embedded tissue areas vary from 25 to 200 mm 2 . In general, 3 × 5 μm sections are suitable for a single extraction using the smaller volumes of reagents proposed in the standard protocol.

7. The forceps can be cleaned using an RNase decontamination solution (e.g., RNaseZAP, Ambion). After applying RNaseZAP, wipe thoroughly with a paper towel, rinse with water, and dry with a clean paper towel.

8. To minimize further RNA degradation, the cut tissue needs to be extracted within 24 h of sectioning.

9. The first step of de-paraffinization can be achieved using several methods including De-paraffinization Solution (Qiagen); organic solvents such as heptane, xylene, limonene, and CitriSolv; or melting. We use xylene at our laboratory, which is economical and can be used in large volumes to ensure thorough de-paraffinization.

10. The microcentrifuge that we used allows a maximum speed of 25,000 × g.

11. The pellet is not very solid. Avoid touching the pellet. If necessary, leave a small volume of xylene that will be washed by the ethanol. Steps A1 and A2 can be repeated if the deparaffinization seems incomplete (the solution is turbid in this case).

12. We used the smaller volumes for all reagents and followed the standard protocol for the RNeasy FFPE kit (Qiagen), except for ethanol (step 10), which is available in unlimited quantities.

13. We used a minimal volume of RNase-free water for RNA elution to obtain the highest possible concentrations.

14. RNAs extracted from FFPE samples are generally significantly degraded. The RNA integrity number (RIN) is not informative for the utility of the RNA extracted from FFPE tissues [17]. Typically, we obtain a RIN around 2.2-2.3 but have no problems with the downstream analysis. The final qPCR results are evaluated for all samples, but when the housekeeping genes are detected at low levels (e.g., C T > 30) and the genes of interest are undetectable (C T > 36), samples need to be considered with caution or excluded from the analysis (also see Subheading 3.6).

15. The RT Enzyme Mix remains liquid at -20 °C so it does not need to be thawed. 16. The total number of reactions should contain 2-4 (depending on the number of effective reactions) more than required to compensate for the volume loss caused by repeated pipetting.

17. Mean sizes of the RNA fragments extracted from FFPE samples are about 100-200 nucleotides. To achieve accurate quantification of fragmented mRNA expression, the most important thing is to choose primers amplifying small amplicons (the amplified region of the cDNA) that are preferentially less than 70 bp. We use commercial TaqMan Gene Expression Assays (Applied Biosystems). When choices are limited, assays that generate amplicons longer than 70 bp may also be tested.

Each assay needs to be validated first using a series of control samples (similar tissues but not part of the study) that are of the same quality as those included in the research study.

18. Increase proportionally the number of cDNA copies for each gene in each sample so the initial relative quantities of individual genes across samples will remain the same in the final products after pre-amplification.

19. Each TaqMan assay is 20× concentrated (to be diluted 20-fold in the final classical PCR reaction). For pre-amplification, a final concentration of 0.05× is required, so the assay needs to be diluted 400-fold for this step. For example, for a 50 μL preamplification reaction, we need 12.5 μL of a diluted TaqMan assay mixture (0.125 μL of a 20× assay each; this volume is too small to be pipetted, so we need an intermediate dilution). For different cDNA samples, the same TaqMan assay mixture will be used, so we prepare a sufficient amount of this mixture by simply mixing equal volumes of each assay before diluting them together to a concentration of 0.2×.

20.

RNase-free water is suitable for replacing the Tris-EDTA (TE) buffer proposed in the standard protocol for the TaqMan PreAmp Master Mix kit (Applied Biosystems).

21. For one 50 μL of pre-amplification, the allowable quantity of cDNA (corresponding to initial RNA) is 1-250 ng, which needs to be adapted according to the quality and the total available amounts of the RNA. For FFPE tumor samples, we determined 100 ng to be a good compromise between the TLS-Related Gene Detection in FFPE Human Samples RNA quantity variations (obtained from a single extraction) across samples and the quality of gene quantification using pre-amplified cDNA.

22. The number of pre-amplification cycles can be adjusted depending upon the number of genes to be quantified. Ten cycles produce sufficient material for 50 × 20 μL or 20 × 50 μL final PCR reactions. Fourteen cycles produce material for 200 × 20 μL or 80 × 50 μL PCR reactions. We used 10 cycles in our experiments for quantifying less than 15 genes.

23. Dilute 5-fold for 10 cycles and 20-fold for 14 cycles, respectively. The diluted, pre-amplified cDNA can be stored at -20 °C in aliquots by minimizing freeze-thaw cycles.

24. Universal housekeeping genes are hard to identify with exceptions to uniform expression often detected in large data sets.

To minimize the risk, it is recommended that no less than three housekeeping genes are used for gene expression analysis in total tumor tissues that contain a mixture of cell types. In addition, the extent of RNA degradation can vary between individual FFPE blocks, which may differentially affect gene detection in each sample. We selected our housekeeping genes [POLR2A, TMBIM4 (="CGI-119" in ref. 18) and TBP] from publications [18,19] and commercially available TaqMan Gene Expression Assays designed for housekeeping genes (Applied Biosystems). The homogeneity of their natural expression levels was validated in a series of 88 frozen BC samples (with high-quality RNA) from a microarray data set [20] (Fig. 3a). The combination (mean values) of the three housekeeping genes further improved the homogeneity. We analyzed 60 BC with available FFPE samples using qPCR after pre-amplification. Exceptionally, reduced detection levels (inversely proportional to C T values) were found in 4 out of 60 (6.7%) samples for the mean of housekeeping genes (Fig. 3b, blue frame). Nevertheless, a linear correlation was obtained for the genes of interest (e.g., CXCL13) between the microarray data analyzing frozen samples and qPCR results from FFPE samples (CXCL13 was normalized to the mean of the housekeeping genes in FFPE samples) (Fig. 3c). It is possible that including other reliable housekeeping genes would improve these results.

25. Due to the large number of samples in these studies, it is better to include the maximum number of samples (rather than genes) on the same plate. This avoids errors coming from preparation of different mixtures for each gene and improves inter-experimenter reproducibility. Typically, we organize our experiments so that one or two (maximum) genes in triplicate are run on a single plate. 26. Centrifugation at high speed could affect enzyme efficiency. This speed is sufficient to push the drops to the bottom of the well.

27. A Section-Transfer-System with an integrated water bath and a disposable blade system optimizes the paraffin ribbon retrieval process. The sections are gently transferred by a permanent laminar water flow into the water bath providing more useable sections from less material.

28. Dried FFPE sections can be stored at 4 °C for 1 week.

29. Incomplete removal of paraffin can lead to poor staining of the tissue section. From this point onwards, at no time should the tissue sections be allowed to dry. Drying will cause nonspecific antibody binding and therefore high background staining.

30. Most formalin-fixed tissues require an antigen retrieval step due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigen epitopes including those recognized by antibodies used for IHC. The two methods of antigen retrieval are heat mediated using sodium citrate buffer or EDTA, and enzymatic reagents. For the dual-CD3/CD20 staining, sodium citrate buffer is used.

31. TLS were scored by two experienced immunopathologists in our institute examining CD3/CD20 IHC-stained full-face breast tumor sections.

32. The PCR amplification curves are carefully examined. The threshold can be adjusted based on the data obtained but generally 0.2 is optimal in our laboratory. It is important to use the same threshold for analyzing data from different plates that quantify the same gene. Occasionally, obviously imperfect results showing artifact amplification curves should be deleted before data analysis. If this occurs in more than one of the triplicates for the same gene and the same sample, this data will need to be verified by redoing a PCR reaction. For sample exclusion, also see Note 14.

33. The expression level of housekeeping genes should ideally be proportional to the total RNA amount and/or the total number of cells of a given sample.

Introduction

Immunoglobulins (Ig) or antibodies are a central component of the immune system. They consist of two identical heavy chains (IgH) and two identical light chains (Igκ or Igλ), each of which contains a variable domain for antigen recognition and a constant region for the effector functions. The IgH locus consists of variable (V), diversity (D), and joining (J) gene segments. The Igκ/λ locus consists only of V and J gene segments. Assembly of V(D)J gene segments by V(D)J recombination process is the central mechanism that leads to Ig antigen receptor diversity. The Ig variable domain consists of three short regions, named complementarydetermining regions (CDR), which are embedded into four conserved framework regions (FR). Starting from the amino terminus of both VH and VL, the CDR are called CDR1, CDR2, and CDR3. The FR1, 2, and 3 together with the CDR1 and CDR2 are encoded by the V gene segments. The FR4 is encoded by the J gene segment. Additional diversity in the Ig antigen receptor is introduced in structures called germinal centers (GCs) where somatic hypermutation and affinity-based selection take place bringing to the generation of memory and plasma cells which produce high-affinity antibodies. In healthy individuals, GCs form in secondary lymphoid organs (lymph nodes, spleen, mucosal associated lymphoid tissues). During infection, autoimmune diseases, cancer, transplant rejection, and ectopic lymphoid structures (ELS), also called tertiary lymphoid structures, resembling GCs can form at the site of inflammation supporting the local production of autoantibodies [1,2]. For instance, 40% of patients with rheumatoid arthritis (RA), an inflammatory autoimmune disease, develops in their joint ELS which can acquire features of secondary lymphoid organs supporting a GC response [3,4]. Interestingly, these structures have been shown to support locally antibody diversification, and thus production of autoreactive B cells [1,5]. However, the nature and fine specificities of (auto)antigens driving B-cell autoimmunity and the exact pathogenic role of autoantibodies in RA but also other autoimmune diseases such as Sjögren's syndrome (SS) have not been elucidated yet. One of the limitations in order to address these important aspects is the technical challenge of analyzing the fine specificity of autoreactive B cells isolated from the inflammatory milieu. Moreover, the possibility to analyze B-cell autoreactivity from lesional B cells of autoimmune patients at single-cell level is a good tool to better understand the frequency and Ig gene repertoire underlying the development of autoimmunity.

Here, we describe a method which allows the cloning and expression of recombinant monoclonal antibodies (rmAbs) from single lesional B cells isolated from RA synovial tissue characterized by functional ELS [1,6]. This technique is a modified version of the strategy developed some years ago in the Nussenzweig's laboratory [7][8][9]. The protocol gives a detailed guide of all the steps involved in the generation of rmAbs from single B cells obtained from RA synovial tissue, applicable to other inflammatory tissues (e.g., salivary glands of SS patients), starting from isolation of mononuclear cells, single-B-cell sort by flow cytometry, amplification of the Ig genes, cloning, and in vitro expression of full IgG1 rmAbs (Fig. 1). The rmAbs generated can then be used for downstream analysis including (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo. 13. Trypan blue solution 0.4%: Use at a final concentration of 0.2%, diluted in 1× PBS. 17. 100 bp DNA ladder ranging from 100 to 1517 bp.

Mononuclear Cell Isolation from Synovial Tissue

18. 3′ Primers for sequencing: 3' Cμ CH1 int, 3' Cμ IgG int, 3' Cμ CH1-2 int (Table 1).

1. Sequencing service company.

2. International ImmunoGeneTics (IMGT) free online database (www.imgt.org/vquest).

3. PCR cooler suitable for 96-well PCR plates.

4. PCR thermal cycler suitable for 96-well PCR plates skirted and half-skirted.

5. Gel tank for DNA agarose gel.

6. 96-Well plates, half-skirted.

7. Reservoir reagent 100 mL sterile polystyrene.

8. Adhesive plate seals and adhesive PCR seals suitable for hightemperature incubations. 9. Nuclease-free water.

10. Ethanol, RNase, and DNase Away decontamination solution.

Gene Sequence Analysis and Gene-Specific PCR

Methods

Mononuclear cells are isolated from synovial tissue specimens obtained from joint replacement surgery or biopsy characterized by the presence of lymphocytic infiltration. The extracellular matrix in animal tissues is made by collagens and other extracellular matrix proteins such as glycoproteins and proteoglycans. To isolate single cells without disrupting the cellular structure a combination of proteolytic enzymes is necessary to dissociate the tissue (see Note 22). All steps are performed under a tissue culture laminar hood.

1. Put the synovial tissue into a petri dish with RPMI-1640 (Fig. 2) (see Notes 23 and 24).

2. Cut the synovial tissue into small pieces using scissors (see Note 25).

3. Prepare 5 mL round-bottom tube(s) containing the digestion buffer (see Notes 26 and 27).

Recombinant Monoclonal Antibody Production 2.7 Purification of Recombinant Monoclonal Antibodies

Mononuclear Cell Isolation from Synovial Tissue

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4.

Transfer the cut synovial tissue into 5 mL round-bottom tubes with the digestion buffer (see Note 28).

5. Add one magnetic stirring bar to each 5 mL round-bottom tube using tweezers.

6. Start the digestion for 1 h at 37 °C under shaking in a water bath (Fig. 3) (see Note 29).

7. Add 15 μL of 0.5 M EDTA to each tube to stop the reaction.

8. Filter each sample through 40 μm cell strainer to remove undigested tissue and centrifuge for 10 min at 400 × g. The filtered solution contains the mononuclear cells (Fig. 3). 9. Resuspend the mononuclear cells in RPMI-1640 and count the cells.

10. Proceed to the immunofluorescence labeling for flow cytometry for single-cell sorting (see Note 30).

Purified mononuclear cells isolated from synovial tissues are stained with fluorescent-labeled anti-human CD19 and anti-human CD3, in order to isolate single CD19 + CD3 -B cells (Fig. 4) (see Note 31).

1. Aliquot purified mononuclear cells into 1.5 mL tubes and centrifuge at 400 × g for 4 min at 4 °C. 3. Resuspend the cell pellet in 100 μL of FACS buffer staining solution containing all the antibodies at the appropriate final concentration.

Isolation of Single Human B Cells by Fluorescence-Activated Cell Sorting (FACS) into 96-Well Plates

4. Incubate the cells for at least for 30 min in the dark at 4 °C.

5. Wash the cells with 1 mL FACS buffer and resuspend in FACS buffer at a final concentration of 10 7 cells/mL.

6. Add DAPI to exclude dead cells.

7. Keep samples on ice until single-cell sorting is performed.

8. Sort the cells into 96-well PCR skirted plates containing 4 μL of single-cell lysis solution per each well (see Note 32).

Immediately after the sorting, seal the plate with the adhesive aluminum foil and put on dry ice. If not immediately used for the steps described below, store the plates at -80 °C (see Note 33).

Complementary DNA is synthesized in a total volume of 14.5 μL per well in the original 96-well sorting plate. Before starting all steps in this section, it is important to clean the PCR hood or the laboratory bench and laboratory pipettes with ethanol, RNase Away, and DNase Away decontamination solution in order to work in RNase/DNase-free conditions. Perform all the steps on ice or on a PCR cooler.

1. Thaw a plate of single-sorted B cells on ice and spin down before removing the adhesive aluminum foil. 3. Transfer the plate on a hot plate for 1 min at 68 °C and put back on ice or onto the PCR-cooler. 8. Send the second PCR product of the expected size (IgH, Igκ, and/or Igλ) for sequencing with the respective 3′ primer.

Single-Cell RT-PCR and Immunoglobulin V Gene Amplification by Nested PCR

Single-Cell RT-PCR

Ig nucleotide sequences are analyzed using the IMGT/V-QUEST database in order to identify the V, D, and J genes and alleles in rearranged V-J and V-D-J sequences by alignment with germline Ig gene and allele sequences of the IMGT directory. It defines the FR and CDR regions and provides a detailed characterization of the query sequence. The algorithm used to identify the closest V, D, and J genes and alleles is based on global pairwise alignment followed by a similarity evaluation. Determining the Ig gene usage is essential for performing the gene-specific PCR (Fig. 5).

1. Determine for each PCR product (IgH and IgL (κ/λ)) the Ig-V and Ig-J gene usage in order to set up the gene-specific second nested PCR.

2. Define the gene-specific primer using Table 3.

3. Clean the PCR hood or the laboratory bench and laboratory pipettes with ethanol, RNase Away, and DNase Away decontamination solution.

Ig V Gene Sequence Analysis and Gene-Specific PCR

Elisa Corsiero et al.

4.

Dilute gene-specific primers to a working concentration of 5 μM in nuclease-free water.

5. In a 96-well plate deposit 2 μL of the matched 5′ and 3′ primers for one PCR product into the same well. Cover the plate with adhesive plate seal and spin down.

6. In the same plate with the primers, add 33.8 μL of PCR master mix in each well. 10. Purify gene-specific PCR products using a PCR cleanup kit following the manufacturer's instruction. Elute the PCR products in nuclease-free water in a final volume of 60 μL.

11. PCR products can be stored at -20 °C before performing the expression vector cloning. Restriction enzyme digestion is used to generate compatible ends on the PCR product that can be ligated into a specific expression vector DNA. In order to generate compatible ends, all the primers used in the gene-specific PCR are characterized by the presence of restriction sites which are compatible with the expression vector that will be used to express the rmAbs. 4. Purify the digested PCR products using a PCR cleanup kit following the manufacturer's instruction. Elute the digested PCR products in nuclease-free water in a final volume of 60 μL.

5. Digested PCR products can be stored at -20 °C before performing the ligation and transformation step.

1. Set up the following reaction in a 0.2 mL PCR tube on ice (see Note 38). 8. Check the size of the insert, check PCR products by running a 2% agarose gel. Expected sizes are Igγ1 vector = 650 bp, Igκ1 vector = 700 bp, and Igλ2 vector = 590 bp. Load 5 μL of the PCR product mixed with 5 μL of loading dye into the agarose gel containing GelRed and run for 30 min at 120 V. Visualize the DNA bands using a UV transilluminator. 9. Sequence the insert check PCR product using the 5′ Ab sense primer as sequencing primer to confirm the identity with the second PCR product. Check also that the PCR product is in frame within the plasmid. Pick up the colonies with 100% of identity from the backup plate using an inoculating loop and inoculate 4 mL LB medium containing 100 μg/mL ampicillin in 14 mL round-bottom tube. Loosely close the tube and incubate at 37 °C overnight under vigorous shaking (250 rpm) (see Note 45).

Expression Vector Cloning: PCR Product Restriction Enzyme Digestion, Ligation, and Bacteria Cell Transformation

PCR Product Restriction Enzyme Digestion

Ligation

10. After incubation, check for growth which is characterized by a cloudy medium appearance (see Note 46).

11. Use some of the inoculating medium for preparing a glycerol stock for long-term storage of the bacteria before starting the plasmid mini-prep (see Note 47).

12. Isolate plasmid using a nucleic acid purification plasmid kit (plasmid mini-prep) following the manufacturer's instruction. Elute DNA plasmid in 75 μL nuclease-free water.

13. Measure the concentration of the DNA plasmid with a spectrophotometer at A260nm. Determine the purity of the DNA plasmid by the ratio 260/280 nm (see Note 48).

Recombinant monoclonal antibodies are expressed using the Expi293F™ Expression System that allows a rapid and high-yield protein production in mammalian cells. Expi293F cells are specifically adapted to grow in suspension at high density and exhibit higher protein production compared to classical 293 cells. The medium used in this system is chemically defined and serum-and protein free that, in combination with the Expi293 Transfection Reagent, enables a high-efficiency transfection.

1. Add 7.5 × 10 7 Expi293F cells to 25.5 mL of Expi293 Expression medium in a 125 mL Erlenmeyer flask (8% CO 2 incubator at 37 °C and 125 rpm on an orbital shaker) (see Notes 49 and 50).

2. Dilute 30 μg of plasmid DNA (15 μg IgH plasmid and 15 μg IgL (κ or λ) plasmid) in Opti-MEM to a total volume of 1.5 mL. Mix gently.

Recombinant Monoclonal Antibody Production

Elisa Corsiero et al.

Dilute 81 μL of ExpiFectamine

Reagent in Opti-MEM to a total volume of 1.5 mL. Mix gently and incubate for 5 min at room temperature.

4. Add the diluted DNA to the diluted ExpiFectamine Reagent for a total volume of 3 mL. Mix gently.

5.

Incubate the mixture for 20 min at room temperature.

6. Add 3 mL mixture to the flask. Put flask back to the incubator.

7. After 20 h, add 150 μL of ExpiFectamine Transfection Enhancer 1 and 1.5 mL of Enhancer 2 to the flask.

8. Six days after transfection, sediment cells by centrifugation for 10 min at 800 × g at 4 °C. 9. Filter the supernatant through 0.2 μm syringe filter and store at 4 °C (see Note 51).

Recombinant monoclonal antibodies are purified from the culture supernatant by affinity chromatography using Protein A Sepharose beads.

1. Equilibrate 100 mg of Protein A Sepharose beads with cold 1× PBS. Centrifuge for 10 min at 4000 × g at 4 °C. Aspirate supernatant and resuspend beads in 1 mL of 1× PBS.

2. Add the equilibrated Protein A beads into the culture supernatant and incubate overnight at 4 °C under rotation.

3. Separately, add 0.5 M NaOH into the chromatography column and leave the solution overnight to clean and sanitize the column (see Notes 52 and 53).

4. Discard the NaOH solution. Rinse and equilibrate the column with abundant cold 1× PBS.

5. Load culture supernatant/Protein A beads onto the chromatography column and discard flow-through (see Note 54).

6. Wash the beads with 2 × 50 mL of cold 1× PBS and discard the flow-through (see Note 54).

7. Elute recombinant antibody adding elution buffer. Collect three fractions of 500 μL in microcentrifuge tubes containing 50 μL of neutralization buffer (see Note 55).

8. Measure antibody concentration at A 280nm using a UV spectrophotometer.

9. Purified antibody may be stored at 4 °C. Sodium azide may be added for long-term storage (see Note 56).

10. Analyze purity and integrity of recovered immunoglobulin by loading 500 ng/lane in a SDS-PAGE followed by a protein staining technique (see Note 57).

Purification of Recombinant Monoclonal Antibodies Using Protein A Beads

Notes

1. The synovial membrane or synovium is a loose connective tissue which is found between the joint capsule and the joint cavity of arthrodial joints. The normal synovium is composed mainly by two distinct layers: the intimal lining layer and the synovial sub-lining layer. The intimal lining layer is mainly composed by fibroblast-like synoviocytes and macrophages. The sub-lining layer is made of connective tissue containing blood vessels, fibroblasts, adipocytes, and some resident immune cells like macrophages and mast cells [10]. During chronic inflammation, the intimal lining layer is characterized by marked hyperplasia, whereas the sub-lining layer is the site of massive infiltration of immune cells. In order to generate recombinant monoclonal antibodies, only synovial tissue with infiltration of lymphoid cells is used.

2. Prepare a stock concentration at 100 mg/mL and 10 mg/mL in 1× PBS for collagenase D and DNase I, respectively. Aliquot and store at -20 °C. Use heat-inactivated FBS throughout all the protocol.

3. It is important to use skirted PCR plates to avoid tilting of the plates during the single-cell FACS sorting.

4. This protocol has been designed to isolate single CD19 + CD3 - B cells but the sorting strategy can be modified accordingly to the cell population of interest: CD19 antibody (clone SJ25C1) and CD3 antibody (clone HIT3a).

5. Random hexamer primers and NP-40 are diluted at their final concentration in nuclease-free water.

6. The 100 mM dNTP set consists of four deoxynucleotides (dATP, dCTP, dGTP, dTTP), each at a concentration of 100 mM. Prepare aliquots of dNTP mix adding 50 μL dATP, 50 μL dCTP, 50 μL dGTP, and 50 μL dTTP from the 100 mM dNTP set. Store aliquots at -20 °C.

7. Prepare each primer to a working dilution of 50 μM. For the primer mix, combine equal volume of each primer. Primer mixes can be prepared in advance and stored at -20 °C. All the primers used are the same as previously published with the exception in the use of the Cμ internal primer (GGGAATTCTCACAGGAGACGA) in the second round of the nested PCR [7,8].

8. The HotStart Taq DNA polymerase has an error rate of 2 × 10 -5 /nucleotide and cycle and it has been chosen instead of high-fidelity enzymes which introduce less error because the HotStar Taq DNA polymerase has a high amplification efficiency for low-copy templates when starting from single cell.

Elisa Corsiero et al.

9.

GelRed is a safe and ultrasensitive fluorescent nuclei acid dye which is used to replace the toxic ethidium bromide for staining the dsDNA in agarose gels. Use according to the manufacturer's instruction.

10. Dilute the loading dye to a working dilution of 1× in distilled water before use.

11. For Igλ 3′ primer, use the primer 3' XhoI Cλ for all the PCR products.

12. Use the following primers for sequencing: for IgH = 3' Cμ CH1 int or Cγ IgG int or Cα CH1-2 int; for Igκ = 3' Ck494; for Igλ = 3' XhoI Cλ.

13. 10× NEB CutSmart buffer includes already BSA.

14. IgH γ1, IgL κ1, and IgL λ2 expression vectors are characterized by the ampicillin-resistant gene. The original vectors are from Dr. Hedda Wardemann's group, as published in [7,8]. Human IgG heavy-and light-chain expression vectors are also commercially available. Please check that the cloning sites are compatible with the primers containing restriction sites in use in this protocol.

15. Cool autoclaved LB agar medium to below 50 °C (when you can hold it easily) before adding ampicillin which is a heatsensitive antibiotic. Pour LB agar medium into a 100 mm petri dish next to a Bunsen burner. Dry plates by leaving them upside down with lids on at room temperature overnight.

Store plates inverted at 4 °C in a darkroom or wrapped in aluminum foil to preserve light-sensitive ampicillin. Do not store for longer than 1 month as ampicillin may degrade.

16. Genotype DH10B E. coli cells: Δ(ara-leu) 7697 araD139 ΔlacX74 galK16 galE15 e14-ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC).

Competent cells are supplied with a control vector pUC19 used as positive control during the transformation reaction. 17. Prepare each primer to a working dilution of 50 μM: 5′ Absense primer (GCTTCGTTAGAACGCGGCTAC); 3′ IgG internal (GTTCGGGGAAGTAGTCCTTGAC) for IgH γ1 vector; 3′ Cκ 494 (GTGCTGTCCTTGCTGTCCTGCT) for IgL κ1 vector; and 3′ Cλ (CACCAGTGTGGCCTTGTT GGCTTG) for IgL λ2 vector.

18. Standard adherent HEK293T cells can also be used for the production of the recombinant monoclonal antibodies together with alternative transfection reagents such as polyethylenimine (PEI), as reported in the section "Recombinant Antibody Production" in [8].

19. To produce lower amount of recombinant antibodies, it is possible to reduce the quantity of plasmids. In this case, scale down all required solutions. 20. For smaller volume, disposable polypropylene spin columns (1.2 mL bed volume) can be used.

21. Alternatively, protein G beads can also be used.

22.

Collagenase is an enzyme which is used for the dissociation of tissues since it breaks down the native collagen that holds tissues together. In particular, collagenase D is used to preserve functionality and integrity of cell-surface proteins. Enzyme activity is inhibited by the addition of EDTA.

23. Synovial tissue is the pink layer on the fat tissue and once it is in the medium it forms telltale fronds-like structure (Fig. 2). Do not (1) use tissue that has been burnt during cauterization which is indicated by black coloration and (2) take materials from around the artery entrance.

24. Add the RPMI-1640 medium as much as you need to cover the synovial tissue in order not to dry out the tissue during the cutting.

25. Be aware that the synovial tissue is a quite gluey and elastic tissue.

26. The number of 5 mL round-bottom tubes depends on the amount of synovial tissue pieces prepared.

27. For one tube = 1.5 mL RPMI-1640, 2% FBS, 37 μL collagenase D, and 2 μL DNase I.

28. 1-2 Pieces for tube.

29. Set the water bath at 37 °C before starting all the steps.

30. Be aware that single-cell sorting is associated with considerable cell loss up to 95%. Therefore, it is better starting the sorting from at least 1 × 10 6 cells.

31. From the total cell number, depending on the final yield, use 0.5/1 × 10 6 cells for each of the following controls.

Control 1: Use single staining for setting up the flow cytometer and to perform fluorescence compensation. Use compensation beads, according to the manufacturer's instruction.

Control 2: Use the fluorescence minus one (FMO) controls to set appropriately the cell population gates.

32. Prepare the PCR sorting plate(s) in advance and keep at 4 °C. After the addition of the lysis buffer, spin down the plate and seal with adhesive plate seal.

33.

Transferring the PCR plate on dry ice quickly is a critical step for maintaining a high RNA integrity.

Elisa Corsiero et al.

34.

For one plate, prepare a master mix for 110 wells in order to have a surplus of reagents.

35. Preparing an enzyme digestion master mix is necessary to avoid loss of enzyme due to small pipetting volumes.

36. Depending on the number of PCR products to digest, this step can be performed in single-PCR 0.2 mL tubes or into 96-well PCR plate.

37. Digestion can be performed either in a water bath or in a PCR cycler.

38. T4 DNA ligase should be added last.

39. For the IgH PCR product use the human IgHγ1 linearized plasmid vector; for Igκ PCR product use the human IgLκ1 linearized plasmid vector; for Igλ PCR product use the human IgLλ2 linearized plasmid vector.

40. Do not vortex the tubes. For maximum transformation efficiency, the mixture should be incubated at least for 30 min.

Reducing the incubation time decreases the transformation efficiency.

41. Normally, the manufacturer's instruction suggests to use 50 μL for each transformation. However, scaling down of 1:10 for each transformation is also possible bringing to a good transformation efficiency.

42. Incubate for at least 1 h. Reducing the incubation time decreases the transformation efficiency.

43. LB agar plates can be used warm or cold, wet or dry, without affecting the transformation efficiency. However, working with warm plates is easier and allows a more rapid colony formation.

44. Set up also a positive and negative control mix.

Reagent Positive control

Negative control

Nuclease-free water 3 μL 3 μL 46. Use as control LB media with ampicillin without any bacteria inoculated. In this tube, you should not see the appearance of a cloudy medium after inoculation overnight.

Expression plasmid vector

47. Prepare a glycerol stock: Add 500 μL of the overnight bacteria culture to 500 μL of 60% glycerol in a cryovial tube and gently mix. Make 60% glycerol solution by diluting 100% glycerol in deionized water and autoclave before use. Freeze the glycerol stock tube at -80 °C.

48. A good purity ranges from 1.80 to 2.00.

49. Expi293F cells are grown according to the manufacturer's instructions. Cells should be passaged every 3-4 days when the density reaches 3-5 × 10 6 cells/mL, and split to 0.3-0.5 × 10 6 cells/mL. 50. Transfection of Expi293F cells is also possible in smaller volume. In this case, scale down all required solutions.

51. Collect and filter the supernatant under the tissue culture hood to keep the supernatant on sterile conditions, particularly if you need to use the antibodies for in vivo assays.

52. Sodium hydroxide is effective in removing proteins and nucleic acids. It is also effective for inactivating most viruses, bacteria, yeasts, and endotoxins.

53. For smaller supernatant volumes, more suitable chromatography columns can be used, like spin disposable chromatography columns (BioRad).

54. The flow-through can be collected to check if the binding capacity of the Protein A Sepharose beads has not been saturated, and thus for residual presence of antibody in the supernatant.

55. Fractions may be pooled after determining protein concentration.

56. Alternatively, use a desalting column or dialysis to exchange the purified antibody into a more suitable buffer.

57. In parallel, sample can be prepared using a nonreducing loading buffer to check the integrity of the antibody. Molecular weight Ig in reducing condition: HC (each chain) = 50 kDa; LC (each chain) = 25 kDa. Molecular weight Ig in nonreducing condition = 150 kDa.

Introduction

During the last decades, the immune microenvironment has been extensively characterized by immunohistochemistry in many solid tumors [1]. It has been shown that T lymphocytes are able to infiltrate the stroma and tumor islets [2], whereas B lymphocytes are mainly located at the invasive margin of the tumor, in ectopic lymphoid aggregates called tertiary lymphoid structures (TLS), displaying a similar architectural organization to secondary lymphoid organs [3]. They are composed of a B-cell zone adjacent to a T-cell zone. The prognostic value of TLS was reported for the first time in NSCLC patients [4].

Given that T cells are the key players in immunosurveillance, many studies have been conducted to deeply characterize their phenotype and function within the tumor microenvironment. Indeed, a high density of T lymphocytes and more particularly cytotoxic and memory CD8 + T cells was shown to be correlated with a favorable clinical outcome in the most frequent solid tumor types [5]. However, the clinical impact of other T-cell subpopulations, such as conventional CD4 + T cell and regulatory T cells (Treg), or unconventional T cells, such as γδ T cells, remains a matter of debate. For instance, in ovarian cancer, it has been demonstrated that a high Treg infiltrate was correlated with a poor clinical outcome [6], in opposition to head and neck cancer [7], follicular lymphoma [8], and colorectal cancer [9], for which the increased density of Tregs was associated with a good clinical outcome. Surprisingly, in some cases, such as anal carcinoma, glioma, and glioblastomas, the Treg infiltrate was not correlated with any clinical outcome [10][11][12]. Therefore, in order to better elucidate the function of Tregs in cancer, we developed an optimized method, which permits the sorting of CD4 + T-cell subsets, i.e., Tregs and non-Treg CD4 + T cells (mainly conventional CD4 + T cells), from the lung tumor, non-tumoral distant lung, and blood of NSCLC patients. The method is designed to obtain highly pure T cells (purity 98-100%), which can be used for further cellular and molecular biology applications such as functional assays or gene expression analysis. This in particular allows a better understanding of the potential suppressive activities of Tregs in NSCLC patients.

Another population of unconventional T lymphocytes, called gamma delta (γδ) T cells, is also a key player in the antitumor immune response. γδ T cells express a particular T-cell receptor (TCR) composed of a γ chain and a δ chain [13]. In opposition to conventional T cells (CD4 + and CD8 + T cells), γδ T cells are able to recognize a broad range of antigens independently of the classical major histocompatibility (MHC) molecules. In human, there are two major subsets of γδ T cells: Vδ2 + cells that are mainly present in the circulation and Vδ2 -cells (among which Vδ1 + cells) mainly present in epithelial tissues with a wider TCR diversity and less known antigen specificity. Vδ2 + T cells always associate to the Vγ9 chain in adults and mainly recognize phosphorylated nonpeptidic molecules called phosphoantigens that are metabolic intermediates of the isoprenoid biosynthesis, and could be produced by tumor cells [14]. It has been shown that Vδ1 T lymphocytes from human lung tumors are able to selectively lyse autologous malignant cells ex vivo [15]. Furthermore, the administration of Vγ9Vδ2 + T cells at suitable intervals after chemotherapy and zoledronate treatment was demonstrated to increase the cytotoxic function and IFN-γ production by γδ T cells followed by a complete lysis of tumor cells in different malignancies [16]. Therefore, these cells seem to be a promising treatment tool. But their physiopathological relevance is not well known and different parameters concerning human γδ T-cell function and phenotype within tissues are still not well understood. For this aim, a deeper characterization of γδ T-cell-associated biomarkers and function is required to better understand their prognostic value and their contribution to the antitumor immune response in NSCLC patients. Given that these cells are rare and could be activated by anti-TCR antibodies added during the classical cell sorting, their isolation for further assays is challenging. The techniques described in this chapter are useful to obtain "untouched" γδ T cells for functional assays but also to sort Vδ2 + and Vδ2 -T-cell subsets for gene expression analysis. Using these strategies, the sorting of γδ T-cell subsets could be optimized, undesired activation prior to functional assays avoided, and other cellular contaminants such as other immune cells, dead cells, and tumor cells eliminated. This will permit a better understanding of the contribution of intra-tumoral γδ T cells in the development of the antitumor immune response and ultimately may reconcile some studies in which results seemed contradictory.

Finally, the role of tumor-infiltrating B cells (TIL-B) in the tumor microenvironment is still controversial. On the one hand, the pro-and antitumor functions of B cells were reported in various studies and on the other hand the manipulation of B cells seems to be a promising therapeutical approach, as reviewed in [17]. The first studies have shown that the depletion of B cells in several mouse models could decelerate the tumor progression [18][19][20]. In another study in mice, it has been shown that B cells were recruited among leukocytes into the tumor [21]. These cells were able to produce lymphotoxins, thus promoting the proliferation and survival of cancer cells [21]. However more recent studies have reported an antitumor role of B cells in several human cancers such as in ovarian cancer [22], breast cancer [23], and hepatocellular carcinoma [24] and revealed that patients having high density of tumor-infiltrating B cells have a better clinical outcome than patients having few B-cell infiltrate. These cells are mainly located at the invasive margin of the tumor, more precisely in TLS. Our team has previously demonstrated that a high density of TLS-B cells is correlated with a higher overall survival in NSCLC patients [25,26]. Furthermore, it has been shown that germinal center (GC)-B-cell formation and TLS density are reduced by corticosteroid treatment in lung squamous cell carcinoma [27]. These findings highlight the prognostic importance of GC-B in TLS during tumor development and treatment. Given these observations, we hypothesize that TLS-B cells may be key players in the antitumor immune response generated in NSCLC cancer and aimed to better characterize their phenotype and functional relevance. To address this question, we developed a novel flow cytometry-based approach to isolate untouched B cells from the tumor tissues, too. 3. Penicillin (10,000 U/mL)/streptomycin (10,000 μg/mL) (Gibco).

4. l-Glutamine (200 mM) (Gibco).

5. Sodium pyruvate (100 mM) (Gibco).

6. β-Mercaptoethanol (50 mM) (Gibco).

HEPES (1 M) (Gibco).

3 Methods

1.

Place the tumor and non-tumoral distant lung (NTDL) in a petri dish using a forceps and keep the complete RPMI medium used for tumor collection (see medium composition in Subheading 2.6).

2. Dilacerate the tumor using a scalpel and a forceps (make pieces as small as possible).

3. Pour the tumor pieces directly from the petri dish to a 50 mL Falcon.

4. Rinse the petri dish with 20 mL of Cell Recovery Solution, close well the Falcon (surround the cap with "parafilm"), mix well (by inversion), and incubate for 1 h at 4 °C under gentle agitation.

5. After incubation, filter the suspension on a cell strainer placed onto a 50 mL Falcon; use a 10 mL syringe plunger to crush the pieces.

6. Rinse the filter first with the complete RPMI medium in which the tumor was collected, and then with 8-10 mL of washing buffer.

7. Spin the filtered suspension for 10 min at 300 × g at room temperature (along with available blood tubes).

8. After spinning, collect the plasma from blood samples (make aliquots of 500 μL in Eppendorf tubes and store them at -80 °C).

9. Resuspend blood cells, tumor, and NTDL pellets in 30 mL of washing buffer.

10. Load each sample suspension on 15 mL of lymphocyte separation medium.

Obtainment of a Cell Suspension from Non-tumoral Distant Lung, Tumor Tissue, and Peripheral Blood of NSCLC Patients

Sorting of Tertiary Lymphoid Structure-Immune Cells 11. Spin for 20 min at 800 × g, at room temperature, without acceleration and break.

12. Collect the ring of peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in a 50 mL Falcon; take an aliquot of 20 μL of each sample for cell counting.

13. Count the cells; dilute the 20 μL aliquot (1/10) in trypan blue, and load 10 μL on a Kova slide.

14. Fill up the tubes containing the collected cells to 50 mL with washing buffer and centrifuge for 10 min, at 300 × g, and at 4 °C. . Fill up to a total volume of 2.5 mL by adding the washing buffer to the cell suspension.

6. Mix the cells in the tube by gently pipetting up and down 2-3 times.

7. Place the tube (without cap) in front of the magnet.

8. Incubate for 5 min at room temperature. The magnetically labeled unwanted cells will remain bound inside the original tube.

Enrichment of CD4 + T Cells and γδ T Cells Using Negative Selection Kits

Enrichment of CD4 + T-Cell Populations

Priyanka Devi-Marulkar et al.

9.

Use sterile pipette to gently take out the unlabeled cells in suspension. Transfer the cells to the 50 mL Falcon tube for further processing.

10. Wash the untouched cells twice by filling up the tube with 50 mL of washing buffer, and centrifuge for 10 min, at 300 × g, and at 4 °C.

1. Use the EasySep™ Human Gamma/Delta T cell Isolation Kit (StemCell Technologies, catalog #19255) for immunomagnetic isolation of untouched γδ T cells (follow the manufacturer's instructions).

2. Collect the negative fraction of each sample (i.e., tumor, NTDL, and blood) in new 5 mL FACS tubes with caps.

3. Count the cells.

4. Fill up the tube with washing buffer and centrifuge for 10 min at 300 × g, at 4 °C.

5. After centrifugation, collect the negative fraction pellet.

6. Check the purity of the isolated population by flow cytometry (see Table 5 for the antibodies) or proceed to a further cell sorting with the appropriate antibodies (see Notes 1 and 2) (see Table 3 to sort γδ T cells for functional assays and Table 4 to sort cells for gene expression analysis of γδ T cells). 2. Without washing out the blocking buffer, add the appropriate antibodies recognizing the cell surface antigens (according to Tables 1, 2, 3, 4, 5, and 6) with or without CD62L staining (as a specific TLS marker), and based on the application (cell culture, gene expression analysis, phenotypic analysis). A previous titration should determine the optimal volume of antibody to be added to the cell suspension.

3. Vortex the antibodies and incubate with the cells for 30 min at 4 °C in the dark.

4. Fill up with 10 mL of washing buffer, mix, and centrifuge at 400 × g for 10 min at 4 °C to eliminate unfixed antibody.

5. Aspirate the supernatant and resuspend the cells in 1 mL of washing buffer.

6. Filter the cell suspension with 30 μm filter.

7. Add 1 μL/mL of DAPI to the cell suspension in order to exclude dead cells during the cell sorting.

Stainings for the Sorting of T-and B-Cell Subsets

Sorting of Tertiary Lymphoid Structure-Immune Cells 8. Keep the cells on ice (or at 4 °C) in the dark before the flow cytometry analysis and/or the cell sorting (see Notes 1-3).

1. When you proceed to cell sorting, gate on lymphocytes according to SSC and FSC values and exclude the doublets (Fig. 1).

2. Options: For gene expression analysis, gate on live cells (DAPI -) expressing both CD3 and CD4 to select CD3 + CD4 + T cells. For functional immunosuppression assays, gate on live cells (LDCY -) expressing both CD4 and CD2 in order to avoid undesired activation through the binding of anti-CD3 antibody on T cells. LDCY is a viability marker and refers to LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation (Thermo Fisher).

3. The sorting of CD4 + Treg and non-Treg populations among total CD4 + T cells will be based on the differential expression of CD25 and CD127. Indeed, gate on CD4 + CD25 hi CD127 -/ lo cells for the sorting of the CD4 + T Treg population, and on CD4 + CD25 +/-CD127 + cells for conventional CD4 + T non-Treg cells.

Gating Strategy for B-and T-Cell Subset Isolation

For CD4 + Treg and Non-Treg Populations

4. The sorting of lymphocytes infiltrating TLS and non-TLS areas of the tumor will be performed based on the differential expression of CD62L molecule. TLS-T cells selectively express the CD62L marker whereas non-TLS T cells do not [2,28]. Thus, four populations could be sorted in this case from fresh tumor and non-tumoral tissue specimens and blood, namely CD62L + TLS Tregs, non-TLS CD62L -Tregs, CD62L + TLS conventional CD4 + non-Treg cells, and CD62L -non-TLS conventional CD4 + non-Treg cells (Fig. 1).

1. Gate on the lymphocyte population according to SSC and FSC values.

2. Select live DAPI -cells.

3. Gate on live cells expressing the CD45 marker.

4. Gate on CD45 + CD14 -CD56 -cells.

5. Then, gate on cells negative for epithelial, CD66b, CD11c, CD36, CD43, CD2, and CD16 markers.

6. Remove cell doublets.

7. Check the B-cell purity by adding an anti-CD19 antibody to an aliquot of the B-cell fraction (Fig. 2).

1. Gate on the lymphocyte population based on SSC and FSC parameters.

2. Exclude the doublets.

3. Sort the CD2 + LDCY -γδ T-cell population (Fig. 3) for cell culture assay (see Subheading 3.7).

1. Collect the sorted cells for gene expression per 2 mL freezing vials containing 300 μL of RLT and 1% β-mercaptoethanol.

2. Directly freeze the cells in a vial in a small container of liquid nitrogen, and then store them at -80 °C.

1. For cell culture, collect the cells in Eppendorf tubes containing 200 μL of washing buffer (γδ T cells) or 500 μL of the pure filtered FCS (CD4 + T-cell subsets and B cells).

2. After sorting, centrifuge the collected cells at 300 × g for 5 min at 4 °C.

3. Discard carefully the supernatant.

4. Resuspend the pellet in the appropriate volume of cell culture medium.

Collection of Sorted Cells for Gene Expression Analysis

Collection of Sorted Cells for Functional Assays

Sorting of Tertiary Lymphoid Structure-Immune Cells 2. Wash the excess of antibody with 200 μL/well of PBS. 1. The use of the magnetic bead-based enrichment kit is insufficient for achieving best purity of CD4 + and γδ T cells from tissue because of the presence of epithelial contaminants. Thus, we added a further step of the cell sorting using appropriate antibodies (see Tables 1,2, 3, and 4). This was useful to better study these immune cells in gene expression analysis and culture assays.

γδ T-Cell Culture Assay

2. Do not use anti-CD3, anti-γδ TCR, or anti-CD19 antibodies to avoid cellular activation before cell culture. Anti-CD2 antibody can be used instead of anti-CD3 for the sorting of the T-cell subsets.

3. For some experiments as co-cultures, the positive selection of B cells is not recommended. Numerous research articles collected B cells using untouched isolation kits, which are very effective to collect human B cells from peripheral blood by negative selection (purity >95%). However, negative selection using these kits is not a good strategy to collect B cells from tissues such as tumors due to a strong presence of contaminating epithelial and stromal cells. Given the heterogeneity of tumors, it is infeasible to design and to modify these kits each time. Based on these results, the use of this negative selection strategy using a cell sorter seems to be more appropriate. The addition of anti-CD4 and anti-CD8 antibodies makes possible the sorting of not only untouched B lymphocytes but also T lymphocytes in order to perform a B-T coculture assay. may participate in TLS formation and maintenance through their capacity to secrete CXCL13, sustaining a local microenvironment controlling tumor growth [21,22].

In this chapter, we describe complementary multiplex methods, i.e., two methods of immunofluorescence (IF) tissue staining (three-and five-color) on formalin-fixed paraffin-embedded (FFPE) tissue sections, and one method of flow cytometry analysis of cell suspensions prepared from fresh tumor samples, to efficiently detect in situ TLS-associated Tfh using NSCLC and hepatocellular carcinoma (HCC) as examples. The first two parts are dedicated to immunofluorescence. The last part described the characteristics of the primary antibodies used for flow cytometry. Abbreviations: UC unconjugated and NA not available

Materials

Methods

1. Dry the slides for a minimum of 30 min (or overnight) in a drying oven at 37 °C.

2. Deparaffinize the slides: Under a laboratory fume hood, immerse the slides in three successive baths of 100% xylene (or Clearene), for 5 min each, then in one bath of absolute ethanol for 5 min, one bath of 90% ethanol for 5 min, one bath of 70% ethanol for 5 min, one bath of 50% ethanol for 5 min, and one bath of distilled water for 5 min (use glass containers for slides) at room temperature (RT).

3. Retrieval of antigens: Immerse the slides in a bath of prewarmed antigen retrieval solution (TRS, pH 6) for 30 min at 97 °C (in a water bath; use a plastic container for slides).

4. Take out the plastic container from the water bath and let it cool for 30 min at RT, on the bench.

5. Wash the slides in 1 TBS for 5 min under gentle agitation at RT (use a glass container for slides) (see Note 1).

Visualization of TLS-Associated Tfh on FFPE Tumor Tissue by Triple-CD20/CD3/ PD-1 Immunofluorescence Staining

Follicular Helper T Cells Detection Within Tumors 38. Wash the slides twice in cold distilled water.

39. Mount the slides with glass coverslips using ProLong ® Gold mounting medium without DAPI.

40. Scan some fields in the slides using a confocal microscope (LSM710 Zeiss or equivalent).

41. View digital images using the Zen (black) imaging software (Fig. 2) (see Notes 12-14).

1. Place the tissue in a petri dish using forceps.

2. Dilacerate the tumor tissue in pieces of up to 2 mm using a scalpel in complete RPMI medium without FBS (about 1 mL for 500 mg of tissue).

3. Transfer the tissue pieces with the medium into a sterile jar.

4. Add collagenase IA and DNase I to reach a final concentration of 1.25 mg/mL and 100 μg/mL, respectively, in a final

Tfh Identification by Multiparametric Flow Cytometry from Fresh Tumor Tissue

Follicular Helper T Cells Detection Within Tumors 5. Proceed to enzymatic digestion by incubating tissue pieces for 45 min at 37 °C under magnetic agitation in a cell incubator with 5% CO 2 (see Note 17).

6. Transfer the digested material in a 50 mL Falcon tube and centrifuge for 8 min at 350 g.

7.

Remove the supernatant and resuspend the pellet in 10 mL of complete RPMI medium.

8. Filter the cell suspension through a 70 μm cell strainer and use a 10 mL syringe plunger to crush the remaining tumor fragments.

9. Rinse the cell strainer and complete with RPMI medium up to 50 mL.

10. Centrifuge for 8 min at 350 g at RT.

11. Resuspend the pellet in 1-10 mL of complete RPMI medium depending on pellet size.

12. Dilute 10 μL of cells in 90 μL of Türk's solution which destroys red blood cells to count them with a FastRead slide or equivalent.

13. Resuspend 6 × 10 6 of cells per tissue of interest in 5 mL of complete RPMI medium.

14. Incubate cells for a minimum of 1 h at 37 °C in a water bath to allow re-expression of CXCR5 on the cell surface (see Note 18).

15. Prepare the antibody mix and the corresponding isotype controls (see

Notes

1. After antigen retrieval, it is possible to let the slides in a bath of 1× TBS overnight at RT before continuing the experiment.

2. When drying the slides with absorbent papers, pay attention to the tissue. Also, treat the slides one by one: do not let the slides dry for more than 2 min before adding the following reagent.

3. To better control DAPI intensity, it is possible (after step 19) to incubate the slides with a DAPI solution (working concentration: 50 μg/mL in 1× TBS, Life Technologies), for 2 min (or less) at RT, and then to perform step 20 using a mounting medium without DAPI (ProLong ® Gold Antifade Reagent, Life Technologies).

4. Tfh cells correspond to PD-1 bright cells within CD20 + B-cell follicles of LN and TLS from NSCLC patients (Fig. 1b, f, yellow arrows). However, some PD-1 +/dim cells can also be observed outside of the GC (Fig. 1b, f, white arrows).

5. Depending on the tissue, a lot of autofluorescence (collagen fibers) can be observed, especially in the FITC, and to a lesser extent in the Cy3, channel (Fig. 1e-h). For this reason, it is

Coline Couillault et al.

advised to associate easily recognizable CD20 + B-cell follicles and CD3 + T cells with FITC and Cy3 channels, respectively, and to associate PD-1 with the AF647 channel.

6. Protocol can be performed on all Ventana machines (Ultra, Benchmark). However, optimizations may be necessary.

7. Multiplexing IF could be replaced by multiplexed immunohistochemistry with appropriate tyramide components. By IHC, the washing of the slides has to be completed with one bath of 50% ethanol followed by one bath of 70% ethanol, then one bath of 90% ethanol, one bath of absolute ethanol, and finally two baths of 100% xylene. This step is really important to remove residual paraffin which could interfere with staining analysis. Then, mount the slides with Cytoseal 60 toluenebased medium (Richard-Allan Scientific).

8. All antibodies work with Cell Conditioner #1 (CC1) in this case, but it is possible to perform another antigen retrieval step with CC2 (pH 6 citrate buffer).

9. Reconstitute prior to use tyramide coupled to Alexa Fluor reagent with 50 μL of DMSO (100× stock solution) according to the manufacturer's protocol.

10. The denaturation step is crucial to strip the preceding primary and secondary antibodies. Tyramides bind covalently to the tissue, so the denaturation step does not remove the signal.

11. Avoid using the ProLong ® mounting medium with DAPI to get a better nucleus staining.

12. It is important to perform single staining for each fluorochrome to record reference emission spectra (library) in order to carry out spectra deconvolution. During image analysis, it is important to find an area with all stainings to avoid poor deconvolution.

13. Control tissues with expected positive staining for all markers must be included for each experiment. Human tonsil or LN is recommended to detect GC zones and Tfh cells, as a positive control of the staining.

14. The combination of CD3 and CD4 markers is essential to detect Tfh cells, as CD4 + cells could also represent macrophages or dendritic cells.

15. Steps 1-3 in Subheading 3.3 have to be carried out in RPMI without FBS to avoid the inhibition of enzyme action.

16. Collagenase 1A can be used to digest the extracellular matrix of different tissue with addition of DNase I to eliminate DNA of dead cells during the digestion. Timing and concentration might have to be adjusted depending on the tissue. The protocol described here works for breast, ovarian, hepatic, and NSCLC tumors.

Follicular Helper T Cells Detection Within Tumors 17. Use of a water bath is preferable to maintain, over a short period, a constant temperature in the tube.

18. Tissue digestion with Collagenase IA leads to the shaving of CXCR5 which is one of the main markers allowing Tfh identification (Fig. 3). Re-expression of surface CXCR5 is optimally obtained after 2.5 h of incubation at 37 °C, but 1 h is enough to identify Tfh cells (see Fig. 4).

19. A negative control tube contains all the antibodies with the exception of antibodies directed against PD-1, CXCR5, ICOS, and Bcl6 that are replaced by matched isotype controls.

Fig. 3 Gating strategy for Tfh cell detection in hepatocellular carcinoma. After excluding debris and cell doublets, dead (Zombie Yellow + ) cells were gated out. Then, on the CD45 + immune cell population, Tfh cells were identified as CD3 + CD4 + PD1 hi CXCR5 + T cells (blue gate). Tfh cell phenotype was confirmed with Bcl6 and ICOS expression. PD1 lo cells were used as a negative internal control for Bcl6 and ICOS expression Fig. 4 Enzymatic digestion hampers surface CXCR5 detection by flow cytometry. Cells obtained by enzymatic digestion of tonsils were incubated at 37 °C in a water bath for 1 h and 2.5 h or were kept at 4 °C, and were then stained with the antibody mix (Table 1). Culture of cells prepared by enzymatic tissue digestion restores surface expression of CXR5 and allows identification of bona fide PD1 hi CXCR5 hi Tfh by flow cytometry at a frequency similar to that observed after mechanical dissociation alone (or in the absence of enzymatic digestion)

Coline Couillault et al.

Introduction

Tertiary lymphoid structures (TLS) are ectopic lymphoid structures whose architecture is closely similar to that of secondary lymphoid structures (SLO) (i.e., spleen and lymph nodes). TLS were first depicted as lymphoid aggregates called bronchus-associated lymphoid tissue (BALT) in rabbit [1,2] and then in humans [3]. They include a central B-cell zone that can be evidenced by labeling B-cell follicles with an anti-CD19 or anti-CD20 antibody and a marginal adjacent T-cell zone stained by an anti-CD3 antibody.

In the latter zone, one can detect high endothelial venules (HEV) that are specialized blood vessels that allow peripheral lymphocyte transportation and recruitment into TLS. TLS contain germinal center (GC) in the B-cell follicles and follicular dendritic cells (FDC) that display antigens as immune complexes [4]. TLS are not detected in normal lungs in the absence of inflammation. These structures are present in the lungs of patients with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension, idiopathic pulmonary fibrosis, asthma, or pulmonary complications of rheumatoid arthritis [5][6][7][8][9]. Also, they have been found in patients with non-small cell lung cancer (NSCLC) [10] and then in a number of other tumor types (among which melanoma, bladder cancer, colorectal cancer, and in some breast cancers) [11]. In cancer patients, their presence has been associated with an improved survival and correlates with a more favorable clinical outcome. TLS have also been detected in the lungs of patients with cystic fibrosis (CF) [12]. Cystic fibrosis is a genetic disease due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ion channel expressed in epithelial cells. The CFTR dysfunction leads to an increase in mucus viscosity, persistent inflammation, and chronic bacterial infection, which contribute to the development of bronchiectasis. The prognosis of the disease depends on the respiratory impairment that is related to chronic bacterial infections. Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are the two main bacteria that infect bronchi of adult patients with cystic fibrosis. These chronic infections are associated with a strong airway inflammation and severe decline in pulmonary function of CF patients. In France, about 39% of CF patients are chronically infected with PA and 61% with SA [13]. In the USA, the CF Foundation Patient Registry indicates a 46% prevalence of PA infection and a 26% prevalence of methicillin-resistant SA (MRSA) and more than half of the patients having at least one culture positive for methicillin-sensitive SA (MSSA) in 2016 [14]. In mice, it has been shown that repeated nasal instillations of inactivated PA induce TLS [15] as well as acute infection of lungs with bacteria such as Chlamydia pneumoniae [16]. These experimental settings have made it possible to identify chemokines involved in the recruitment of immune cells that are present in TLS such as CXCL12, CXCL13, and CCL19 [15]. The role of TLS is still largely unknown. Although they appear beneficial in a large number of tumors [11] with one exception [17], whether their presence can be detrimental in diseases such as cystic fibrosis remains an open question. The precise cellular components of TLS could be essential in determining their role. This has been suggested by works showing that TLS represent a microniche for tumor progenitor cells in a genetically engineered mouse model of hepatocellular carcinoma [17] and that the presence of regulatory T cells (Treg) in TLS inhibits antitumor responses in a mouse model of lung adenocarcinoma [18]. Moreover, the persistent inflammatory tissue environment that is associated with TLS in most cases could impair the adaptive responses against cancer cells or pathogens and provoke the destruction and remodeling of tissues, such as lungs.

Clearly, the development of animal models making it possible to study TLS induction and maintenance and their relationship with chronic inflammation, tumor control and escape, and acute and chronic infection is therefore needed. We describe below a technique to explore a murine model of chronic infection, where P. aeruginosa or S. aureus strains are used as inducers of TLS [19]. This model could give useful information about the role and mechanisms of action of TLS in patients with cystic fibrosis and could bring new insights into the role of chronic inflammation in the induction and maintenance of TLS in infected or tumor tissues. Notably, this model can be used to decipher the role of B cells present in these ectopic structures and to evaluate the clinical risks and biological events associated with anti-B-cell therapy given to CF patients with autoimmune diseases or before lung transplantation [20]. The technique relies on the use of agarose beads where live bacteria (PA or SA) are entrapped. After intratracheal instillation of the beads, animals develop a chronic pulmonary infection marked by the presence of TLS. TLS induced by a bronchial persistent infection in mice are shown in Fig. 1. These infected animals can then be manipulated using various immune intervention protocols to define the role of immune cell subsets in the formation, maintenance, and functions of TLS. A protocol for depleting TLS in Chronically Infected Lungs immune cells in these infected animals is also given here as an example of immune intervention.

Materials

Access to a safety-level microbiology L2 laboratory and to an animal facility with a dedicated room for PA and SA experiments on rodents is needed. 12. Disposable individually ventilated cages.

Materials and Supplies Necessary

TLS in Chronically Infected Lungs cal strain isolated from the sputum of an 8-year-infected CF patient from the adult CF center at the Cochin Hospital (Paris, France).

3. A Staphylococcus aureus (SA) clinical strain isolated from the sputum of a 10-year-infected CF patient at the adult CF center at the Cochin Hospital (Paris, France). This SA strain is methicillin sensitive (MSSA) (see Note 1) and makes it possible to closely mimic the infection observed in a large number of CF patients in Europe (where the prevalence of MSSA strains is higher than MRSA, i.e., methicillin-resistant SA).

4. Bacteria strains are kept frozen at -80 °C in 1.8 mL cryotube vials.

All procedures performed on mice should be approved with the local animal ethic committee and carried out in accordance to the rules enacted by the relevant regulatory agencies of the country where the experiment is performed. See the ILAR and European guidelines [21,22] for the care and use of laboratory animals.

1. House 6-week-old C57Bl/6J female mice in conventional conditions (see Note 2).

2. Monitor regularly mice for infection by typical pathogens according to the FELASA recommendations [23].

Animals

Methods

The murine model of chronic infection is based on the use of a single intratracheal nontraumatic instillation of agarose beads containing live PA or SA. This instillation induces a long-term bronchopulmonary infection (see Note 3) and does not require to be repeated. The infection kinetics in the instilled mice can be followed up by in vitro bacterial cultures of lung homogenates of sacrificed mice at different time points post-instillation. PA or SA infection can be detected up to 2 months after instillation of C57Bl/6 mice.

1. PA or SA colonies are isolated on Petri dishes [cultured on trypticase soy (TS) agar].

2. Bacteria are then cultured in broth TS medium for 72 h at 37 °C under constant agitation in a 50 mL culture tube.

Cultures are then centrifuged and the bacterial pellet is resuspended in PBS to obtain a bacterial suspension of 10 9 -10 10 CFU (colony-forming unit)/mL. The latter is immediately used for preparing PA-or SA-entrapped beads.

PA or SA is entrapped into agarose beads, as described by van Heeckeren and Schluchter [24] with some minor modifications.

1. Heat 150 mL heavy mineral oil in a water bath up to 50 °C.

2. In parallel, agarose (high EEO) is melted at a 100 °C water bath (0.3 g agarose in 15 mL PBS). Cool the solution down to 50 °C under sterile conditions.

3. Keep equilibrated both solutions at 50 °C under sterile conditions.

4. Mix 8.5 mL agarose with 1 mL bacteria in culture medium (10 9 -10 10 CFU/mL) and 0.5 mL undiluted Indian ink (Indian ink will make it possible to visualize beads when resected lungs will be examined).

5. The agarose-bacteria-Indian ink mix (10 mL) is then added to mineral oil (150 mL) and the resulting solution is stirred first for 6 min at room temperature (RT) and then cooled on ice with continuous stirring for 20 min using a stirrer (IKA RW 16 Basic Labo Technic) equipped with sterile plastic-covered blades (speed: 4) (see Note 4).

Lung Removal and Lung Tissue Homogenates

Preparation of Bacteria Stock Solutions

Agarose Bead Preparation

Bead washing and resuspension in PBS:

The mix is then centrifuged at 3000 × g at 4 °C for 20 min (this speed is required due to the viscosity of the heavy mineral oil in the mix). After removing supernatant, 0.5% sodium deoxycholate (1.5 g sodium deoxycholate in 300 mL PBS) is added and the bead pellet is gently resuspended. After centrifugation at 1200 × g for 20 min at 4 °C, the bead pellet is resuspended in 0.25% sodium deoxycholate (0.75 g sodium deoxycholate in 300 mL PBS) and centrifuged at 1200 × g for 20 min at 4 °C. Beads are then washed similarly twice in PBS.

7. To get a homogeneous size of beads (median size: 300 μm), the bead solutions are first filtered through a 400 μm stainless steel test sieve (gently add PBS with a 10 mL sterile plastic pipette to allow all the bead solution to be filtered). The flow-through is then filtered through a 300 μm stainless steel test sieve to remove beads <300 μm from the bead preparation (make sure that agarose beads are not drying by gently adding PBS with a 10 mL sterile plastic pipette) (see Note 5). Beads remaining in the upper part of the sieve with a size ranging between 300 and 400 μm are then gently collected with a 5 or 10 mL pipette and diluted in sterile PBS to a 5% final stock solution at 4 °C. They can be stored for up to 3 days before use. All steps require a gentle manipulation to avoid disruption of the agarose beads.

8. To estimate the number of bacteria entrapped within beads, aliquots of bead suspensions are homogenized and plated on TS agar for bacterial culture and CFU are estimated. 9. Control beads are prepared as described above without the addition of PA or SA. They should be found sterile when cultured on TS soy agar.

Intratracheal instillation bypasses the initial host immune response conducted by the respiratory epithelium of the upper airways.

1. 7-8-Week-old mice (see Note 6) are anaesthetized by intraperitoneal (i.p.) injection (20 G needle) of a ketamine/xylazine solution (0.1 mL per 10 g of weight) (light anesthesia).

2. Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain surgical plane.

3. Monitor the mouse vital signs during anesthesia (respiratory rate and effort, color of mucous membranes, and reflected eye color (in albino animals) at regular intervals (<15-min intervals).

4. Mice are then intubated with a 20 gauge (20 G) cannula attached to a 1 mL syringe.

Mouse Intratracheal Instillation

TLS in Chronically Infected Lungs 5. Introduce cannula as described by Das et al. [25]: place the anesthetized mouse on a vertical support, suspended by its upper incisors. The best visualization is with the ventral side of the mouse facing the investigator. Very gently pull out the tongue and hold with thumb and forefinger. The middle finger is placed between the neck and plastic support. Traction on the tongue with the index finger and thumb allows the opening of the mouth. The angle of the head is adjusted with the middle finger behind the neck to straighten the insertion of the cannula into the trachea.

6. Lay the mouse down. The cannula is secured using a piece of tape. Be careful not to move the cannula.

7. Connect the Luer end of the cannula to the 1 mL syringe. This method is given as an example of immune intervention aimed at deciphering the role of immune cells in the generation, maintenance, and functions of TLS. This part of the protocol can be adapted to use any other depleting antibody targeting a specific immune cell subset.

1. In the tail vein of C57Bl/6J mice, perform a single intravenous (i.v.) injection of 250 μg/mouse of an anti-mouse CD20 antibody or of an isotype control (in 200 μL sterile PBS) 1

B-Cell Depletion

week before the instillation of PA-or SA-entrapped beads. A heating lamp with a 250 W (infrared) bulb can be used to warm up mice until the tail vein is clearly dilated (at about 1 ft. distance). A tail veiner restrainer can be used to make the i.v. injection into the tail vein easier.

2. Collect blood (at day 2 after anti-CD20 injection) by submandibular puncture into capillary tubes containing heparin (as anticoagulant) with a 23 or 25 gauge needle slightly behind the mandible, but in front of the ear canal. This procedure requires gas anesthesia (with isoflurane in an induction chamber).

3. Apply gentle pressure to the blood collection site with a gauze sponge until bleeding has stopped.

4. Lyse red cells of the collected blood with ACK buffer according to the manufacturer's recommendations. Wash cells once in 1× PBS and adjust at 10 6 /mL before phenotyping.

5. Analyze B-cell presence in the cell preparation by flow cytometry using anti-mouse CD19-PE and anti-mouse CD45R (B220)-APC antibodies (double labeling). Compare with untreated mice using the same procedures.

This assay makes it possible to control the success of instillation of PA-or SA-entrapped beads. Only one mouse/experiment is evaluated as the technique of instillation is very robust and reproducible.

Bacterial Analysis of Lung Tissue Homogenates

Histological Analysis of Resected Lungs

TLS in Chronically Infected Lungs 24. Adhere a coverslip to the tissue section. An organic mounting media such as the limonene mounting medium can be used when DAB is used as substrate (a chromogen resistant to organic solvent). Add 2-3 drops of mounting medium. Apply coverslip carefully, avoiding air bubbles.

25. Negative controls are obtained by omission of primary antibodies and incubation with irrelevant immunoglobulins. The execution of every step is identical.

Quantification of TLS and calculation of the area of lung tissue sections can be performed using dedicated image algorithms that generate mathematical descriptions of expression and morphological patterns in tissues. The steps presented below correspond to a manual quantification that can be performed if no imaging software is available.

1. The antibodies used to detect the various immune cell subsets present in TLS (peribronchial and perivascular) are listed in Table 1.

2. Count manually TLS under photonic microscopy [26]. All peribronchial TLS detected in one lung section are counted. For a given mouse, count two sections separated by at least 300-400 μm.

Microphotograph lung sections (×10).

4. Overlay a counting grid with regularly disposed points on microphotographs.

5. Count every point intersecting lung parenchyma.

6. For each lung section, calculate the total lung surface according to the formula [Σcounted points × (2.36)] 2 /[Final magnification] 2 . The number of lymphoid aggregates is then expressed by square centimeter of lung. Lung surface is evaluated using the point counting method.

Detection and Quantification of TLS

TLS in Chronically Infected Lungs

Notes

1. SA methicillin resistance is due to the acquisition of the mecA gene that encodes a penicillin-binding protein. It leads to an increased resistance to antibiotics treatment, making treatments more difficult, although it is not considered as a virulence factor. Thus, the pathogenicity of the bacteria is not modified [27]. SA persistence in bronchial tree of the patients is due to the capacity of the bacteria to adapt themselves in front of the patient immune system, antibiotics pressure, and interspecies competition when several pathogens are colonizing the tree [28].

2. It has been reported that mice older than 4 months are more susceptible to an infection with Pseudomonas aeruginosa than younger mice that are more or less resistant [29]. Whether the sensitivity of mice to intratracheal instillation of PA-or SAentrapped beads is also dependent on their age has not been documented so far. Use C57Bl/6 mice for intratracheal instillation of PA-entrapped beads. It has been demonstrated that DBA/2 and C57Bl/6 are susceptible to P. aeruginosa infection while BALB/c mice are resistant [30].

3. Direct intratracheal instillation of live PA or SA bacteria (without trapping into agarose beads) does not induce chronic infection. It provokes an acute pneumonia that leads to a rapid animal death or to the cure of infection with a whole clearance of bacteria [24].

4. The size of the beads depends on the speed of the stirring and of the rate of cooling.

5.

One has to produce beads with a diameter larger than 300 μm. This threshold allows beads to stay within bronchi and bronchioles, and thus prevent them to reach alveoli.

6. Animals should be kept housed without any experimental manipulation for 1-2 weeks after purchase and delivery to the animal facility.

7. A rapid intraluminal bronchia infiltration of inflammatory cells (mostly neutrophils and, to a lesser extent, macrophages) can be detected 24 h after instillation of PA-or SA-entrapped beads and is still detectable at day 14. Immunochemistry analysis also shows the recruitment of subepithelial B lymphocytes around bronchia exhibiting PA-or SA-entrapped agarose beads as early as 24 h after instillation. During the following days, round B-cell aggregates are detected and become surrounded by T cells at days 4-7. Fully structured TLS are observed at day 14.

8. To confirm that persistent airway infection is present after instillation of PA-or SA-encapsulated beads, control animals can be sacrificed at day 7. Tenfold dilutions of left lung homogenates are then prepared and plated on trypticase soy agar. Bacterial counts should confirm that a persistent airway infection is present. No bacteria should be detected in lung homogenates of animals that are instilled with sterile beads (data not shown).

9. Homogenate from one lung is diluted in sterile 500 μL saline (0.9% NaCl solution).

10. The lungs will slightly inflate. It will transition from a red/pink color to a completely white aspect when the procedure is complete.

11. Inflation of the lungs with PFA is critical to some histological analysis. The volume of PFA required to properly inflate the lung tissue is usually ≤1.5 mL. To prevent overinflation of the lungs, the infusion step can be completed with the lungs in situ, with PFA infusion stopping when the lung volume completely fills the thoracic cavity.

12. Instillated agarose beads appear as tiny black spots that can be observed macroscopically at the surface of the resected lungs. It shows that the instillation has been successfully performed. If no spot is observed, the instillation experiment should be performed again (see Subheading 3.3, step 1).

13. Keep the slides in the tap water until ready to perform antigen unmasking (retrieval antigen). The slides should not be allowed to dry. Drying out will cause nonspecific antibody binding, leading to high background staining.

14. Antigen unmasking is required to improve the quality of staining when polyclonal rabbit antibodies to the endothelial marker von Willebrand factor (vWF) (Dako) are used to identify peribronchial blood vessels.

15. Most of these antibodies are supplied in lyophilized form and can be reconstituted with 1 mL sterile distilled H 2 O 2 . With some exceptions the recommended dilution for most applications is 1:200.

Introduction

It is well established that the immune system plays an important role in cancer immunosurveillance and tumor eradication. A high density of CD8 + T lymphocytes infiltrating solid tumors is commonly associated with favorable clinical outcomes and patient survival (reviewed in [1][2][3]). Cytokine infusion, checkpoint blockade inhibition, vaccination, and adoptive transfer strategies have all been used to increase the representation of intratumoral CD8 + T lymphocytes and bolster antitumor immunity [4][5][6][7]. However, only a small fraction of patients respond favorably to these immunotherapies. Gene expression profiling of patients who respond favorably to these immunotherapies has been shown to have a preexisting immune infiltrate prior to treatment [8][9][10]. This suggests that the limited representation of intratumoral CD8 + T lymphocytes is a fundamental barrier to the success of current cancer immunotherapies. One potential strategy to increase the representation of CD8 + T lymphocytes in solid tumors is by increasing their exogenous infiltration through vaccination or alterations in tumorassociated vasculature. An alternative potential strategy is to enhance the intratumoral immune response by promoting the development of tertiary lymphoid structures (TLS). TLS were initially identified as ectopic accumulations of immune cells that develop in and/or near chronically inflamed non-lymphoid tissues in association with microbial infections, graft rejection, and autoimmune disorders [reviewed in [11]]. Some of these TLS have considerable morphological similarities to conventional secondary lymphoid organs. For example, TLS often exhibit organized T-and B-cell compartments, a stromal infrastructure that produces homing chemokines and survival factors, lymphatic vessels, and high endothelial-like vessels that express peripheral node addressin (PNAd) [12][13][14]. Alternatively, TLS-like structures found in adipose tissue display all of the above characteristics expect for discernable T-and B-cell compartmentalization [15,16]. TLS have been documented in association with a wide variety of primary and metastatic solid tumors in humans. Their presence is almost always a favorable prognostic indicator for patient survival [17,18]. Higher densities of TLS correlate with higher representation of tumor-infiltrating CD8 + T lymphocytes [19,20]. Additionally, the presence of TLS associates with infiltrates that display an activated and cytotoxic immune signature [21]. These observations suggest that tumor-associated TLS serve as productive sites for in situ activation of CD8 + T lymphocytes, which in turn control tumor growth.

The availability of animal models that could enable a more comprehensive understanding of the functionality of TLS in solid cancers has been limited. Recently, we demonstrated that naïve CD8 + T cells can directly infiltrate tumors formed from two transplantable murine cell lines: B16-F1 melanoma and Lewis lung carcinoma. Once in the tumor, these naïve cells become activated, proliferate, and differentiate into immune effectors [22]. This infiltration is dependent on the development of tumor-associated blood vessels that express PNAd and CCL21, the ligands that engage CD62L and CCR7 on naïve and central memory T cells, respectively, and enable them to enter lymph nodes (LN) [23]. Interestingly, tumors growing in the peritoneal cavity, but not subcutaneously, develop intratumoral TLS in association with this PNAd + /CCL21 + LN-like tumor vasculature. In this chapter, we describe methods for the development, identification, and characterization of intratumoral TLS in intraperitoneal B16 melanoma tumors. Additionally, we describe an adoptive transfer method to demonstrate the function of the PNAd + /CCL21 + LN-like tumor vasculature in promoting naïve T-cell infiltration.

Add 5 mL cold complete medium and transfer cell suspension

to a 15 mL conical tube. Rinse flask with 5 mL cold complete medium and add to the 15 mL conical tube. Centrifuge for 5 min at 600 × g at 4 °C.

4. Aspirate supernatant, resuspend in 5 mL cold complete medium, and centrifuge for 5 min at 600 × g at 4 °C.

5. Aspirate supernatant, resuspend in 5 mL cold PBS, and centrifuge for 5 min at 600 × g at 4 °C. Repeat once more. 12. Pipette 200 μL 0.5 μg/mL of DyLight550-conjugated streptavidin in staining buffer uniformly across sections. Incubate for 1 h at room temperature. Wash as in step 5.

13. Pipette cell surface marker antibody cocktail in staining buffer (Table 1) (see Note 7) uniformly across sections. Incubate for 1 h at room temperature. Wash as in step 5.

14. Apply ProLong Gold DAPI Antifade Mountant and apply coverslip.

Acquisition and analysis of immunofluorescent images can be performed with any fluorescent microscope and image software package, respectively. We use a Zeiss AxioImager with Apotome and an AxioCam MRm camera, and Zeiss Zen image analysis software. We define intratumoral TLS to have the following features:

1. Aggregates of 50 or more B220 + B lymphocytes in juxtaposition to PNAd + CD31 + vasculature: Note that such aggregates are present in intraperitoneal tumors (Fig. 1e-h), but lacking in subcutaneous tumors (Fig. 1a-d), despite the presence of We utilize Beckman Coulter CytoFlex S units for acquisition, and imported FCS files into FlowJo software (TreeStar) for analysis. While flow cytometry does not enable TLS-associated cells to be directly distinguished from non-TLS-associated cells in tumors, we have found significant quantitative differences between IP and SC tumors in certain populations that are TLS-associated. For comparably sized tumors, the number of CD31 + endothelial cells is actually lower in IP tumors (Fig. 3a). However, the number of podoplanin + fibroblasts is substantially higher (Fig. 3b). While the podoplanin/CD31 ratio in SC tumors is slightly less than 1, the ratio in IP tumors is about 3.5, consistent with the presence of the reticular network that is evident by immunofluorescence (Fig. 3c). This provides a method to further delineate the characteristics of both populations in these different tumor microenvironments. Similarly, the average number of B lymphocytes in IP tumors is about tenfold greater than in SC tumors (Fig. 3d), and these are largely concentrated in TLS. The number of T cells in both tumors is comparable, about 10 6 cells. 12. Eighteen hours after adoptive transfer (see Note 12), tumorinfiltrating CD8 + T lymphocytes can be isolated and quantitated as described in Subheading 3.5 using the antibody cocktail described in Table 3, with the addition of anti-Thy1.1. Typical results are shown in Fig. 4. 1. We use a B16-F1 melanoma cell line (ATCC) transfected to stably express chicken ovalbumin (B16-OVA). These cells present the OVA 257 epitope on H2-K b [24]. This was determined by flow cytometry after incubating cultured B16-OVA cells overnight at 37 °C with 100 U/mL of murine recombinant IFNγ, staining with anti-mouse SIINFEKL-H-2K b (BioLegend), and evaluating by flow cytometry.

Identification and Characterization of TLS in B16

2. We utilize congenic Thy1.1 mice to distinguish exogenous versus endogenous CD8 + T lymphocytes. An alternative congenic marker, such as CD45.1, can be substituted.

3. We use tribromoethanol to minimize movement during subcutaneous tumor implantation. This maneuver diminishes spreading of cells after injection, and results in more regularly formed tumors.

4. These conditions lead to subcutaneous tumors that are 2 mm in each of the two measurable dimensions. Intraperitoneal tumors grow somewhat faster and may be harvested earlier for that reason. Since intraperitoneal tumors cannot be measured directly, ensure that mice do not gain more than 5-10% in bodyweight and do not show any signs of discomfort.

5. Overnight fixation provides excellent preservation of tumor morphology. However, some cell surface markers become undetectable (i.e., CD31). We've determined that 1-h fixation still provides excellent tumor morphology and detection of all markers described.

6. PNAd is expressed at relatively low levels on tumor vasculature when compared to LN-HEV. Tyramide signal amplification enables successful visualization.

7. We use B220, CD31, and PNAd to identify TLS, CD3 and CD11c to identify T cells and antigen-presenting cells, respectively, and podoplanin to identify fibroblasts. Specific fluorochromes for each are shown in Table 1, and representative staining combinations are shown in Figs. 1 and2.

8. The steps in Subheadings 3.5 and 3.6 should be carried out as quickly as possible. Isolated endothelial cells and fibroblasts from lymphoid organs and tumors are fragile and significantly lose viability over a few hours. Awesome MACS buffer provides with nutrients to maintain their survival for the duration of the experiment. Enrichment steps and staining of isolated stromal cells should always be carried out in this buffer. 12. Our lab and others have reported that 18 h after adoptive transfer of T lymphocytes is a sufficient amount of time to visualize their infiltration into tumors [23,[25][26][27][28]. We have also observed substantially similar levels of infiltration in 1 h [23].

Introduction

Tumor-associated tertiary lymphoid structures (TA-TLS) are lymph node-like aggregates that develop at sites of chronic inflammation, and have been identified in many types of human cancers. The increased presence of TLS has been correlated with improved patient outcomes in various cancer types including non-small cell lung cancer (NSCLC) [1], colorectal [2], pancreatic [3], and ovarian cancer [4]. TA-TLS share many features with secondary lymphoid tissues and are thought to play an important role in regulating the antitumor immune response [5]. Therefore, TA-TLS represent an exciting potential therapeutic target in cancer therapy. Regardless, the mechanisms by which TA-TLS impact the local and systemic immune response have not been fully elucidated.

Even less is known regarding when and how TA-TLS are generated and maintained within tumor microenvironments. In large part, this is due to the dearth of appropriate preclinical models in which to study the development of TA-TLS in the context of a fully intact immune system. Immunocompetent mouse models of cancer can be divided into two main types: injected syngeneic mouse models and genetically engineered mouse models (GEMMs). Both syngeneic mouse models and GEMMs of cancer possess caveats which provide insight regarding the absence of TA-TLS. In syngeneic models, tumor tissue or tumor cell lines are implanted into mice with the same genetic background. In these cases, tumors develop within weeks and progress rapidly, often so rapidly that the mice succumb to disease before the development of a chronic inflammatory environment present in human disease [6]. It is likely that this rapid progression from initiation of tumors to humane endpoint does not allow for the full development of TA-TLS. GEMMs, on the other hand, develop tumors over several months, and more closely mirror the histopathological features of cancer progression in humans [7]. Nevertheless, GEMMs have been shown to have a very low mutational burden compared to human cancers. In particular, oncogene-driven GEMMs of lung adenocarcinoma have been shown to harbor ~50fold fewer somatic mutations compared to their human counterparts [8], and therefore are thought to be less "immunogenic," easily evading immune recognition [9]. To overcome these restrictions, we have designed a novel GEMM of lung adenocarcinoma in which the expression of neoantigens by tumor cells can be programmed using lentiviruses [10]. Intriguingly, the induction of neoantigen expression in tumors results in the formation of TA-TLS, highlighting the role of tumor antigens in this process.

To date, several different strategies have been used to identify and quantify the presence of TLS in human tumors. These studies assess the density of TA-TLS by identifying the presence of various immune cell populations, with the use of immunohistochemistry and/or fluorescence-activated cell sorting (FACS), or by gene expression assays to identify TLS-related gene signatures. In order to enumerate and characterize the TA-TLS in our murine model, we use a combination of immunofluorescence staining of frozen sections (IF-F) and FACS. IF-F of sequential tumor sections allows us to assess the presence and relative location of TLS in relation to the tumors themselves while concurrently determining the size of immune cell compartments within these structures. Additionally, we are able to determine the activation status of certain cell populations within TLS, such as tumor-specific CD8 + T cells, by FACS of the single-cell suspensions from these lung tumors. Using these complementary techniques, we previously demonstrated that regulatory T cells (T regs ) within TA-TLS actively suppress antitumor immune responses. Furthermore, immune-mediated tumor destruction is enhanced following the depletion of T regs [10]. Combining these techniques to investigate TA-TLS in our clinically relevant mouse model of lung adenocarcinoma permits an in-depth interrogation of the formation, maintenance, and functions of TA-TLS.

Fluorescence-Activated Cell Sorting (FACS)

Methods

For these studies, Kras Lox-STOP-Lox (LSL)-G12D p53 flox/flox (KP mice) mice were used (see Note 8). As an additional tool to track tumor cell fate, KP mice were crossed with Rosa26 LSL-tdTomato(tdT) mice to generate "KPT" mice [11] (see Note 8). In KPT mice, Creinducible tdT expression [12] is useful for identifying tumor cells by immunofluorescence as transformed cells fluoresce red (see Fig. 1). Tumors were initiated by intratracheal administration of 2.5 × 10 4 to 5 × 10 4 PFU of non-replicating lentiviruses (LVs) which co-express LucOS (firefly luciferase fused to a portion of ovalbumin and the antigenic 2C peptide) and Cre-recombinase [10]. Lungs were harvested 8-20 weeks after infection. All mice were handled according to IACUC-approved protocols and were humanely sacrificed prior to natural expiration. If working with infected tissues, all work should be performed in a tissue culture hood. Appropriate personal protective equipment (PPE) should be worn for handing BL2 materials. Use safety caps for any centrifugation steps. Allow pipette tips and waste to soak for 30 min in 10% bleach solution inside hood, and then discard. Wipe hood surfaces with 10% bleach to disinfect, followed by 70% ethanol. Lungs should inflate and turn white while perfusion occurs. Allocate lung lobes for staining.

2. Preserve tissue by overnight fixation at 4 °C with PLP.

3. Perfuse and cryoprotect PLP-fixed tissues by injecting 30% sucrose solution into lung lobes and incubating lobes in 30% sucrose solution for 4-6 h at 4 °C (see Note 9).

4. Inject tissue with 30% OCT compound solution.

5. Place tissue in cryomolds and cover with 100% OCT compound and freeze by placing on dry ice.

6. Once frozen (OCT will be white and hardened), store at -80 °C.

All steps, unless otherwise noted, are done at room temperature (RT).

1. Cut sections (ranging 20-70 μm thick) at -20 °C using cryostat (see Note 10). 3. While lung tissues are incubating, lymph nodes and 5 mL RPMI-1% should be transferred from 15 mL tubes into petri

Isolation and Preparation of Tumor-Bearing Lungs for IF-F

Immunoflu orescence Staining

Tissue Isolation and Preparation for FACS

Tissue Dissociation and Staining for FACS

the identification of T cells and B cells, respectively. In place of one of the previously mentioned primary antibodies, we occasionally stain with a primary antibody against NK2 homeobox 1 (NKX2.1), also known as thyroid transcription factor 1 (TTF-1), in order to stain for tumor cells. TTF-1 is expressed strongly by lung adenocarcinoma cells [13] and is therefore a good marker to distinguish tumor cells from normal in IF-F sections. For a more thorough analysis of TLS, you may consider adding antibodies against the following proteins to your sections: CXCL13 (B cell chemokine), CR1 (follicular dendritic cell marker), CCL21 (T-cell chemokine), ER-TR7 (follicular reticular cell marker), CD31 (endothelial cell marker), and/or PNAd (HEV marker).

4. Secondary antibodies should be carefully chosen based on both the fluorescence spectra of chosen fluorophores and the configuration of your microscope. We commonly use goat anti-rabbit Alexa Fluor-568 (1:500) and anti-FITC Alexa Fluor-488 (1:500) in combination with the primary antibodies mentioned above (see Note 3).

5.

To 500 mL of PBS, add 2.5 mL of thawed, heat-inactivated FBS and 2.5 mL of 20% sodium azide solution. Store at 4 °C.

6. From 16% PFA ampule, add 125 μL 16% PFA per 1 mL FACS buffer. Make fresh each use.

7.

See Table 1 for detailed information regarding the antibodies used. Conjugated fluorophores should be carefully chosen based on both the fluorescence spectra of chosen fluorophores and the configuration of your flow cytometer. For our work, stained cells were run using 9-11-color FACS on an LSR II (BD) with 405, 488, 561, and 635 lasers.

8. "KP" mice were produced by crossing two commercially available mouse strains: Kras LSL-G12D (Jackson Laboratory; Stock No: 008179) and p53 LoxP (Jackson Laboratory; Stock No: 008462). To obtain "KPT" mice, "KP" mice were crossed with commercially available Ai14 Cre reporter mice (Jackson Laboratory; Stock No: 007914). 9. Staining for the presence of B and T cells in mouse spleens serves as a good positive staining control. Having a positive control is essential for correct interpretation of microscopy images.

10. Tumors span several hundred microns. Therefore, TLS within tumors can easily be underrepresented or missed altogether if thin tumor sections (5 μm) are analyzed. For our studies, we have found that cutting 30-μm-thick sections is ideal for identification of TLS [10].

Tumor-Associated Tertiary Lymphoid Structures for a 1:40 dilution. From this, pipette 10 μL onto hemocytometer with coverslip and, using a compound microscope, bring the etched grid located in the center of the hemocytometer into focus. Count the number of trypan blue-negative, viable cells within the four outer squares of the grid, and divide by four to obtain the average number of cells per square.

Multiply by 40 (trypan blue dilution factor) and divide this by 0.0001 mL (volume of square on hemocytometer) to calculate the number of live cells per mL. In order to calculate the total number of cells, multiply the number of cells/mL by the volume (mL) of the cell suspension you counted.

21. As an alternative to polystyrene tubes, cells can be stained in 96-well U-bottom plates.

22. FBS must be washed out of cells as it will interfere with Live/ Dead stain. Ensure that wash is performed with PBS only.

23. If staining with tetramers to identify antigen-specific T cells, tetramer staining can occur at the same time as staining with directly conjugated antibodies (see Subheading 3.5, step 20), depending on available fluorophores. If you wish to stain cells with tetramers, the tetramers should be thawed on ice and diluted in FACS buffer. Resuspend cells in 50 μL for lymph nodes, or 150 μL for lung tissue/tumors, and incubate for 15 min at 4 °C in the dark. Always fix tetramer-stained cells, even when planning to run samples on cytometer immediately. For a more thorough analysis of TLS, you may consider adding antibodies against the following proteins to your panel in order to assess the presence and activation status of other cell types within TLS: CD11c, CD11b, MHC class II, Thy1.2, CD103, KLRG1, GITR, CD62L, CD39, CTLA-4, and CD80.

24. Optimal dilutions of antibodies and tetramers should be determined prior to experimental FACS. This is most commonly achieved by making a series of dilutions of each individual antibody/tetramer and performing titration experiments.

25. Some fluorophores, like PE-Cy5, are sensitive to fixation in PFA. Therefore, when PE-Cy5 is included in your panel we suggest running samples fresh on the same day as staining. For the fluorophores included in this chapter, we did not see a measurable effect of fixation on fluorophore brightness. This is due to a relatively short fixation period (10 min).

26. Ideally, run samples on cytometer within 1 week of fixing for optimal detection of tetramers and conjugated stains. Samples that are not fixed should be run as soon as possible on the same day as staining.

Introduction

Conventional cancer therapies target the tumor tissues and can increase survival in some cancer patients. However, despite initial responses, many tumors relapse and develop resistance after prolonged treatment [1]. In contrast, immunotherapy targets the immune system to provoke a systemic response against tumors. Recent clinical trials with cancer immunotherapy have shown unprecedented durable responses in patients with a variety of advanced cancers, representing one of the major breakthroughs in oncology [2,3].

In an antitumor immune response, antigen-presenting cells (APCs) uptake tumor antigens from tumor tissues, migrate to draining lymph nodes (LNs), and present processed antigens to T cells, leading to T-cell activation [4]. In many cancers, the presence of tumor-infiltrating lymphocytes (TILs) correlates with better prognosis, indicating the important roles of lymphocyte infiltration and immune responses [5]. During the persistent inflammation seen in tumors, the migration and positioning of immune cells form patterns similar to secondary lymphoid organs (SLOs). Such structures are known as tertiary lymphoid structures (TLS). TLS can be induced both inside and adjacent to tumors. Compared to draining lymph nodes, TLS may be a better place for T-cell priming (Fig. 1) [6]. As TLS are close to tumor tissues, it takes less effort for APCs to migrate to TLS and to present antigenic peptides to T cells than to emigrate to the draining LNs, knowing that each step is not exclusive of the other. After activation, effector T cells can go to the tumor tissues with fewer barriers. In addition, tumor antigen load may be higher in TLS than in draining LNs [7]. The microenvironment in TLS can induce better T-cell priming and prevent exhaustion or anergy. Since TLS can support effective antigen presentation and lymphocyte activation, it has become an intriguing target for cancer immunotherapy [8]. Strategies aiming at inducing TLS have shown antitumor effects in several different mouse tumor models. In one study, systemic treatment with an agonist antibody against lymphotoxin beta receptor (LTβR) increases lymphocyte infiltration and antitumor immune responses in both mouse and human xenograft models [9]. In another study, a lymphotoxin alpha (LTα) fusion protein induces the formation of high endothelial venule (HEV)-like structures in a mouse model of melanoma [10]. Further, LTα therapy inhibits the growth of primary tumors as well as lung metastasis [10]. Our group and others have shown that recombinant proteins targeting the TNF superfamily member LIGHT can induce the formation of TLS, normalize blood vessels, support lymphocyte trafficking to tumor tissues, and lead to better antitumor responses [7,[11][12][13]. Clinically, the presence of TLS usually correlates with active antitumor immunity and better prognosis [14][15][16]. In colorectal carcinoma patients, TLS are associated with a higher number of CD3 + T-cell infiltration and improved survival [17][18][19]. A 12-chemokine gene expression signature, most of which are critical for the formation of SLOs and TLS, correlates with better survival in melanoma and colorectal carcinoma patients [17,20].

One major characteristic of TLS is the unique organization of immune cells, primarily dendritic cells (DCs), B cells, and T cells [21]. The organization of TLS is highly dependent on the expression of chemokines such as CCL19, CCL21, and CXCL13 [8,16]. In this chapter, we describe the general approaches to established mouse tumor models and their applications in evaluating immunotherapies targeting TLS.

Notes

1. Different models vary in their tumor microenvironments, and thus may have different responses to therapy. Both B16F10 and Ag104Ld cell lines characteristically have a low level of spontaneous T-cell infiltration. They are more sensitive to therapies that are able to manipulate lymphocyte infiltration.

2. Different therapeutics have been developed to induce the formation of TLS and have shown an antitumor activity. These include, but are not limited to, antibodies [23,24], recombinant cytokines/chemokines [11,13,25,26], and antibodycytokine fusion proteins [10,27,12].

3. It is important to use cells in good condition for inoculation (viability >90%). Using cells in the log phase of proliferation curve will increase tumor take rate in mice.

4.

For our experiments, we usually use 5 × 10 5 for both B16F10 and Ag104Ld cell lines. However, the tumor growth curve may vary depending on various factors, such as condition of the cells, different tumor models, animal facility, and genetic background of the mice. Thus, it is recommend to titrate tumor cell numbers before starting any experiment.

5. While it is not necessary to anesthetize mice during tumor inoculation, mice may struggle and thus doing so can improve the reproducibility of the tumor growth curve in vivo.

6. If using syringes with permanently attached needles, cells should be drawn and injected slowly. Do not aspirate cells with the needle as this may damage the cells, leading to low tumor take rate.

7. While restraining mouse with the nondominant hand, hold one rear foot with ring and baby fingers. Mouse often tries to kick back during injection. This will help to keep the injection site steady especially during i.p. injection.

8. The time to start therapy may vary depending on the purposes.

It usually takes about 7 days for the adaptive immune responses to take place. So, it is recommended to start treatment at least 7 days after the tumor inoculation when studying T-cell response.

9. Drugs may leak out quickly from the tumor after intra-tumoral injection. To increase retention, drugs can be mixed with Matrigel (Corning) before its injection.

10. Digestion time varies depending on tumor types. Soft tumors such as B16F10 take a short time to digest. In contrast, rigid tumors such as Ag104Ld take longer. However, do not overdigest as it may result in loss of cell surface markers or even cell death.

Fig. 1

 1 Fig. 1 CNS-lymphatic system drainage. Schematic illustration of the CNS-draining route via lymphatic vasculature for antigens and immune cells. Macromolecules and antigen-presenting cells are proposed to exit via (1) the lymphatics of the nasal region crossing the cribriform plate or (2) the meningeal lymphatic network. Abbreviations: APC Antigen-presenting cell, CSF Cerebrospinal fluid, RMS Rostral migratory stream

Fig. 2

 2 Fig. 2 Peripheral versus CNS tertiary lymphoid structure. Schematic illustration of the different organizations of peripheral and CNS tertiary lymphoid structures

Fig. 1

 1 Fig. 1 TLS visualization and quantification through three different double IHC. (a, b) IHC staining of a FFPE section from NSCLC tumor with anti-human CD20 (in red) and anti-human CD21 (in blue). (a) Visualization of four TLS-associated B-cell follicles, subsequently identified by Calopix software (b) during automatic quantification. (c-e) IHC staining of a FFPE section from NSCLC tumor with anti-human DC-LAMP (in red) and antihuman CD3 (in blue). (c and e) Visualization of DC-LAMP + mDCs forming clusters with CD3 + T cells within a TLS. (d) DC-LAMP + mDCs are manually identified (small numbers "1") and counted by two independent observers using Calopix software. (e) Illustration of the difficulty of performing an automatic counting of DC-LAMP + cells with Calopix software in NSCLC tumors, as type II pneumocytes are also visualized with DC-LAMP staining. (f) IHC staining of a FFPE section from NSCLC tumor with anti-human CD20 (in red) and anti-human PD-1 (in blue), for T FH cell visualization within two TLS. (a-f) Scale bar = 100 μm. Abbreviations: IHC Immunohistochemistry, FFPE Formalin-fixed paraffin-embedded, mDC Mature dendritic cell, NSCLC Non-small cell lung cancer, TLS Tertiary lymphoid structure

  TLS-associated B-cell follicles % ( )

Fig. 2 (Fig. 2

 22 Fig. 2 (continued) DC-LAMP/anti-human CD3 IHC, and (c) double anti-human CD20/anti-human PD-1 IHC. Regions of whole-slide image (WSI) were extracted at 20× resolution and registered, as described in Subheading 4.3.4. (d-f) An area containing a TLS was then cropped from these registered images. (g-l) Stainings were separated using the color deconvolution function of ImageJ, contrast inverted (g-k), and then overlaid (l). (a-c) Scale bar = 2.5 mm. (d-l) Scale bar = 250 μm. Abbreviation: IHC Immunohistochemistry

Fig. 1

 1 Fig. 1 Multispectral image analysis workflow. Schematic representation of the main four steps of multispectral image analysis using the Inform ™ software. Representative images of lung cancer tissues undergoing each processing step are shown

Fig. 2

 2 Fig.2MSI of seven-color immunostained human tissues for the analysis of TLS. Tissue sections were immunostained to detect B cells (CD20), T cells (CD3), mature dendritic cells (DC-LAMP), high endothelial venules (PNAd), follicular dendritic cells (CD21), and germinal centers (CD23) in combination with the nuclear stain DAPI using the Opal ™ 7-color Manual IHC Kit (PerkinElmer). Multispectral images were acquired with the automated microscopy system Vectra 3.0 ™ (PerkinElmer). Autofluorescence profile was determined in unstained human lung tumor tissue and each individual marker signal was unmixed using Inform ™ software (PerkinElmer). (a) Human tonsil tissue was used as a positive control, and each individual marker staining is displayed as a pseudo-bright-field image and the composite image depicting the tissue expression of all analyzed markers. (b) A representative image of a GC-positive TLS in human lung tissue from a lung cancer patient with autofluorescent erythrocytes (left); image overlaid with tissue segmentation map (middle) and cell phenotype map (right)

Fig. 1

 1 Fig. 1 Duplex (a) and triplex (b) IHC staining of serial sections of a non-small cell lung carcinoma with TLS. (a) PD1 (brown) is combined with PD-L1 (purple) and hematoxylin counterstaining. (b) CD3 (purple) is combined with CD68 (brown), pan-cytokeratins (yellow), and hematoxylin counterstaining. PD1 + lymphocytes are intensely labeled in the germinal center (GC) of the TLS and are also present in the parafollicular zone of the TLS and in other regions of the tumor microenvironment. PD-L1 + cells are co-localized with PD1 + cells in the GC, but are more prominent in the tumor region (Tu), to include neoplastic cells and some infiltrating macrophages. Both CD3 + lymphocytes and CD68 + macrophages are present in the GC, parafollicular zone, and elsewhere in the tumor microenvironment, including a number of macrophages infiltrating tumor islands. A raft of intensely PD-L1 + cells (arrowhead) is present in the TLS; in the serial section, these cells are also cytokeratin positive, demonstrating their epithelial origin. Original magnification: 200×

Fig. 2 ReferencesFig. 3 A

 23 Fig. 2 Manual annotations of different tumor regions for a larger portion of the NSCLC shown in Fig. 1. Shown are several TLS (blue) in the tumor region beyond the invasive margin (green) and tumor center (red). Original magnification: 40×

Fig. 1

 1 Fig. 1 HEV can be detected in mouse fibrosarcomas after regulatory T-cell depletion in FoxP3 DTR mice using diphtheria toxin. Frozen tumor sections were stained with anti-PNAd (MECA-79, red) + anti-pan-EC (MECA-32, green) antibodies (A and B) and anti-PNAd (MECA-79, red) + anti-MAdCAM-1 (MECA-367, green) antibodies (C and D). Cell nuclei were counterstained with DAPI (blue). Low-power images are shown in panels A and C, and high-power images of boxed regions are shown in panels B and D. Scale bars are shown in white

Fig. 2 L

 2 Fig.2L-selectin-dependent homing of T cells in mice. L-selectin-sufficient (CD62L +/+ ) and L-selectin-deficient (CD62L -/-) donor T cells were labeled with either CFSE or CMTMR and mixed 1:1 (pre-transfer). 1-3 h following intravenous administration to recipient mice, organs were analyzed for CD62L +/+ and CD62L -/-T cells and T cell homing identified as either CD62L dependent or CD62L independent. Bar chart shows relative percentages of CD62L +/+ and CD62L -/-T cells pre-transfer and recruited into inguinal (Ing), axillary (Ax), and mediastinal (Med) LN, spleen, and peripheral blood (PB). T-cell homing to peripheral LN such as inguinal and axillary is L-selectin dependent. In contrast, T-cell homing to the mucosal associated mediastinal LN is not exclusively dependent on L-selectin. HEVs are not found in the spleen so not dependent on L-selectin for T-cell entry. Peripheral blood is analyzed to determine whether changes in the ratio of T-cell populations inside tissues result from a change in the ratio in the bloodstream

Fig. 3

 3 Fig. 3 Localization of homed T lymphocytes in HEV-containing tissues. (A) CFSE-labeled naïve T lymphocytes were injected into naïve mice and after 60 min peripheral lymph nodes analyzed for infiltrating T cells (green) and HEVs stained using MECA-79 (red). Representative image showing CFSE-labeled (green) T lymphocytes in the process of transmigration and inside HEV (arrows) and CFSE-labeled T lymphocytes which have completed transmigration and outside HEV (arrow heads). Bar, 50 μm. (B) Naïve T lymphocytes were split into two and labeled with either CFSE or CMTMR, mixed 1:1, and injected into naïve mice. After 60 min, peripheral lymph nodes were stained for HEV using MECA-79 (blue) and the ratios of CFSE (green) to CMTMR (orange) T cells inside HEV (white arrows) and outside HEV (arrowheads) determined

14 .

 14 Fluorescently conjugated secondary reagents diluted in 1% BSA in PBS: To detect MECA-79, a fluorescently conjugated anti-rat IgM may be used. Other primary antibodies can be detected with fluorescently conjugated species-specific secondary antibodies or fluorescently conjugated streptavidin reagents. The fluorochromes chosen should be compatible with the specific capabilities of the microscope being used to analyze the tissues, that is, the laser systems and detectors. The Alexa Fluor dyes are excellent conjugates since they are very bright and resistant to photobleaching; Alexa Fluor 488, 594, and 647 emission spectra are sufficiently separated to provide minimal overlap between fluorochromes and are compatible with most standard wide-field and confocal fluorescent microscopes. 15. Optional: Nuclear counterstain. Example Hoechst or DAPI (Sigma) diluted in PBS. 16. Mounting media: Example: Vectashield (Vector) or Prolong gold (Thermo Fisher Scientific).

2 .

 2 Process in tissue processor (Leica) as follows: (a) 70% Ethanol-1 h at 45 °C (b) 80% Ethanol-1 h at 45 °C (c) 95% Ethanol-1 h at 45 °C (d) 100% Ethanol-1 h at 45 °C (e) 100% Ethanol-1 h at 45 °C (f) 100% Ethanol-1 h at 45 °C (g) 100% Xylene-1.5 h at 45 °C (h) 100% Xylene-2 h at 45 °C (i) 100% Xylene-2 h at 45 °C (j) Wax-1.5 h at 65 °C (k) Wax-2 h at 65 °C (l) Wax-2.5 h at 65 °C (m) Embed in wax and cool 3. Store tissue blocks in a cool dry place until ready to cut.

(a) Cycle 1 : 7 .

 17 10 min at 95 °C (b) Cycle 2: 15 s at 95 °C for 40 cycles (c) Cycle 3: 1 min at 60 °C for 40 cycles (d) Cycle 4: hold at 4 °C 6. Start the run. Calculate the Ct values for each sample and the reference housekeeping genes and average the value per replicates.

Fig. 1

 1 Fig. 1 Representative TLS in human BC tissue. FFPE tumor sections were dual-IHC-stained for CD3 (T cells in brown) and CD20 (B cells in red). TLS are characterized by a dense, compact B-cell zone (similar to B-cell follicles in SLOs) adjacent to a more expansive T-cell zone (variable in cell density). (a) A TLS containing a large T-cell zone. (b) A TLS with more B cells than T cells. Images are at ×400 magnification

Fig. 2

 2 Fig. 2 Spearman correlations between CXCL13 (a) or LTB (b) gene expression and TLS scores on dual-CD3/CD20 IHC-stained BC tissue sections. Gene expression levels were quantified using qPCR following cDNA pre-amplification. Data are from a series of 80 FFPE BC samples. P-values (two-tailed) <0.05 are considered significant. Degrees of significance: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****)

Fig. 3

 3 Fig. 3 Gene expression in frozen and FFPE BC tissues. (a) Expression levels of individual and mean values for three housekeeping genes (POLR2A, TMBIM4, and TBP) in frozen BC samples from a microarray data set (n = 88) [20]. SD standard deviation and VAR variance. (b) qPCR C T values for the mean of these housekeeping genes in FFPE BC samples from 60 patients in (a) [11]. The CXCL13 gene is shown for comparison. (c) Pearson correlation between frozen microarray and FFPE qPCR data for the CXCL13 gene (n = 60). P-values (twotailed) <0.05 are considered significant. Degrees of significance: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****)

Elisa Corsiero et al. 1 . 4 . 9 .

 149 Hot plate. 2. Glass beaker. 3. Synovial tissue obtained from joint replacement or biopsy (see Note 1). Petri dishes 100 mm Ø × 15 mm H. 5. 5 mL Polypropylene round-bottom tubes. 6. Forceps and tweezers. 7. 40 and 70 μm nylon mesh cell strainers. 8. 5 mL Syringe plungers. Magnetic stirring bars, micro (5 mm × 2 mm). 10. 1× Phosphate-buffered saline (PBS) without calcium and magnesium (pH 7.3-7.5). 11. Ultrapure 0.5 M EDTA, pH 8.0 (sterile filtered solution). 12. Tissue digestion buffer: Roswell Park Memorial Institute (RPMI)-1640 Medium supplemented with 2% heat-inactivated fetal bovine serum (FBS), collagenase D (from Clostridium histolyticum), deoxyribonuclease I (DNase I) from bovine pancreas (see Note 2)

Fig. 1

 1 Fig. 1 Summary of the strategy to generate RA synovial recombinant monoclonal antibodies. The method starts from (1) mononuclear cell preparation from synovial tissue followed by (2) isolation of single B cells by flow cytometry, (3, 4) Ig V gene amplification, (5) expression vector cloning, and (6) monoclonal antibody production

7 . 8 .

 78 Elution buffer: 0.1 M Glycine-HCl, pH 3.0. Neutralization buffer: 1 M Tris base, pH 9.0. 9. 4-20% SDS polyacrylamide gel: Reducing and nonreducing loading buffer for SDS-PAGE gel. 10. Color protein standard broad range (11-245 kDa).

Fig. 2 Fig. 3

 23 Fig. 2 Representative picture showing synovial tissue dissected out from a joint. The image shows an example of synovial tissue before and after the cutting out from an RA joint

Fig. 4

 4 Fig. 4 Sorting gating strategy example. Mononuclear cells are surface labeled with fluorochrome-coupled anti-CD19 and anti-CD3 antibodies. The sorting gate strategy for single CD19 + CD3 -B cell is shown

7 .

 7 Check the size of the second PCR products by running a 2% agarose gel. Expected sizes of the second PCR are IgH = 450 bp, Igκ = 510 bp, and Igλ = 405 bp. Load 5 μL of the PCR product mixed with 5 μL of loading dye into the agarose gel containing GelRed and run for 30 min at 120 V. Visualize the DNA bands using a UV transilluminator.

9 .

 9 Check the size of the gene-specific PCR products by running a 2% agarose gel. Expected sizes are IgH = 450 bp, Igκ = 510 bp, and Igλ = 405 bp. Load 5 μL of the PCR product mixed with 5 μL of loading dye into the agarose gel containing GelRed and run for 30 min at 120 V. Visualize the DNA bands using a UV transilluminator.

Fig. 5

 5 Fig. 5 Schematic representation of the first, second, and gene-specific PCR. (1) First round of nested PCR starting from total Ig cDNA and using a mix of primers. (2) Second round of nested PCR starting from the first PCR product and using a mix of primers. (3) Gene-specific PCR starting from the first PCR product and using gene-specific primers

  Inoculation volume is used for preparing mini-prep. For larger preps, bigger volumes of LB inoculating medium are necessary.

5

 5

3. 2 . 2

 22 Enrichment of Total γδ T Cells

Fig. 1

 1 Fig. 1 Gating strategy to sort Tregs and non-Treg CD4 + T-cell subsets for gene expression analysis. Human CD4 + T-cell subsets sorted from the tumor, non-tumoral lung tissue, and blood of NSCLC patients. The gating strategy shows initial selection of live, single CD3 + CD4 + CD8 -T lymphocytes. Tregs and non-Treg CD4 + T cells defined according to the differential expression of CD25 and CD127 are further sorted as TLS and non-TLS cells based on the CD62L expression. The dot plots are representative of the sorting of the cells from one patient

3. 4 . 2

 42 For Total B Cells 3.4.3 For Total γδ T Cell

Fig. 3 (Fig. 2

 32 Fig. 3 (continued) (blood, panel (b) and tumor, panel (c)) negative selection. The FACS plots show a high percentage of tumor cells remaining in the cell suspension obtained from the tumor, indicating the necessity to perform the further cell sorting of CD2 + LDCY -γδ T population

Fig. 3

 3 Fig. 3 γδ T-cell purity by flow cytometry after negative selection. Flow cytometry analysis on the isolated γδ T cells using EasySep™ Human Gamma/Delta T cell Isolation Kit (StemCell) before (blood, panel (a)) and after

Fig. 1

 1 Fig. 1 Triple-IF stainings of FFPE sections from NSCLC-derived LN (a-d) or NSCLC tumor tissue (e-h) using antibodies against human CD20 (panels a-b, d, e-f, h), CD3 (panels a, c-d, e, g-h), PD-1 (panels b-d, f-h), and DAPI (panels c and g). (e-h) Focus on a TLS within NSCLC tissue. Yellow and white arrows (b, f) indicate PD-1 bright cells and PD-1 +/dim cells, respectively. Magnification: ×200

Fig. 2

 2 Fig. 2 Confocal imaging of a FFPE NSCLC tumor section using multiplex immunofluorescence. (a) Numerous CD3 + (green) CD4 + (red) T cells can be identified

Fig. 2 (

 2 Fig. 2 (continued) (in yellow) within the CD20 + B-cell zone (purple) of TLS. (b-e) Visualization of Tfh cells within TLS. A higher magnification of Panel (a) enables the visualization of (b) CD3 + (green) and (c) CD4 + (red) cells. (d) Merge of (b)-(c) shows that some CD3 + CD4 + T cells (yellow) express Bcl6 (white), a master regulator of Tfh differentiation. (e) These Bcl6 + cells (white) are visualized within the CD20 + B-cell zone of TLS (purple), but do not express the B-cell marker CD20

Fig. 1

 1 Fig. 1 Mouse instillation with bacterium-entrapped beads (here Pseudomonas aeruginosa) induces the neogenesis of peribronchial tertiary lymphoid structures (TLS) (detectable 10-14 days after instillation). Preparation and instillation of Pseudomonas aeruginosa-entrapped beads were performed as outlined in Subheadings 3.2 and 3.3. Representative photomicrographs of immunostainings (brown color) in lung tissues from mice sacrificed at day 14 after instillation with sterile (upper panels) and Pseudomonas aeruginosa-entrapped (lower panels) beads (Bd) are shown. Pseudomonas aeruginosa-infected mice (but not mice instillated with sterile beads) develop peribronchial TLS displaying from left to right segregated B-cell areas (anti-CD20 mAb, first panel) containing follicular dendritic cells (anti-CD21 Ab, second panel) as well as germinal centers (PNA, data not shown), and peripheral T-cell areas (anti-CD3 mAb, third panel) containing high endothelial venules (anti-PNAd Ab, fourth panel, marked with arrows)

Fig. 2 A

 2 Fig.2A B16-OVA tumor growing in the intraperitoneal cavity of a C57BL/6 mouse was harvested, histologically prepared, and stained for immunofluorescent microscopy (a-d), as outlined in Subheadings 3.1-3.3. Tumor sections were stained with antibodies of the indicated specificities (Table1). Scale bar = 100 μm. (f-g) Area measurements were done by drawing a perimeter around TLS and podoplanin + structures with the polygon tool in Zen software. Frequency of TLS (e) and individual immune subsets (h) was assessed by using the manual count tool in Zen software. Abbreviation: IP intraperitoneal. Error bars represent mean ± SEM. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values in (h) were calculated using the Mann-Whitney test

Fig. 3 8 . 9 .

 389 Fig. 3 Intraperitoneal and subcutaneous B16-OVA tumors grown in C57BL/6 mice were prepared for flow cytometry, as outlined in Subheadings 3.1 and 3.4. Endothelial cells were gated as live singlet CD45 neg Ter119 neg CD31 pos . Fibroblasts are gated as live singlet CD45 neg Ter119 neg podoplanin pos . B cells are gated as live singlet CD45 pos CD3 neg CD19 pos . Abbreviations: SC subcutaneous and IP intraperitoneal. Error bars represent mean ± SEM. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were calculated using the Mann-Whitney test

Fig. 4

 4 Fig. 4 Naïve CD8 + T cells from congenic Thy1.1 mice were intravenously injected into C57BL/6 mice with established subcutaneous (a) and intraperitoneal (b) B16-OVA tumors. Tumors were harvested 18 h later and infiltrating Thy1.1 pos T cells were enumerated by flow cytometry. Numbers on plots indicate the percentage of Thy1.1 pos cells out of the live singlet CD45 pos CD3 pos CD8α pos parent gate. Abbreviations: S.C. subcutaneous and I.P. intraperitoneal

9 .

 9 Liberase TM and DNase I at the concentrations specified in working buffer enable high-yield isolation of tumor-associated stromal cells from B16 without substantial loss of CD3, CD8, TLS in Murine Melanoma CD4, and B220 on tumor-infiltrating lymphocytes. If you are assessing alternative markers or tumors, optimizing enzyme concentrations and incubation times is recommended. 10. Cell suspension should initially be pipetted several times with a cut 1000 μL pipette tip. After 10 min, aggregates should easily be pipetted with an uncut 1000 μL tip. If not, prepare a singlecell suspension using a Dounce homogenizer. 11. Pooled lymph nodes and spleen from a single congenic Thy1.1 mouse provide 4.0 × 10 6 naïve CD8 + T cells, sufficient for two tumor-bearing animals. It is recommended to use three congenic Thy1.1 mice for a maximum of seven tumor-bearing animals.

1 .

 1 Following euthanasia, perform cardiac lung perfusion to flush out all blood cells from circulation: Pin ribs down to expose the lungs and heart. Gently immobilize heart with forceps. Using a 20 mL syringe and 23 gauge needle, inject 10-20 mL PBS into the right ventricle of the heart (the right ventricle is the bottom left portion of the heart from your perspective).

Fig. 1 1 .

 11 Fig. 1 Expression of the fluorescent protein Tomato in transformed lung epithelial cells. Schematic illustrating Cre recombinase-mediated DNA deletion which results in the induction of tdTomato expression by tumor cells. tdTomato provides a strong signal and facilitates the detection of small tumor cell clusters with the use of a dissecting microscope and immunofluorescence (IF) sections

Fig. 2 Fig. 3

 23 Fig. 2 Visualizing TA-TLS associated with LucOS-expressing tumors in whole tumor-bearing lung lobe 20 weeks postinfection. (a) Schematic illustrating the serial sectioning of whole lung lobe and reconstruction of IF sections in silico. (b) Artistic rendering of a reconstructed lung lobe stained for T cells (anti-CD3; orange), B cells (anti-B220; green), and tumor cells (anti-NKX2.1; blue) (left). Rendering of an individual tumorassociated tertiary lymphoid structure showing distinct clusters of T and B cells (right)

Fig. 1

 1 Fig. 1 Immune responses mediated by tumor-associated tertiary lymphoid structures. Circulating lymphocytes are recruited from HEV to tumor-associated TLS through a gradient of lymphoid chemokines. Intra-tumoral DCs uptake antigens and present processed antigens to specific T cells in the T-cell-rich areas of TLS, leading to T-cell (re)activation and differentiation. Effector T cells migrate to tumor nests for destruction of malignant cells. Tumor-infiltrating B cells are activated within the B-cell follicle of TLS and mediate antibody production. Abbreviations: B B cell, DC dendritic cell, HEV high endothelial venule, T T cell, and TLS tertiary lymphoid structure

Table 1 Antibodies and reagents used in IHC protocols Antibody or reagent Conjugate Host Clone or reference Source Dilution

 1

			IHC-based TLS Visualization and Quantification
	Human CD20	UC	Mouse	L26	Agilent/Dako	1/250
			IgG2a
	Human CD21	UC	Mouse	1F8	Agilent/Dako	1/30
			IgG1
	Human DC-LAMP	UC	Rat IgG2a 1010 E1.01 Dendritics	1/80
	(cross-reactivity with canine,
	feline, murine, and sheep
	DC-LAMP)
	Human CD3	UC	Rabbit IgG A0452	Agilent/Dako	1/80
	Human PD-1	UC	Mouse	NAT105	Roche	Pre-
			IgG1			diluted
	Mouse IgG2a	Biotin	Goat IgG	115-065-	Jackson	1/100
				206	ImmunoResearch
	Mouse IgG1	UC	Goat IgG	115-005-	Jackson	1/30
				205	ImmunoResearch
	Rat IgG	Biotin	Donkey	712-066-	Jackson	1/500
			F(ab′) 2	153	ImmunoResearch
	Rabbit IgG	AP	Goat	111-056-	Jackson	1/300
			F(ab′) 2	045	ImmunoResearch
	Streptavidin	HRP	NA	P039701	Agilent/Dako	1/300
	APAAP	AP	Mouse	D0651	Agilent/Dako	1/30
			IgG1
	Abbreviations: AP Alkaline phosphatase, APAAP Alkaline phosphatase anti-alkaline phosphatase, HRP Horseradish
	peroxidase, NA Nonapplicable, UC Uncoupled

  6. Heat-resistant slide trays (glass jars with glass slide holders or plastic 50 mL tubes, depending on the number of slides being processed simultaneously) for antigen retrieval.

	7. Regular slide trays for washing steps.
	8. Washing buffer (WB): 1× Phosphate-buffered saline (PBS)
	containing 0.1% (v/v) Triton X-100.
	9. Rocking platform.
	10. PAP pen, for example ImmEdge Hydrophobic Barrier PAP
	Pen (Vector Laboratories) (see Note 4).
	11. 3% H 2 O 2 (v/v) in double-distilled water (ddW).
	12. Wet chamber (a tray with a lid containing a support for hold-
	ing slides and water).
	1. Blocking buffer (BB): 4% Bovine serum albumin (w/v), 4%
	normal donkey serum (v/v) in WB.
	2. 37 °C Incubator.

3. Antibody-dilution buffer (ADB): 1% Bovine serum albumin (w/v), 1% normal donkey serum (v/v) in WB. 4. Primary antibodies (Table 1): Rabbit anti-human CD3 (clone SP7), mouse anti-human CD20 (clone L26), rat anti-mouse/ human PNAd (clone MECA-79), rat anti-mouse/human DC-LAMP (clone 1010E1.01), rabbit anti-human CD23 (clone SP23), mouse anti-human CD21 (clone 2G9) (see Note 5).

Table 1 Immunostaining reagents for the analysis of TLS in human tissues Maker Antibody clone

 1

			Antibody	Producer, catalogue	HRP-conjugated secondary
			dilution	number	antibody a
	CD21	2G9	1:5000	Leica,	Donkey anti-mouse IgG
				NCL-L-CD21-2G9
	DC-LAMP 1010E1.01	1:1000	Dendritics	Donkey anti-rat IgG
	CD23	SP23	1:1000	Abcam, ab16702	Donkey anti-rabbit IgG
	PNAd	MECA-79	1:5000	Biolegend, 120801	Goat anti-rat IgM
	CD20	L26	1:5000	Dako	Donkey anti-mouse IgG
	CD3	SP7	1:1000	Thermo Fisher	Donkey anti-rabbit IgG
	a

Table 2 The detection of each fluorescently stained marker by Vectra 3.0 ™

 2

	Maker	Fluorophore, dilution	Excitation (nm)	Emission (nm)	Detection filter
	Nucleus	DAPI, 1:10	358	461	2
	CD21	Opal-520, 1:100	494	525	3
	DC-LAMP	Opal-540, 1:100	523	536	4
	CD23	Opal-570, 1:100	550	570	4, 5
	PNAd	Opal-620, 1:150	588	616	5
	CD20	Opal-650, 1:150	627	650	6
	CD3	Opal-690, 1:50	676	694	6

  Depending on the size of the tissue section, 50-150 μL/slide is sufficient.

		Analysis of TLS by Quantitative Pathology
		4. Place slides in a wet chamber with tissue facing upwards, apply
		the first HRP-conjugated secondary antibody (anti-mouse
		IgG in ADB), and incubate for 1 h (see Note 6). Depending 19. Repeat steps 3-13, but use donkey anti-mouse IgG, donkey on the size of the tissue section, 50-150 μL/slide is anti-rabbit IgG, and donkey anti-rat IgG antibodies with sufficient. tyramide-fluorophore conjugates Opal-650, Opal-690, and
		5. Decant the secondary antibody mix and wash in WB for Opal-540 to detect CD20, CD3, and DC-LAMP antigens,
		3 × 5 min on a rocking platform. respectively.
		6. Place slides in a wet chamber with tissue facing upwards, apply 20. Place slides in a wet chamber with tissue facing upwards, apply
		the first tyramide-fluorophore conjugate Opal-520 diluted in the DAPI solution (2 drops/1 mL PBS), and incubate for
		the amplification diluent buffer, and incubate for 10 min to 5 min. Depending on the size of the tissue section, 100-
		detect the CD21 antigen (see Note 16). 300 μL/slide is sufficient.
		7. Decant the tyramide-dye mix and wash in WB for 5 min on a
		rocking platform.
		8. Place slides in a wet chamber with tissue facing upwards, apply
	3.1.2 Immunostaining	3% H 2 O 15. Place the tray in a 95 °C water bath for 10 min to remove the
		bound antibodies.
		16. Wash the slides in WB for 10 min on a rocking platform.
		17. Place slides in a wet chamber with tissue facing upwards, apply
		BB, and incubate for 15 min at 37 °C. Depending on the size
		of the tissue section, 100-300 μL/slide is sufficient.

2 , and incubate for 30 min to block the activity of the first HRP-conjugated secondary antibody (see Note 17). 9. Decant the H 2 O 2 solution and wash the slides in WB for 15 min on a rocking platform. 10. Place slides in a wet chamber with tissue facing upwards, apply the second HRP-conjugated secondary antibody (donkey antirabbit IgG in ADB), and incubate for 1 h (see Note 6). Depending on the size of the tissue section, 50-150 μL/slide is sufficient. 11. Repeat steps 5-9, but use the tyramide-fluorophore conjugate Opal-570 to detect CD23 antigen.

12. Place slides in a wet chamber with tissue facing upwards, apply the third HRP-conjugated secondary antibody (goat anti-rat IgM in ADB), and incubate for 1 h (see Note 6). Depending on the size of the tissue section, 50-15 μL/slide is sufficient.

13. Repeat steps 5-7, but use the tyramide-fluorophore conjugate Opal-620 to detect PNAd antigen.

14. Place slides in a heat-resistant tray containing ARB such that tissues are covered and the tray is not filled to the brim.

18. Decant BB; place slides back in the wet chamber; apply the second primary antibody mix in ADB containing mouse antihuman CD20, rabbit anti-human CD3, and rat anti-mouse DC-LAMP antibodies; and incubate overnight at 4 °C (see Note 5).

Table 1 Antibodies used for immunofluorescence staining and flow cytometry

 1 Alternatively, MECA-79 hybridoma supernatant can be prepared in-house and titrated on LN sections before staining HEV in tumor sections.

					HEV and TLS in Cancer
						Working
						concentration
	Antigen	Conjugate	Clone	Isotype	Company	(μg/mL)
	Primary antibodies for immunolabeling frozen sections
	PNAd	Purified	MECA-79	Rat IgM, κ	Biolegend	2.5
	Pan-endothelial	Biotinylated	MECA-32	Rat IgG2a, κ	Biolegend	2
	cell (pan-EC)
	MAdCAM-1	Biotinylated	MECA-367 Rat IgG2a, κ	Biolegend	2
	CD3	Purified	-	Rabbit polyclonal DAKO	2
	CD45R/B220	Biotinylated	RA3-6B2	Rat IgG2a, κ	Biolegend	2
	Follicular dendritic	Purified	FDC-M1	Rat IgG2c, κ	BD	2
	cell (FDC)				Biosciences
	Secondary antibodies/reagents for immunolabeling frozen sections
	Anti-rat IgM	Alexa Fluor	-	Goat polyclonal	Invitrogen	1
		594
	Streptavidin	Alexa Fluor	-	-	Invitrogen	1
		488
	Streptavidin	Alexa Fluor	-	-	Invitrogen	1
		647
	Anti-rabbit	Alexa Fluor	-	Goat polyclonal	Invitrogen	1
		594
	Anti-rat IgG2c	Alexa Fluor	MARG2c-5 Mouse IgG2a	Invitrogen	1
		FITC
	Isotype control antibodies for immunolabeling frozen sections
	Rat IgM κ	Purified	RTK2118	Rat IgM, κ	Biolegend	2
	Rat IgG2a, κ	Biotinylated	RTK2758	Rat IgG2a, κ	Biolegend	2
	Rabbit polyclonal Purified	-	Rabbit polyclonal Abcam	2
	Rat IgG2c, κ	Purified	RTK4174	Rat IgG2c, κ	Biolegend	2
	Primary antibodies for immunolabeling paraffin sections
	PNAd	Purified	MECA-79	Rat IgM, κ	Biolegend	2.5
	CD45R/B220	Purified	RA3-6B2	Rat IgG2a, κ	Biolegend	2
	CD3	Purified	-	Rabbit polyclonal DAKO	2
	Secondary reagents for immunolabeling paraffin sections
	Immpress anti-rat HRP	-	Goat	Vector	-
	Immpress	HRP	-	Horse	Vector	-
	anti-rabbit
						(continued)

9. If using formaldehyde or PLP to fix sections then prepare 0.3 M glycine (Thermo Fisher Scientific) in PBS, pH 7.4, to quench free aldehydes.

10. Optional: Avidin/biotin blocking kit if a biotinylated primary or secondary antibody is used.

11. Serum blocking solution to block nonspecific binding of antibodies. 2.5-5% serum of the species in which the secondary antibody is raised is often used. Other sera, for example horse or mouse serum, may also be used.

12. Primary antibodies diluted in 1% bovine serum albumin (BSA, Sigma) in PBS: Rat anti-mouse PNAd antibody (clone MECA-79, rat IgM) is used at a working concentration of 2.5 μg/mL to detect HEV (see Notes 2 and 3). working concentration of 2 μg/mL, Biolegend) can be used to detect immature HEV in tumors (Fig.

1

). Example 3: The presence of TLS in tumor tissue, characterized by T/B-cell segregation and follicular dendritic cell (FDC) networks, may

  Vector) and Vector-VIP (purple; Vector) followed by rabbit anti-mouse CD3 (DAKO) detected with anti-rat Immpress reagent (Vector) and Vector-SG (grey; Vector).23. Counterstain in hematoxylin (10-20 s) and blue inScott's tap water (10 s) (see Note 9). Cull T-cell donor mice by CO 2 inhalation and/or cervical dislocation and pin out on dissecting board ventral side up. Swab ventral surface with 70% alcohol to sterilize and wet fur, and open abdominal cavity by midline incision using scalpel. Remove spleen carefully using forceps and scissors and avoid damaging the surface. Collect spleen into PBS in labeled tubes and transport on ice to biological safety cabinet (see Notes 10 and 11).

	24. Dehydrate in ascending alcohols (70, 90, 100, and 100%) for
	3 min each and 3× 100% xylene washes for 5 min each.
	25. Mount in mounting media and coverslip.
	1.
	14. Block nonspecific protein binding with blocking solution for
	30 min at RT.
	15. Drain slides (no need to wash).
	16. Incubate with MECA-79 antibody or rat IgM isotype control
	in 1% BSA/PBS overnight at 4 °C or for 1 h at RT in a humid-
	ified chamber.
	17. Wash slides three times in PBS over 5 min.
	18. Incubate with anti-rat Immpress reagent (Vector) or other
	HRP conjugated secondary antibody and incubate at RT for
	30 min.
	19. Wash slides three times in PBS over 5 min.
	20. Incubate with freshly prepared DAB Chromogen solution and
	monitor under a microscope until color develops.
	21. Rinse with deionized H 2 O and then wash in fresh deionized
	H 2 O.

22.

Optional: It is possible to sequentially stain the same tissue section with other antibodies in order to identify other cells and structures using different colored chromogens (see Note 8). For example, the presence of TLS-containing organized B-and T-cell zones can be verified with rat anti-mouse CD45R/B220 (Biolegend) detected with anti-rat Immpress reagent (

  EmmaJones et al. 100 μg/mL isotype control (clone MAC193, rat IgG2a) for 30 min at 4 °C (see Note 13). 9. Inject 0.2 mL of T cells plus antibody (5 × 10 6 cells) intravenously into experimental mice (see Notes 10, 15, and 16). 10. Cull mice after 1, 4, or 24 h (see Note 17) by CO 2 inhalation and/or cervical dislocation. Collect blood by cardiac puncture from the left ventricle using a 1 mL syringe with 27G needle into 1.5 mL heparinized Eppendorf tubes containing 100 μL of 10 units/mL heparin or citrate-EDTA. Store carcasses on ice and prepare for dissection, as described in step 1. 11. Collect tumor, spleen, peripheral (axillary, brachial, and inguinal) LN, and mucosal (mesenteric or mediastinal) LN, and process as follows: cut tumor, spleen, and mucosal LN into two halves, one half to analyze infiltrating T cells by histology and the other half to isolate infiltrating T cells for flow cytometric analysis. Pool right peripheral LN and use to isolate infiltrating T cells. Collect left peripheral LN separately for immunolocalization of infiltrating T cells. Fix LN, spleen, and tumor in 2% formaldehyde/5% sucrose in PBS for 2 h, transfer

to 20% sucrose in PBS for 2 h, snap-freeze in liquid nitrogen, and store at -80 °C for up to 2 years. Collect lymphocytes from one half of a tumor, one half of spleen, and pooled right peripheral LN, as described in steps 2-5 above (see Notes 18 and 19).

  ; arrowheads). Total lymphocytes counts range from 200 to 500 cells. The numbers of CFSE-labeled cells inside and outside HEVs are not significantly different in inguinal and brachial LN of individual mice or between animals in the same experimental group[START_REF]-free): A mouse anti-mouse CD20 mAb can be obtained from companies like Genentech[END_REF]. Results are pooled from inguinal and brachial LN of mice within each experimental group and expressed as means ± SEM CFSE-labeled cells/ mm 2 inside and outside HEVs[34].

Capture Microdissection of TLS from Frozen Tissues 2.3 RNA Extraction from Microdissected TLS 2.4 Assessing RNA Integrity

	1. High-Capacity cDNA Reverse Transcription Kit (Applied
	Biosystems).
	2. Pipet 200, 10, and 2 μL dedicated for RNA work.
	3. PCR thermal cycler.
	1. Applied Biosystems quantitative real-time PCR system (or
	compatible real-time PCR system from other suppliers).
	2. MicroAmp Optical 96-Well Reaction Plate for quantitative
	Real-Time PCR (Thermo Fisher Scientific).
	3. 96-Well MicroAmp Optical Adhesive Film (Applied
	Biosystems).
	4. Compression pad to seal the quantitative real-time PCR plate.
	5. TaqMan Universal PCR Master Mix (Applied Biosystems).
	6. TaqMan Gene Expression Assays (Applied Biosystems).

7. Pipet 200, 10, and 2 μL dedicated for RNA work.

  Allow all reagents from the Agilent RNA 6000 Pico kit (Agilent Technologies): Pico Gel Matrix, Pico Dye Concentrate, Pico Conditioning Solution, and Pico Marker to equilibrate at room temperature and protected from light for at least 30 min before use (see Note 25). Remove an aliquot of the Pico RNA Ladder from the freezer and let it thaw on ice (see Note 26).

	2. Pipet 550 μL of the RNA 6000 Pico gel matrix (red tube) into
	the top of receptacle of a spin filter. Place the spin filter in the
	microcentrifuge and centrifuge at 1500 × g for 10 min at room
	temperature. Aliquot 65 μL of filtered gel into 0.5 mL RNase-
	free microcentrifuge tubes. The filtered gel can then be stored
	at 4 °C for up to 4 weeks.
	3. Pipet 1.3 μL of each RNA samples into an RNase-free 0.5 mL
	tube. Heat denature the RNA samples for 2 min at 70 °C with
	a heat block. Keep then the RNA on ice until further utiliza-
	tion. Before further use, briefly centrifuge the tube to collect
	the RNA at the bottom of the tube.

14. Transfer 1.3 μL of the RNA sample to a new 0.2 mL tube for further evaluation of RNA quality and quantity. Store the samples immediately at -80 °C. 1. 4. Vortex the RNA dye concentrate (blue tube) for 10 s and spin down briefly. Add 1 μL of dye concentrate into a 65 μL aliquot of filtered gel (previously equilibrated at room temperature).

  It is important to assure that the DNase I incubation mix is in contact with the membrane; otherwise the digestion will be incomplete.24.Qiagen supplies the buffer RPE as a concentrate in the kit.Investigator must have prepared a working solution, that is, by adding 4 volumes of 100% ethanol to the concentrate RPE buffer, before starting.25. Dyes are light sensitive and must be kept covered while equilibrating at room temperature. The total RNA concentration for use with the Agilent RNA 6000 Pico kit must be between 500 and 5000 pg/μL. If concentration of the sample is above this range, dilute it with nuclease-free water.26. To avoid repetitive/thaw cycles, the Pico RNA Ladder must have been prepared and aliquoted upon kit arrival. The ladder needs to be denatured by warming for 2 min at 70 °C on a heat block before cooling on ice. Add 90 μL of nuclease-free water, mix well before aliquoting in 0.5 mL RNase-free vials, and store at -80 °C.

	23.
	18. As a confirmation of a proper collection of the microdissected
	TLS, the RLT lysis solution within the flat cap should progres-
	sively turn blue during the LCM step due to the accumulation
	of toluidine blue-stained tissue within the lysis buffer. Absence
	of blue coloration should raise concern about the RNA con-
	centration or the proper collection of the tissue during the
	LCM. It is recommended collecting the microdissected TLS in
	at least two PCR tubes for recovering enough quantity of
	material for posterior RNA extraction.
	19. The steps in Subheading 3.2 should be carried out as quickly
	as possible. Tissue staining and LCM of TLS should require a
	maximum of 60 min (depending on the number of microdis-
	sected tissue sections per slide) to prevent RNA degradation.
	20. Tissue lysates can be extracted for RNA immediately after the
	LCM session or alternatively stored at -80 °C for up to
	6 months. When possible, we recommend performing the
	RNA extraction the same day after performing the LCM.
	21. To avoid ß-mercaptoethanol odors in the lab, discard the RLT
	buffer eluent in a closed 50 mL Falcon tube during Subheading
	3.3, step 3, of the RNA extraction.
	22. DNase I is highly sensitive to physical degradation and risk
	denaturation if vortexed.

  6. Transfer the supernatant to a new microcentrifuge tube without disturbing the pellet. Steps 7-16 are carried out at room temperature. 7. Add 16 μL (see Note 12) DNase Booster Buffer and 10 μL DNase I (reconstituted solution). Mix by inverting the tube. Add 1200 μL (see Note 12) of 96-100% ethanol on the sample and mix well by pipetting. Do not centrifuge. 11. Transfer 700 μL of the sample, including any precipitate that may have formed, to an RNeasy MinElute spin column placed in a 2 mL collection tube. Close the lid gently and centrifuge for 20 s at ≥8000 × g. Discard the flow-through. 12. Repeat the previous step until the entire sample has passed through the RNeasy MinElute spin column.

	Centrifuge briefly to collect residual liquid from the sides of
	the tube.
	8. Incubate at room temperature for 15 min.
	9. Add 320 μL (see Note 12) Buffer RBC to adjust binding con-
	ditions and mix the lysate thoroughly.
	10. 17. Put the RNA on ice.
	18. Quantify RNA using a NanoDrop or equivalent spectropho-
	tometer using 1 μL per sample (see Note 14).
	19. Store the remaining RNA at -80 °C.

13. Add 500 μL of Buffer RPE to the RNeasy MinElute spin column. Close the lid gently and centrifuge for 15 s at ≥8000 × g. Discard the flow-through. 14. Again, add 500 μL of Buffer RPE to the RNeasy MinElute spin column. Close the lid gently and centrifuge for 2 min at ≥8000 × g. Discard the collection tube with the flow-through. 15. Place the RNeasy MinElute spin column in a new 2 mL collection tube. Open the lid of the spin column and centrifuge at full speed (see Note 10) for 5 min. Discard the collection tube with the flow-through. 16. Place the RNeasy MinElute spin column in a new 1.5 mL collection tube. Add 14 μL (see Note 13) of RNase-free water to the spin column membrane (eject water just above the center of the membrane). Close the lid gently and centrifuge for 1 min at full speed (see Note 10) to elute total RNA.

  1. Before starting, thaw the RT Buffer Mix tube at room temperature (see Note 15). After completely thawing, mix by inverting several times, then spin down, and keep on ice.2. Thaw the RNAs in a refrigerator (4 °C). Move them to ice after they are completely thawed. in FFPE samples, detectable and quantifiable segments of the gene transcripts are at very low copy numbers. To use a reasonable quantity of RNA from the FFPE extract to detect a satisfactory number of gene targets, an intermediate step called pre-amplification (also by PCR) is necessary before the final step of real-time PCR for mRNA quantification. To ensure linearity (see Note 18) of the preamplification step, all investigated genes are amplified together with low concentrations (0.05× of the normal concentrations) of the PCR primers and the TaqMan probes (included in commercial TaqMan Gene Expression Assays). Work on ice and avoid exposure to light with the TaqMan Gene Expression Assays.

	3. Up to 2 μg of total RNA can be reverse transcribed per
	reaction.
	4. One reaction contains 10 μL of 2× RT Buffer Mix, 1 μL of 20×
	RT Enzyme Mix, and 9 μL of RNA (adjust with RNase-free
	water if sample volume is less than 9 μL of RNA) in a 0.2 mL
	PCR tube.
	5. Prepare a mixture of RT Buffer Mix and RT Enzyme Mix for
	the total number of reactions (see Note 16).

6. Add suitable volumes of RNase-free water (if required) to the bottom of each PCR tube. Add suitable volumes of total RNA, and briefly mix with water (if applicable) by pipetting once. 7. Distribute 11 μL of the mixture (RT Buffer Mix + RT Enzyme Mix) into each PCR tube, just above the RNA. Mixing by pipetting is permitted but not necessary. Steps 6 and 7 can be reversed. 8. Centrifuge briefly if all the liquid is not at the bottom of the tube. 9. Run the incubation program at 37 °C for 60 min, and 95 °C for 5 min, and then hold at 4 °C on a thermal cycler. 10. Store the cDNA at -20 °C. Due to high levels of RNA degradation (segmentation; see Note 17)

  7. Perform the pre-amplification PCR reaction on a thermal cycler using the following program: 95 °C for 10 min, then 10 cycles at 95 °C for 15 s, and 60 °C for 4 min (see Note 22).

8. Dilute fivefold (see Note 23) the pre-amplification product using RNase-free water (see Note 20). 9. Use 5 μL of pre-amplified, diluted cDNA for each 20 μL PCR reaction of real-time quantification of individual target and housekeeping gene expression (see Note 24). 10. In addition to the cDNA, each 20 μL PCR reaction contains 1 μL of a specific 20× TaqMan Gene Expression Assay, 10 μL of TaqMan Universal Master Mix II with UNG, and 4 μL of nuclease-free water.

Table 1 TaqMan gene expression assays used for FFPE samples Gene name Company Assay ID Amplicon length (bp)

 1

	POLR2A	Applied Biosystems	Hs00172187_m1	61
	TMBIM4	Applied Biosystems	Hs00211390_m1	63
	TBP	Applied Biosystems	Hs00427621_m1	65
	CXCL13	Applied Biosystems	Hs00757930_m1	70
	LTB	Applied Biosystems	Hs00242739_m1	78

Table 1 (continued) Primer Sequence

 1

	Elisa Corsiero et al.

14. Tris/borate/EDTA (TBE) running buffer: 1 M Tris base, 0.9 M boric acid, and 0.01 M EDTA. 15. GelRed Nucleic Acid gel stain (see Note 9). 16. Loading dye for DNA agarose gel: 40% w/v Sucrose, 0.25% bromophenol blue (6× solution) (see Note 10).

Table 2 Reverse primers used to amplify IgH and IgL chain gene transcripts B-cell population IgH PCR reverse primer Igκ reverse primer

 2

	Igλ
	reverse
	primer

Table 3 Gene-specific PCR primers for IgH and IgL (κ/λ) (see Note 11)

 3

	IgλV 5′ Primer IgλJ 3′ Primer	1 3′ XhoI Cλ 5′ AgeI Vλ1 1		2 3′ XhoI Cλ 5′ AgeI Vλ2 2	3 3′ XhoI Cλ 5′ AgeI Vλ3 3	4 3′ XhoI Cλ 5′ AgeI Vλ4/5 4		5 3′ XhoI Cλ 5′ AgeI Vλ4/5 5	6 3′ XhoI Cλ 5′ AgeI Vλ6 6	7 3′ XhoI Cλ 5′ AgeI Vλ7/8 7	8 5′ AgeI Vλ7/8
	5′ Primer IgκJ 3′ Primer	3′ BsiWI Jκ 5′ Age Vκ1-5 1	1/4	3′ BsiWI Jκ 2 5′ Age Vκ1-5 2	3′ BsiWI Jκ 3 5′ Age Vκ1-5 3	3′ BsiWI Jκ 5′ Age Vκ1-5 4	1/4	3′ BsiWI Jκ 6 5′ Age Vκ1-5 5	5′ Age Vκ1-9	5′ Age Vκ1-5	5′ Age Vκ1-5	5′ Age
	IgH V 5′ Primer IgH J 3′ Primer IgκV	1-18 5′ Age VH1-24 1 3′ SalI JH1/2/4/5 1-12		1-2 5′ Age VH1-24 2 3′ SalI JH1/2/4/5 1-16	1-24 5′ Age VH1-24 3 3′ SalI JH3 1-17	1-3 5′ Age VH1-24 4 3′ SalI JH1/2/4/5 1-27		1-45 5′ Age VH1-24 5 3′ SalI JH1/2/4/5 1-33	1-46 5′ Age VH1-24 6 3′ SalI JH6 1-37	1-58 5′ Age VH1-24 1-39	1-69 5′ Age VH1-24 1-5	1-8 5′ Age VH1-24 1-8

  4. Remove cautiously the adhesive PCR seal and add 7 μL RT mix to each well. Cover the plate with adhesive PCR plate seal and spin down. 5. Perform the reverse transcription at 42 °C for 5 min, 25 °C for 10 min, 50 °C for 60 min, and 94 °C for 5 min. 6. cDNA can be stored at -20 °C before performing the immunoglobulin gene amplification PCR. For each cell, IgH and corresponding IgL chain (Igκ and Igλ) gene transcripts are amplified independently by nested PCR starting from 2 μL of cDNA as template. For the IgH gene, three independent nested PCR are performed in order to discriminate between IgH constant regions Cμ, Cγ, and Cα. The nested PCR is a modification of the common PCR in which two sets of primers are used in two consecutive rounds of PCR. The second set of primers bind the first PCR product internally ensuring that the product from the second PCR has little contamination, thus increasing the sensitivity and specificity of the PCR. All PCR reactions are performed in 96-well half-skirted plates in a total volume of 40 μL per well. Prepare a PCR master mix for the second round of nested PCR (or second PCR), as shown below. Add 36.5 μL of PCR master mix 2 and 3.5 μL of first nested PCR product to each well of a new 96-well plate. Cover with adhesive PCR seal plate and spin down. 6. Perform the second PCR at 94 °C for 15 min; for 50 cycles at 94 °C for 30 s, 58 °C (IgH/Igκ) or 60 °C (Igλ) for 30 s, 72 °C for 45 s; 72 °C for 10 min.

		Monoclonal Antibodies from RA Synovial Tissue
		4. Reagent-PCR	1-Well	96-Wells (μL)
		master mix 2	(μL)	(see Note 34)
		Nuclease-free water 31.66	3482.6
		PCR buffer	4.00	440.0
		5′ Primer mix	0.13	14.3
		3′ Primer mix	0.13	14.3
		dNTP	0.40	44.0
		HotStart Taq	0.18	19.8
	3.3.2 Immunoglobulin V
	Gene Amplification by Nested PCR	5.
		Reagent-PCR	1-Well	96-Wells (μL)
		master mix 1	(μL)	(see Note 34)
		Nuclease-free water 32.16	3647.6
		PCR buffer	4.00	440.0
		5′ Primer mix	0.13	14.3
		3′ Primer mix	0.13	14.3
		dNTP	0.40	44.0
		HotStart Taq	0.18	19.8
		2. Add 38 μL of PCR master mix 1 and 2 μL of cDNA to each
		well of a new 96-well plate. Cover with adhesive PCR seal plate
		and spin down.

1. Prepare a PCR master mix for the first round of nested PCR (or first PCR), as shown below. 3. Perform the first PCR at 94 °C for 15 min; for 50 cycles at 94 °C for 30 s, 58 °C (IgH/Igκ) or 60 °C (Igλ) for 30 s, 72 °C for 55 s; 72 °C for 10 min.

  7. In a new 96-well PCR plate, add 4.2 μL of unpurified first PCR into the correct well corresponding to the matched primers. Add the mix containing the PCR master mix and primers. Spin down and seal the plate with adhesive PCR plate. 8. Perform the second round of nested PCR at 94 °C for 15 min; for 50 cycles at 94 °C for 30 s, 58 °C (IgH/Igκ) or 60 °C (Igλ) for 30 s, 72 °C for 45 s; 72 °C for 10 min.

Table 4 Enzyme digestion mix for IgH, Igκ, and Igλ PCR product IgH PCR product Igκ PCR product Igλ PCR product

 4 Thaw competent cells on ice. Mix gently and transfer 5 μL of cells into a 0.2 mL tube on ice (see Notes 40 and 41). 2. Add 3 μL of ligation product to the cell mixture and leave on ice for 30 min. 3. Heat shock at 42 °C in a water bath for 20 s and place the tubes back on ice for 5 min. 4. Add 200 μL of LB medium and place for 1 h at 37 °C under shaking (200 rpm) (see Note 42). 5. Warm LB agar plates at 37 °C or room temperature, spread 200 μL of the mixture onto the plate, and incubate overnight at 37 °C leaving the plate upside down. Label each plate with the corresponding PCR product name in order to distinguish between the IgH, Igκ, and Igλ individual clones (see Note 43). Monoclonal Antibodies from RA Synovial Tissue 15 min; for 27 cycles at 94 °C for 5 min, 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s; 72 °C for 10 min.

	Elisa Corsiero et al.
	Mix 1	5.9 μL Nuclease-free	3.95 μL Nuclease-	5.9 μL
		water	free water	Nuclease-
				free water
		0.05 μL AgeI-HF	0.05 μL AgeI-HF	0.05 μL
				AgeI-HF
		0.05 μL SalI-HF		0.05 μL
				XhoI
	Mix 2		1.7 μL Nuclease-free
			water
			0.2 μL NEBuffer 3.1
			0.1 μL BsiWI

Table 1 Antibodies for human Tregs and non-Treg CD4 + T-cell sorting for gene expression analysis

 1 Sorting of Tertiary Lymphoid Structure-Immune CellsThe table shows the different antibodies used for human Tregs and non-Treg CD4 + T-cell sorting, with their specificity, conjugates, clones, supplier, and volumes for gene expression analysis

	Specificity	Clone	Conjugate	Supplier	Volume (μL/10 6 cells)
	CD3	UCHT1	AF700	BD Biosciences	1.5
	CD4	OKT4	BV605	BioLegend	2
	CD8	RPA-T8	FITC	BD Biosciences	1.5
	CD25	BD+	PE-cy7	BD Biosciences	2
	CD127	A019D5	BV650	BioLegend	3
	CD62L	DREG-56	APC	BD Biosciences	2
	DAPI (viability marker)				1 μL/mL cells

Table 2 Antibodies for human Tregs and non-Treg CD4 + T-cell sorting for functional assays

 2

	Specificity	Clone	Conjugate	Supplier	Volume (μL/10 6 cells)
	CD2	S5.2	APC	BD Biosciences	5
	CD4	OKT4	BV605	BioLegend	2
	CD8	RPA-T8	FITC	BD Biosciences	1.5
	CD25	BD+	PE-Cy7	BD Biosciences	2
	CD127	A019D5	BV650	BioLegend	3
	LDCY (viability marker)			Thermo Fisher	0.6

Table 3 Antibodies for human γδ T-cell isolation for functional assays Specificity Clone Conjugate Supplier Volume (μL/10 6 cells)

 3

	CD2	S5.2	APC	BD Biosciences	5
	LDCY (viability marker)			Thermo Fisher	0.6

Table 4 Antibodies for human γδ T-cell isolation for gene expression analysis

 4

	Specificity	Clone	Conjugate	Supplier	Volume (μL/10 6 cells)
	CD45	J.33	Pacific Blue Beckman Coulter 1.5
	CD3	UCHT1	AF700	BioLegend	1.5
	Pan-γδ	IMMU510 PE	Beckman Coulter 5
	Vδ2	IMMU389 AF 647	Beckman Coulter 2
	LDCY (viability marker)			Thermo Fisher	0.6

Table 5 Antibodies to check human γδ T-cell isolation purity by flow cytometry

 5

	Specificity	Clone	Conjugate Supplier	Volume (μL/10 6 cells)
	CD227	HMPV	FITC	BD Biosciences	1
	Epithelial antigen	Ber-EP4	FITC	DAKO	1
	CD3	UCHT1	AF700	BioLegend	1.5
	Pan-γδ	IMMU510	PE	Beckman Coulter	5
	CD2	S5.2	APC	BD Biosciences	5
	LDCY (viability marker)			Thermo Fisher	0.6

Table 6 Antibodies for negative selection of human B cells for functional assays Specificity Clone Conjugate Supplier Volume (μL/10 6 cells) Excluded cell types

 6

	CD14	HCD14	PercP	BioLegend	3	Macrophages, monocytes
	CD2	RPA	APC-H7 BD Biosciences 2.5	T cells
	CD16	3G8	APC-H7 BD	2	Neutrophils
				Pharmingen
	CD56	NCAM16.2 APC	BD	1	NK, NKT cells
				Pharmingen
	CD66b	80H3	FITC	Beckman	20	Neutrophils
				Coulter
	CD227	Ber-EP4	FITC	Dako	1	Epithelial cells
	CD11c	KB90	FITC	Dako	10	Dendritic cells
	CD36	FA6.152	FITC	Beckman	10	Endothelial cells,
				Coulter		macrophages, monocytes
	CD43	DFT1	FITC	Immunotech	10	Leukocytes
	CD45	HI30	PE-Cy7	BD	4	Nonimmune cells
				Pharmingen
	DAPI (viability				1 μL/mL of	Dead cells
	marker)				cells

Table 1 List of reagents used to detect Tfh by immunofluorescence and flow cytometry

 1

		Follicular Helper T Cells Detection Within Tumors
	2.1 Samples	1. Five-μm-thick FFPE TLS-associated NSCLC tumor and lymph
		node (LN) sections.
		2. Fresh HCC tumor samples.
	2.2 Buffers	1. 100% Xylene or Clearene (Leica).
	2.2.1 Visualization	2. 100% Absolute ethanol.
	of TLS-Associated Tfh	3. Distilled water.
	on FFPE Tumor Tissue by Triple-CD20/CD3/PD-1 Immunofluorescence Staining	4. Ethanol solutions at 90, 70, and 50%. 5. 10× TBS: 47.4 g Trizma ® hydrochloride + 263 g sodium chlo-ride, in 3 L of distilled water; pH adjusted to 7.4.
		6. 1× TBS: 1 Volume of 10× TBS + 9 volumes of distilled water.
		7. TBS-T: 1× TBS + 0.04% Tween 20.
		8. Phosphate-buffered saline (PBS).
	2.2.2 Detection	1. 10× EZ Prep (deparaffinization solution) (Ventana): Needs to
	of TLS-Associated Tfh	be reconstituted in type 2 pure water.
	on FFPE Tumor Sections by Multiplex CD3/CD4/ CD20/Bcl6 IF Staining	2. Pre-diluted Cell Conditioner #1 (CC1) solution: Tris-EDTA buffer pH 8.5 for antigen retrieval (Ventana). 3. 10× Reaction Buffer (Ventana): Needs to be reconstituted in
		type 2 pure water.
		4. Discovery Inhibitor (Ventana): Peroxidase blocking solution
		containing hydrogen peroxide between 1 and 5%.
		5. 10× TBS: 47.4 g Trizma ® hydrochloride + 263 g sodium chlo-
		ride in 3 L distilled water; pH adjusted at 7.4.
		6. 1× TBS: 1 Volume of 10× TBS + 9 volumes of distilled water.
	2.2.3 Tfh Identification	1. Complete RPMI: RPMI medium supplemented with 1% l-
	by Multiparametric Flow	glutamine, 1% penicillin-streptomycin, and 10% heat-
	Cytometry from Fresh	inactivated fetal bovine serum (FBS).
	Tumor Tissue

see Note 11).

	Coline Couillault et al.
	4. Select blocking step to block endogenous peroxidases with one 24. Select "multimer" to incubate the slides with 100 μL of ready-
	drop of Discovery Inhibitor, at RT for 8 min. to-use OmniMap goat anti-rabbit coupled to HRP, at 37 °C
	5. Select "primary antibody" to cover the tissue with 100 μL of for 16 min.
	ready-to-use mouse anti-human Bcl6 (clone GI191E/A8) for 25. Repeat steps 7-9.
	32 min at 37 °C. 26. Dilute both tyramide component coupled to Alexa Fluor 647
	6. Select "multimer" to incubate the slides with 100 μL of ready-(100×) and Solution A at 1/100 in 1× TBS to prepare Solution
	to-use secondary antibody (OmniMap mouse Ventana coupled D (see Note 9).
	to HRP) for 12 min. 27. Incubate each slide with 200 μL of Solution D at RT for
	7. Remove the slides from the Ventana autostainer and wash the 10 min.
	slides extensively using a solution containing detergent. 28. Wash the slides twice in cold distilled water.
	8. Rinse for 2 min, three times. 29. Put the slides in the Ventana Discovery XT autostainer.
	9. Prepare hydrogen peroxide solution at 0.15% by diluting the 30. Select dual sequence and apply antibody denaturation at 90 °C stock 9% H 2 O 2 in distilled water (solution A). during 12 min.
	10. Dilute both tyramide component coupled to Alexa Fluor 546 31. Apply manually 100 μL of mouse anti-human CD20 (clone (100×) and Solution A at 1/100 in 1× TBS to prepare Solution L26, diluted at 1/250 in antibody diluent) in the Ventana B (see Note 9). Discovery XT, at 37 °C for 60 min.
	11. Incubate each slide with 200 μL of Solution B at RT for 32. Apply secondary antibody: ready-to-use OmniMap goat anti-10 min. mouse coupled to HRP, at 37 °C for 12 min.
	12. Wash the slides twice in cold distilled water. 33. Repeat steps 7-9.
	13. Put the slides in the Ventana autostainer. 34. Dilute both tyramide component coupled to Alexa Fluor 488
	14. Select dual sequence and apply antibody denaturation at 90 °C (100×) and Solution A at 1/100 in TBS 1× to prepare Solution
	during 12 min (see Note 10). E (see Note 9).
	15. Select "primary antibody" to cover the tissue with 100 μL of 35. Incubate each slide with 200 μL of Solution E at RT for
	ready-to-use mouse anti-human CD3 (clone 2GV6) for 40 min 10 min.
	at 37 °C. 36. Wash the slides twice in cold distilled water.
	16. As previously, select "multimer" to incubate the slides with 37. Apply 200 μL of DAPI solution (at 1 μg/mL in TBS 1×) for 100 μL of ready-to-use OmniMap goat anti-rabbit coupled to HRP for 12 min. nucleus staining. Incubate at RT for 12 min (
	17. Repeat steps 7-9.
	18. Dilute both tyramide component coupled to Alexa Fluor 594
	(100×) and Solution A at 1/100 in 1× TBS to prepare Solution
	C (see Note 9).
	19. Incubate each slide with 200 μL of Solution C at RT for
	10 min.
	20. Wash the slides twice in cold distilled water.
	21. Put the slides in the Ventana autostainer.
	22. Select dual sequence and apply antibody denaturation at 90 °C
	during 12 min.
	23. Select "primary antibody" to cover the tissue with 100 μL of
	ready-to-use mouse anti-human CD4 (clone SP35) at 37 °C
	for 12 min.

Table 1 and

 1 Note 19).

	16. Centrifuge the cells for 8 min at 350 g at +4 °C.
	17. Discard the supernatant and resuspend the cells in 150 μL of
	the viability marker solution (Zombie Yellow diluted in 1×
	PBS at 1/400).
	18. Transfer 50 μL of the cell suspension in 96-well plate wells
	containing either antibody mix, isotype controls, or staining
	buffer only.
	19. Incubate for 30 min at +4 °C.

  Follicular Helper T Cells Detection Within Tumors 20. Wash the cells with 100 μL of PBS in each well and centrifuge for 5 min at 400 g at +4 °C.21. Discard the supernatant and resuspend the cells in 50 μL of fixation/permeabilization buffer per well.

	22. Incubate for 10 min at +4 °C.
	23. Centrifuge for 5 min at 400 g at +4 °C.
	24. Remove the supernatant. Proceed to intranuclear staining by
	incubating the cells with antibody mix or isotype controls pre-
	viously prepared in 50 μL of permeabilization buffer (Table 1).
	25. Incubate for 20 min at 4 °C in the dark.
	26. Wash the cells twice with 100 μL of permeabilization buffer at
	400 g for 5 min.
	27. Discard the supernatant.
	28. Resuspend the cells with 150 μL staining buffer and transfer
	into Micronic tubes.
	29. Rinse the tubes with 150 μL staining buffer and transfer into
	the same Micronic tubes.
	30. Flow cytometry data are acquired with a Fortessa flow cytom-
	eter (BD Biosciences) within 2 days after staining and ana-
	lyzed using the FlowJo software (Tree Star).

Table 1 Antibodies for detecting TLS in instillated lungs (by immunohistochemistry)

 1

				TLS in Chronically Infected Lungs
				Working	Ag	Secondary
	Specificity	Supplier	Clone	dilution	unmasking	antibody
	CD20 (B cell)	Santa Cruz	Sc-7735	1:100	-	Goat
	CD3 (T cell)	Novus	SP7	1:50	Citrate	Rabbit
		Biological
	CD21 (follicular	Abcam	Ab75985 1:200	Citrate	Rabbit
	dendritic cell)
	PCNA (germinal	Calbiochem	247*261 1:200	Protease	Rabbit
	center)
	PMN a	Santa Cruz	Sc-71674 1:200	Protease	Rat
	PNAd b (HEV)	BD	BD553863 1:50	Citrate	Rat
		Pharmingen
	F4/80	Santa Cruz	Sc-71,085 1:100	Protease	Rat
	(macrophages)

a PMN Polymorphonuclear cells (neutrophils) b PNAd Peripheral node addressin (staining of high endothelial venules, HEV)

  Resuspend in 80 μL cold Awesome MACS buffer and 20 μL CD45 MicroBeads per 10 7 cells. Mix by inverting the 15 mL conical tube and incubate on ice for 15 min. Centrifuge for

	3. Pass suspension through MACS Smart Strainer into a new
		15 mL conical tube. Wash digestion tube with 5 mL cold har-
		vest buffer and pass through filter. Centrifuge for 5 min at
		600 × g at 4°C.
	4. Aspirate supernatant, resuspend in 5 mL cold harvest buffer,
		centrifuge for 5 min at 600 × g at 4°C, and aspirate
		supernatant.
	5. Resuspend in 2 mL Red Blood Cell Lysing Buffer Hybri-Max.
		Incubate for 1 min at 37°C.
	6. Add 8 mL cold harvest buffer, centrifuge for 5 min at 600 × g
		at 4°C, and aspirate supernatant. Repeat this step twice.
	7. Resuspend in 5 mL cold Awesome MACS buffer (see Note 8)
	Melanoma by	and count viable cells. A typical cell yield for a B16-OVA tumor
	Immunofluorescence	is 5 × 10 7 . Centrifuge for 5 min at 600 × g at 4°C and aspirate
		supernatant.
	8.

  5 min at 600 × g at 4°C and aspirate supernatant. Resuspend in cold Awesome MACS buffer to 2 × 10 8 cells/mL. Proceed immediately to magnetic separation. 9. Rinse LS column in a MACS manual magnetic separator with 3 mL of cold Awesome MACS buffer. 10. Pipette cell suspension onto column and allow to flow by gravity. Collect CD45 Neg cells that flow through into a new 15 mL conical tube. Rinse column with 3 mL of cold Awesome MACS buffer three times, collecting CD45 Neg cells that flow though into same collection tube. Count viable cells, cap tube, and keep on ice. A typical cell yield of CD45 Neg cells from a B16-OVA tumor is 4 × 10 7 . 11. Remove column from magnetic separator and insert into a new 15 mL conical tube. Apply 3 mL of cold Awesome MACS buffer and firmly flush out CD45 Pos cells with supplied plunger. Count viable cells, cap tube, and keep on ice. A typical cell yield of CD45 Pos cells from a B16-OVA tumor is 1 × 10 7 . 12. Centrifuge CD45 Neg cells for 5 min at 600 × g at 4°C. Aspirate supernatant, and resuspend in cold Awesome MACS buffer at 10 7 cells/mL. Add biotinylated anti-podoplanin and/or anti-CD31 (Table 2) to a final concentration of 0.5 μg/mL. Incubate on ice for 30 min. Centrifuge for 5 min at 600 × g at 4°C and aspirate supernatant. 13. Resuspend CD45 Neg cells in 80 μL cold Awesome MACS buffer and 20 μL of anti-biotin MicroBeads per 10 7 cells. Mix by inverting the tube and incubate for 15 min on ice. Centrifuge for 5 min at 600 × g at 4°C and aspirate supernatant. Resuspend in cold Awesome MACS buffer to 2 × 10 8 cells/mL. 14. Repeat steps 9-11 with a new LS column using these CD45 Neg cells as input. Discard cells that flow through while the column is in the magnetic separator, and collect the podoplanin + and/ or CD31 + cells that are eluted with the plunger after the column is removed from the separator. 15. Centrifuge the CD45 Pos cells collected in step 11 and enriched stromal fraction collected in step 14 for 5 min at 600 × g at 4°C. Aspirate supernatant and resuspend in 5 mL of cold Awesome MACS buffer. Centrifuge for 5 min at 600 × g at 4°C and aspirate supernatant. 16. Resuspend CD45 Neg cells in 100 μL cold Awesome MACS buffer and pipet entire suspension to a 96-well plate. Resuspend CD45 Pos cells in 1 mL cold Awesome MACS buffer and pipet 100 μL (1 × 10 6 cells) to same 96-well plate. Centrifuge for 5 min at 600 × g at 4 °C and aspirate supernatant. 17. Resuspend both fractions in 100 μL cold Awesome MACS buffer containing 0.5 μg/mL of anti-CD16/32. Incubate for TLS in Murine Melanoma 15 min at 4°C, centrifuge for 5 min at 600 × g at 4 °C, and aspirate supernatant. 18. Resuspend CD45 Pos cells in 100 μL cold Awesome MACS buffer with CD45, CD3, CD8α, and CD19 antibodies, and the CD45 Neg cells in 100 μL of cold awesome MACS buffer with CD45, Ter119, CD31, and gp38 antibodies (Table 3). Incubate for 30 min at 4°C, centrifuge for 5 min at 600 × g at 4 °C, and aspirate supernatant. 19. Resuspend both fractions in 5 mL cold Awesome MACS buffer. Centrifuge for 5 min at 600 × g at 4°C and aspirate supernatant. Repeat this step twice. 20. Resuspend both fractions in 1 mL cold Awesome MACS buffer containing 0.2 μg/mL DAPI. Incubate for 30 min at 4°C, and analyze by flow cytometry.

  1. Follow steps in Subheading 3.1 to grow intraperitoneal or subcutaneous B16-OVA melanoma cells in C57BL/6 mice. 2. Eighteen hours prior to tumor harvest, anesthetize one or more (see Note 11) congenic Thy1.1 mice by intraperitoneal injection of 25 μL tribromoethanol working solution per gram of mouse weight. Euthanize by cervical dislocation and dissect lymph nodes (axillary, brachial, inguinal, cervical, and mesenteric) and spleen with surgical scissors and forceps. Pool tissues into an autoclaved Dounce homogenizer containing 5 mL of cold CTL medium and homogenize to create a single-cell suspension. . Resuspend in 2 mL Red Blood Cell Lysing Buffer Hybri-Max and incubate for 1 min in a 37°C water bath. Add 8 mL cold CTL medium, centrifuge for 5 min at 600 × g at 4°C, and aspirate supernatant. 5. Resuspend in 5 mL cold Awesome MACS buffer, count viable cells, and adjust to 10 8 /400 μL. Add 100 μL of naïve CD8α T-cell biotin-antibody cocktail (supplied with kit) per 10 8 cells. Mix by inverting tube and incubate for 5 min on ice. 6. Add 200 μL cold Awesome MACS buffer, 200 μL anti-biotin Microbeads, and 100 μL of CD44 MicroBeads per 10 8 cells (both supplied with kit). Mix by inverting tube and incubate for 10 min on ice.

	Anthony B. Rodriguez et al.
	3.6 Cellular
	Characteristics of TLS
	Containing B16
	Melanoma Tumors
	by Flow Cytometry
	3.7 Adoptive
	Transfer of Naïve CD8 +
	T Lymphocytes
	into Mice
	with Established
	Tumors

3. Pipet suspension through MACS Smart Strainer into a 15 mL

conical tube. Wash homogenizer with 5 mL cold CTL medium and pipet through filter. Centrifuge for 5 min at 600 × g at 4°C and aspirate supernatant.

4

Table 1 Antibodies for detection of TLS in IF and FACS

 1 Collagenase IV stock (400×): 5 g Collagenase Type 4 (Worthington-Biochemical Corporation) diluted to 1.5 U/ mL (Collagenase IV ≥160 U/mg) in HEPES buffer. Store 100 μL aliquots at -20 °C. 12. DNase I stock (400×): 100 mg DNase I grade II (Roche) diluted to 8 mg/mL in 1× HBSS containing MgCl 2 and CaCl 2 . Store 50 μL aliquots at -20 °C. 13. Collagenase/DNase buffer: 1× HEPES buffer + 1× Collagenase IV and 1× DNase I (in 1× HBSS containing MgCl 2 and CaCl 2 ). Store at -20 °C. 14. Ammonium-chloride-potassium (ACK) lysis buffer. 15. LIVE/DEAD Fixable Dead Cell Stain kit (Thermo Fisher Scientific). 16. 20 mL Syringe and 23 gauge needle.

	Marker	Species Clone	Conjugate	Working dilution Supplier	Method
	CD3	Rabbit	Polyclonal Unconjugated 1:100	Abcam	IF
	B220	Rat	Polyclonal FITC	1:100	BioLegend	IF
	NKX2.1	Rabbit	Polyclonal Unconjugated 1:1000	Abcam	IF
	CD4	Rat	GK1.5	AF647	1:500	BioLegend	FACS
	CD8	Rat	53.6.7	AF421	1:200	BioLegend	FACS
	CD103	Hamster 2.00E+07 APC	1:100	eBioscience	FACS
	CD44	Rat	IM7	BV421	1:200	BD Biosciences FACS
	PD-1	Rat	29F.1A12 AF421	1:200	BioLegend	FACS
	CD45 (in vivo) Rat	30-F11	PECF594	1:200	Invitrogen	FACS

  ). Full information on this antibody is included in Table 1. 2. Euthanize mouse without performing cervical dislocation (see Note 15). 3. To reduce autofluorescence in FACS analysis, perform bronchoalveolar lavage (BAL): Open the peritoneal and thoracic cavity, and expose the trachea. Identify the wider opening between tracheal rings and snip using fine scissors (see Note 16). Insert needleless catheter attached to 3 mL syringe filled with 1 mL PBS, and inject PBS into trachea. Lungs should inflate. Use plunger to pull the liquid back into the syringe. While catheter remains inserted, detach syringe and discard liquid. Flush lungs with an additional 0.5-1 mL PBS and repeat this steps 2-3 times. . Remove mediastinal lymph node: Flip out the lungs and locate mediastinal lymph node, within the thoracic cavity, attached to the back of the trachea. Isolate with curved forceps and add it to a 15 mL conical tube with 5 mL RPMI-1% (see Note 18). 6. Isolate lungs and place lobes for FACS into gentleMACS C-Tubes containing 2.5 mL Collagenase/DNase buffer (see Note 19). 7. Isolate distant non-mediastinal lymph node for staining controls and add to 15 mL conical tube containing 5 mL RPMI-1%. 8. Place samples on ice and stain as soon as possible following isolation. 1. Place gentleMACS C-Tubes containing lungs and Collagenase/ DNase buffer in the MACS dissociator and run on gen-tleMACS program m_Lung_01. Repeat the program if samples get stuck during the first run.

	5

4. Perform cardiac lung perfusion, as previously described (see Subheading 3.1, step 1) (see Note 17). 2. Incubate lung tissue inside gentleMACS C-Tubes at 37 °C on a shaker for 30 min.

4.3.2 Installation of FIJI and Required Plug-insIHC-based TLS Visualization and Quantification

Emma Jones et al.

Chunyan Gu-Trantien et al.

Monoclonal Antibodies from RA Synovial Tissue

Priyanka Devi-Marulkar et al.

TLS in Chronically Infected Lungs

Tumor-Associated Tertiary Lymphoid Structures

Kelli A. Connolly et al.

Acknowledgments

F.B. is an ARUK Senior Fellow and is part of the NIHR Birmingham Biomedical Research Centre.

Acknowledgments

We would like to thank D. Preston for editing the manuscript and L. Louveau for the artwork. Funding: This work was supported by LE&RN Postdoctoral Fellowship Award to A.L.

Acknowledgement

We thank Dr. Tze Heng Tan (Definiens AG, Munich, Germany) for providing the virtual IF rendering included, as Fig. 3.

Acknowledgments

We thank all present and past members of the Laboratory "Cancer, Immune control and Escape" (UMRS1138 INSERM, Cordeliers Research Center), pathologists, and clinicians who have participated in these studies, for their help and valuable discussions. We are grateful to lung cancer patients involved in this research performed in our laboratory. Funding: This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Pierre and Marie Curie University, Paris-Descartes University, the Labex Immuno-Oncology (LAXE62_9UMRS872 Fridman), CARPEM (Cancer Research for PErsonalized Medicine), Fondation ARC pour la Recherche sur le Cancer. Jeremy Goc was supported by a grant from the Fondation ARC pour la Recherche sur le Cancer.

Acknowledgements

The authors thank the Department of Anatomical Pathology, Institut Jules Bordet, Brussels, Belgium, for use of the IHC staining equipment and Mrs. Anais Boisson for excellent technical assistance.

Acknowledgments

We thank all present and past members of the Laboratory "Cancer, Immune control and Escape" (UMRS1138 INSERM, Cordeliers Research Center), pathologists, and clinicians who have participated in these studies, for their help and valuable discussions. We are grateful to lung cancer patients involved in this research performed in our laboratory. Funding: This work was supported by the "Institut National de la Santé et de la Recherche Médicale (INSERM), Pierre and Marie Curie University, Paris-Descartes University, the Labex Immuno-Oncology (LAXE62_9UMRS872 Fridman), CARPEM (Cancer Research for PErsonalized Medicine), Institut National du Cancer (INCa-PLBio 2014-2017 GDSTRESS), and Fondation ARC pour la Recherche sur le Cancer. Priyanka Devi-Marulkar was supported by a grant from the Fondation ARC pour la Recherche sur le Cancer. Hélène Kaplon was supported by a grant from the Ligue Nationale contre le Cancer.

Acknowledgements

We thank Professor Tyler Jacks (Massachusetts Institute of Technology) and Jacks lab members for their generous gift of reagents, advice, and time. Funding: This work was supported by the National Cancer Institute K22 Transition Career Award.

Funding

This work was supported by the Swiss National Science Foundation, the Sciex Foundation, the Science Foundation for Oncology, the Cancer League Zurich, the University Research Priority Program "Translational Cancer Research," and the Swiss Cancer League.

Funding

Hélène Kaplon was supported by a grant from La Ligue contre le Cancer. The work developed by B. Dubois's group was supported by the Ligue Contre le Cancer Comité de Savoie, Fondation ARC pour la recherche sur le cancer, and INCa (PRT-K16). Funding This work was supported by a grant from Association Vaincre la Mucoviscidose (RF201605016 22/1/2/51), by INSERM, and by Paris Descartes and Sorbonne Universities. Funding This work was supported by the United States Public Health Service (USPHS) grants R01 CA78400 and R01 CA181794 (V.H.E.). Additional support was provided by USPHS grant P30 CA0044579 to the University of Virginia Cancer Center. A.B.R. was supported by USPHS training grant AI007496 and the Wagner Fellowship. J.D.P. was supported by USPHS training grant GM007276 and the Farrow Fellowship.

Funding Y.X.F. holds the Mary Nell and Ralph B. Rogers Professorship in Immunology. This work was in part supported by the US National Institutes of Health through National Cancer Institute grants CA141975 and CA97296, CPRIT grant RR150072, grants from the Chinese Academy of Sciences (XDA09030303), and the Chinese Ministry of Science and Technology (2012ZX10002006, 2011DFA31250, and 2012AA020701) to Y.X.F.

Abbreviations

Bcl-6 B-cell lymphoma 6 CXCL13 C-X-C motif chemokine

Introduction

Follicular helper T (Tfh) cells constitute a CD4 + T-cell subset dedicated to provide T-cell help to B cells and mainly found within germinal centers (GC) of B-cell follicles. Tfh cells support GC reactions and provide critical signals to B cells allowing antibody isotype switching, antibody affinity maturation, and B-cell differentiation into memory B cells and plasma cells (PC) [1]. GC Tfh cells are characterized by high surface expression of CXCR5 (C-X-C chemokine receptor type 5), PD-1 (programmed death-1), and inducible costimulator (ICOS); expression of the transcription factor Bcl-6 (B-cell lymphoma 6) in the nucleus; and secretion of IL-21, IL-4, and CXCL13 (C-X-C motif chemokine 13). The expression of CXCR5 by Tfh cells allows their homing to the B-cell follicle in response to gradients of CXCL13. In solid tumors, Tfh cells are detected in lymph node-like structures with segregated T-and B-cell zones, called tertiary lymphoid structures (TLS) [2,4]. The presence of these cells in tumorassociated TLS may suggest an ongoing local B-cell activation process leading to the differentiation of specific memory B cells and PC, whose presence predicts better patient survival in many solid cancer types [2][3][4][5]. Recent studies highlighted that the presence of a Tfh gene signature was associated with better clinical outcome in several cancer types, including breast cancer [6], colorectal cancer [7], and non-small cell lung cancer (NSCLC) [8]. Consistently, a higher risk of relapse has been observed in patients with CXCL13 deletion in colorectal cancer, probably due to lower densities of Tfh cells and B cells [7]. These findings strongly suggest that B-cell differentiation supported by tumorinfiltrating Tfh cells is a critical component of efficient antitumor immunity.

If the presence of TLS has been clearly correlated with a better prognosis in terms of survival in many solid tumors [9], the mechanisms of TLS neogenesis however are still unclear. Several factors seem to regulate their formation such as TNF family members (lymphotoxin [10,11], LIGHT [12], BAFF [13]), cytokines (IL-17 [14], IL-6 [15], IL-22 [16]), and chemokines (CCL19/ CCL21 [17,18], CXCL13 [19,20]). In this context, Tfh cells Coline Couillault et al. 9. Naïve CD8 + T cell isolation kit (Miltenyi Biotec).

Preparation of Tumor Tissue

10. LS columns (Miltenyi Biotec).

11. MACS manual magnetic separator (Miltenyi Biotec).

12. Heating lamp with a 250 W (infrared) bulb.

13. Tailveiner restrainer (BrainTree Scientific, Inc.).

14. 1 mL Tuberculin syringes pre-attached with a 25 G × 5/8 in. needle.

Methods

1. Culture B16-OVA melanoma cells in complete medium in a T75 flask to 60-70% confluency.

2. Aspirate supernatant, rinse flask with 5 mL room-temperature trypsin-EDTA solution, and aspirate again. Add 5 mL trypsin-EDTA solution and tilt to cover the bottom of the flask. Incubate for 2-3 min at 37 °C, or until cells have fully detached. PNAd + vasculature and small numbers of B220 + B lymphocytes.

Implantation of Subcutaneous and Intraperitoneal Murine Melanoma Tumors

T lymphocytes and CD11c

+ antigen-presenting cells intermingled with B lymphocyte aggregates (Fig. 1i-l).

3. A co-extensive reticular network of podoplanin + fibroblasts (Fig. 2a-d).

4. Additional characteristics of TLS in B16-OVA tumors are shown in Fig. 2e-h. 2. Aspirate digestion buffer from weigh boat and mince tumor with scissors into ~1 mm 3 pieces. Flush minced tumor into a 15 mL conical tube with 5 mL of working buffer (see Note 9). Incubate for 30 min in a 37°C water bath. Pipette suspension gently at 5-min intervals to break up aggregates (see Note 10).

Preparation, Enrichment, and Cell Surface Marker Staining of Tumor-Associated Hematopoietic and Stromal Cells for Flow Cytometry

Fig. 1 B16-OVA tumors grown in the subcutaneous (a-d) or intraperitoneal (e-l) cavities of C57BL/6 mice were harvested, histologically prepared, and stained for immunofluorescent microscopy, as outlined in Subheadings 3.1-3.3. Tumor sections were stained with antibodies of the indicated specificities (Table 1). 11. From this step forward, make sure that the sample never fully runs dry. Add 10-20 μL PBS to keep parts of samples hydrated, as needed. Add water to the bottom of black slide-staining trays and cover with lid to keep slides humidified and prevent drying.

12. Imaging sections with confocal microscopy is a labor-intensive protocol that takes about 1 month. We have optimized this process using EVOS FL Auto 2 and reduced the time required per lung lobe to 12 days maximum (see Fig. 2).

13. We have defined TLS as ≥20 aggregated B cells.

14. To ensure that analysis of intratumoral immune cells is not complicated by the presence of immune cells from circulation, we suggest that you first stain all circulatory CD45 + immune cells via i.v. injection of a fluorescently conjugated anti-CD45 antibody prior to euthanasia. In this way, immune cells present in tumor homogenates that were trapped in the vasculature at the time of tissue harvest can be excluded during data analysis (see Fig. 3). 20. To perform a simple hemocytometer count, pipette 10 μL of cell suspension into a tube containing 390 μL of trypan blue

Methods

1. In a 15 mL tube, add 10 mL pre-warmed complete DMEM medium.

2. Thaw the frozen cell line rapidly in a 37 °C water bath.

3. Transfer 1 mL of cells into pre-warmed complete DMEM medium.

4. Centrifuge at 180 × g for 3 min. Then, discard the supernatant. ���������������������� 1, 2, 4, 7, 8, 17, 18, 20-25, 72, 93, 95-104, 106, 108, 109, 112-114, 120, 126, 140, 152, 189, 190, 192, 195, 198, 199, 202, 223

Preparation of Tumor Cells Before Inoculation