P Bousso
email: capucine.grandjean@pasteur.fr

Immunomodulation under the lens of real-time in vivo imaging

Keywords: immunomodulation, intravital imaging, multiphoton microscopy, fluorescent reporters, immunotherapies

Modulation of cells and molecules of the immune system not only represents a major opportunity to treat a variety of diseases including infections, cancer, autoimmune and inflammatory disorders but could also help understand the intricacies of immune responses. A detailed mechanistic understanding of how a specific immune intervention may provide clinical benefit is essential for the rational design of efficient immunomodulators. Visualizing the impact of immunomodulation in real-time and in vivo has

emerged as an important approach to achieve this goal. In this review, we aim to illustrate how multiphoton intravital imaging has helped clarify the mode of action of immunomodulatory strategies such as antibodies or cell therapies. We also discuss how optogenetics combined with imaging will further help manipulate and precisely understand immunomodulatory pathways. Combined with other single-cell technologies, in vivo dynamic imaging has therefore a major potential for guiding preclinical development of immunomodulatory drugs.

Capturing the consequences of immunomodulation with in vivo multi-photon microscopy

The immune system is composed of an extensive network of specialized cells distributed throughout the body, ready to detect and eliminate intruding microbes or transformed cells and that can infiltrate tissues in case of sterile inflammation (e.g. myocardial infarction, stroke) [START_REF] Wang | Visualizing the function and fate of neutrophils in sterile injury and repair[END_REF]. The initiation and orchestration of immune responses is highly regulated in time and space, typically involving the sequential recruitment and activity of distinct cellular subsets. Over the years, strategies to modulate these stages have emerged with the aim to treat patients with a variety of diseases such as infections, cancer, autoimmune and inflammatory disorders. Thus, understanding the spatiotemporal components of immunomodulation is of critical importance and requires cells to be scrutinized in vivo within their physiological environment. To this aim, multi-photon microscopy is a technique of choice as it provides extensive information on single-cell dynamics in vivo (e.g. migration pattern or cellular interactions) and can thus be particularly resourceful to understand the immediate or longer-term consequences of immunomodulators. Importantly, intravital microscopy (IVM) may reveal unexpected cell behaviors or cellular crosstalk otherwise difficult to grasp in a classical "hypothesis-driven" approach. Two-photon microscopy uses high power pulsed laser and infrared wavelengths providing good tissue penetration, intrinsic optical sectioning, and reduced toxicity compared to other microscopy techniques [START_REF] Bousso | Functional immunoimaging: the revolution continues[END_REF]- [START_REF] So | Two-Photon Excitation Fluorescence Microscopy[END_REF]. Tracking of cells requires their fluorescent labelling and various approaches to meet this need exist. For instance, many fluorescent-reporter mice are available to track particular immune subsets such as T cells [START_REF] Schaefer | Observation of Antigen-Dependent CD8+ T-Cell/ Dendritic Cell Interactions in Vivo[END_REF], [START_REF] Singbartl | A CD2-Green Fluorescence Protein-Transgenic Mouse Reveals Very Late Antigen-4-Dependent CD8+ Lymphocyte Rolling in Inflamed Venules[END_REF], dendritic cells (DC) [START_REF] Lindquist | Visualizing dendritic cell networks in vivo[END_REF], macrophages [START_REF] Burnett | Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene[END_REF], [START_REF] Ovchinnikov | Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP doubletransgenic mice[END_REF], natural killer (NK) cells [START_REF] Gazit | Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1[END_REF], [START_REF] Beuneu | Dynamic behavior of NK cells during activation in lymph nodes[END_REF] or neutrophils [START_REF] Faust | Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages[END_REF], [START_REF] Stackowicz | Mouse Models and Tools for the in vivo Study of Neutrophils[END_REF]. To add a level of complexity to our understanding, biosensors (e.g. cell death sensors, calcium sensors, cytokine reporters, phagocytosis reporter etc.) allow cell behavior and functional outcome to be linked [START_REF] Grandjean | Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis[END_REF]- [START_REF] Thestrup | Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes[END_REF]. Moreover, adoptive transfer of fluorescent cells is another strategy to recapitulate some immune responses and is typically used to study cell therapies in vivo (e.g. tumor-specific T cells or CAR T cells) [START_REF] Boulch | A cross-talk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity[END_REF]- [START_REF] Boulch | Tumor Immunosurveillance and Immunotherapies: A Fresh Look from Intravital Imaging[END_REF]. Alternatively, injecting a monoclonal fluorescent antibody (Ab) targeting a specific cell marker can stain a population of interest within minutes in vivo, allowing to quickly evaluate its behavior [START_REF] Grandjean | Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis[END_REF], [START_REF] Yipp | Antibodies against neutrophil LY6G do not inhibit leukocyte recruitment in mice in vivo[END_REF]. However, potential caveats of this method are limited diffusion of Abs in some organs/tissues and possible artifacts generated by unspecific Ab bindings or Fc receptors engagement. The use of fluorescent Fab fragments can somehow limit some of these issues [START_REF] Moreau | Dynamic In Situ Cytometry Uncovers T Cell Receptor Signaling during Immunological Synapses and Kinapses In Vivo[END_REF]. Additionally, important features of tissue architecture can be visualized through imaging of collagen fibers by second harmonic generation or by staining vasculature through intravenous (i.v.) injection of dyes or antibodies [START_REF] Fukumura | Tumor Microvasculature and Microenvironment: Novel Insights Through Intravital Imaging in Pre-Clinical Models[END_REF]. In particular, visualizing blood vessels can provide information on the ins and outs of trafficking cells. One major advantage of IVM is the possibility to image the exact same location within a tissue before and immediately after the injection of an immunomodulator allowing the early timepoints to be characterized (Figure 1). Altogether, IVM is a powerful technique to track multiple cell populations and cell functions in real time before (at steady state) during and after immunomodulation. Such approach should help unravel new mechanisms of action and suggest means to optimize therapeutic efficacy.

Removal of pathological cells using therapeutic antibodies

Using monoclonal antibodies (mAb) to induce the elimination of malignant or autoreactive cells has proven to be a very effective strategy for patients with cancer or autoimmune disorders [START_REF] Pierpont | Past, Present, and Future of Rituximab-The World's First Oncology Monoclonal Antibody Therapy[END_REF]. A classical example is the clinical use of anti-CD20 mAbs (rituximab, obinutuzumab) for treating B cell malignancies (lymphomas, leukemia) or removing self-reactive B cells (for example in the context of multiple sclerosis or rheumatoid arthritis) [START_REF] Pierpont | Past, Present, and Future of Rituximab-The World's First Oncology Monoclonal Antibody Therapy[END_REF], [START_REF] Salles | Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience[END_REF]. Such depleting antibodies are designed to trigger cell elimination by either activating the complement pathway [START_REF] Cragg | Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts[END_REF], [START_REF] Middleton | Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL[END_REF] (figure 2) or triggering antibodydependent cell cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP) [START_REF] Pierpont | Past, Present, and Future of Rituximab-The World's First Oncology Monoclonal Antibody Therapy[END_REF], [START_REF] Uchida | The Innate Mononuclear Phagocyte Network Depletes B Lymphocytes through Fc Receptordependent Mechanisms during Anti-CD20 Antibody Immunotherapy[END_REF]. ADCC and ADCP occur following antibody Fc-recognition by effector cells such as NK, macrophages or neutrophils.

In vitro studies have shown that certain anti-CD20mAb (e.g. obinutuzumab) may be capable of inducing direct cell death, a mechanism known as antibody dependent cell death (ADCD) [START_REF] Tobinai | A Review of Obinutuzumab (GA101), a Novel Type II Anti-CD20 Monoclonal Antibody, for the Treatment of Patients with B-Cell Malignancies[END_REF], [START_REF] Honeychurch | Antibody-induced nonapoptotic cell death in human lymphoma and leukemia cells is mediated through a novel reactive oxygen species-dependent pathway[END_REF]. Yet, the respective contribution of these mode of action (MOA) in distinct anatomical sites remains to be ascertained as they are likely influenced by the type, numbers and phenotype of available immune effectors. Using IVM, depletion of circulating healthy and malignant B cells was visualized in the liver within minutes of anti-CD20 mAb injection as Kupffer cell arrested and engulfed targets through ADCP [START_REF] Montalvao | The mechanism of anti-CD20-mediated B cell depletion revealed by intravital imaging[END_REF]. It was highly efficient as nearly 60% of the circulating B cell compartment was depleted within 2 hours. Kupffer-cell mediated ADCP was also reported in a melanoma and colon tumor mouse model using TA99 (anti-gp75 mAb) or Cetuximab (anti-hEGFR mAb) therapies respectively [START_REF] Gül | Macrophages eliminate circulating tumor cells after monoclonal antibody therapy[END_REF]. These additional models suggest that Kupffer-mediated ADCP might be a common MOA for mAbs depleting circulating cell targets. To boost effector functions and thus therapeutic efficacy, glycomodifications can be applied to the Fc-portion of the mAb [START_REF] Herter | Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity[END_REF]. IVM of two glycoengineered mouse and human anti-CD20 mAbs revealed their superior abilities to trigger Kupffer cell-mediated ADCP compared to their wildtype counterpart [START_REF] Grandjean | Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies[END_REF]. Additionally, Fc modifications may also favor alternative MOA in vivo. By performing IVM of the liver after injection of a glycosylated anti-CXCR3 mAb, Kupffer cells were seen inducing liver iNKT cell apoptosis following nibbling of large cellular fragments, a phenomenon abrogated in Fc-deficient mice or when using a non-glycosylated anti-CXCR3 mAb [START_REF] Liew | Antibody-dependent fragmentation is a newly identified mechanism of cell killing in vivo[END_REF]. Beyond the elimination of circulating targets, ADCP has also been identified in the bone marrow of two independent mouse tumor models using anti-CD20 or anti-CD52 mAbs. In this instance, ADCP was largely limited in time and space (Figure 2) with only a small fraction of tumor B cells being eliminated after antibody injection [START_REF] Grandjean | Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis[END_REF], [START_REF] Pallasch | Sensitizing Protective Tumor Microenvironments to Antibody-Mediated Therapy[END_REF]. This bottleneck was attributed to the largely sessile phenotype of bone-marrow associated macrophages and their reduced presence in tumor-bearing animals [START_REF] Grandjean | Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis[END_REF], [START_REF] Pallasch | Sensitizing Protective Tumor Microenvironments to Antibody-Mediated Therapy[END_REF]. Importantly, this limitation could be circumvented by chemotherapy that triggered macrophage recruitment and enhanced ADCP following anti-CD52 mAb therapy [START_REF] Pallasch | Sensitizing Protective Tumor Microenvironments to Antibody-Mediated Therapy[END_REF]. Macrophages seemed to also undergo a phase of hyporesponsiveness as phagocytosis events were no longer detected a few hours following antibody injection despite high levels of circulating antibody [START_REF] Grandjean | Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis[END_REF]. This additional limitation may be the consequence of FcRs downregulation following target engulfment [START_REF] Pinney | Macrophage hypophagia as a mechanism of innate immune exhaustion in mAb-induced cell clearance[END_REF]. Finally, therapeutic activity in tumor-bearing mice was also limited at later time points possibly due to antibody consumption by tumor cells [START_REF] Grandjean | Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis[END_REF].

Altogether, mAbs designed to induce Fc-dependent cell elimination may face different bottlenecks depending on anatomical features, effector density and responsiveness, antibody diffusion and consumption. By documenting limitations in antibody-mediated mechanisms, IVM can guide the rational design of future therapeutical strategies and their optimal use in clinical settings.

Elimination of suppressive and deleterious immune cells

Targeting suppressive immune cells such as regulatory T cells (Tregs), neutrophils or immunesuppressive macrophages is an attractive strategy to boost immune reactions typically in the context of established tumors. Several studies have visualized the impact of Treg depletion in various mouse models and have identified distinct mechanisms of downregulation of immune responses in vivo. In this respect, IVM of lymph nodes (LNs) showed that Tregs could directly interact with DCs or with DC:T cell clusters in a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)-dependent manner to dampen T cell priming [START_REF] Matheu | Imaging regulatory T cell dynamics and CTLA4-mediated suppression of T cell priming[END_REF], [START_REF] Tang | Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice[END_REF]. Similarly, the elimination of Tregs boosted T cell priming by increasing contact duration between DC and T cells [START_REF] Pace | Regulatory T cells increase the avidity of primary CD8+ T cell responses and promote memory[END_REF], [START_REF] Tadokoro | Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo[END_REF]. Imaging showed that Tregs could also physically impede cytotoxic T lymphocytes (CTLs) infiltration in the tumor by forming a ring-like structure around malignant B16 melanoma cells [START_REF] Qi | Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy[END_REF]. As a consequence, Tregs elimination with chemotherapy, improved CTLs infiltration and increased interactions with tumor cells [START_REF] Qi | Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy[END_REF].

Macrophages are also endowed with immunomodulatory functions and represents an interesting cell population to target and tune immune responses. IVM has helped assess their roles and the consequences of their elimination. For example, IVM of a mammary mouse tumor showed that macrophages could directly interact with CTLs thus mechanically restraining them from infiltrating the tumor mass [START_REF] Peranzoni | Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment[END_REF]. As a result, depletion of macrophage following pexidartinib, a colony stimulating factor 1 receptor (CSF1R) inhibitor, improved CTLs number and infiltration [START_REF] Peranzoni | Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment[END_REF].

Neutrophils are rapidly recruited to tissues and represent another first-line cell candidate to target to modulate immune responses. For example, in a mouse model of leishmania infection, neutrophils were visualized engulfing Leishmania major (L.m.) parasites, which remained viable and which in turn promoted infection. In this instance, neutrophils depletion slowed down Leishmania infection [START_REF] Peters | In Vivo Imaging Reveals an Essential Role for Neutrophils in Leishmaniasis Transmitted by Sand Flies[END_REF]. An extensive review on the importance of IVM in dissecting how neutrophils contribute to modulate immune responses has been recently published elsewhere [START_REF] De Filippo | The Secretive Life of Neutrophils Revealed by Intravital Microscopy[END_REF].

Altogether, direct visualization of these cell subsets in vivo has helped understand how they exert their immunomodulatory functions providing additional clues into their therapeutic activities.

Modulation of T cell activity with immune checkpoint inhibitors

Sustained stimulation of T cells during viral infection or cancer often results in immune exhaustion [START_REF] Robert | A decade of immune-checkpoint inhibitors in cancer therapy[END_REF]. This phenomenon is classically characterized by the loss of functional capacities and the upregulation of negative regulatory molecules such as Programmed cell death protein 1 (PD-1) or CTLA-4. Immune checkpoints blockade (ICB) used to treat cancer patients include monoclonal antibodies that target PD-1 (nivolumab, pembrolizumab, cemiplimab), PD-L1 (atezolizumab, avelumab, durvalumab) or CTLA-4 (Ipilimumab) molecules with the strategy to unleash T cell cytotoxic functions. [START_REF] Robert | A decade of immune-checkpoint inhibitors in cancer therapy[END_REF]. Yet, what factors contribute to their efficacy and how they finely modulate the dynamics of immune responses in vivo remains unclear.

Several imaging studies have highlighted their possible role in modulating T cell motility. In an infectious model of lymphocytic choriomeningitis virus (LCMV), virus-specific T cells that had arrested in the splenic red pulp after chronic exposure to viral antigen, regained motility upon PD-1 and PD-L1 treatment [START_REF] Zinselmeyer | PD-1 promotes immune exhaustion by inducing antiviral T cell motility paralysis[END_REF]. This phenotype correlated with increased T cell function and IFN-g production [START_REF] Zinselmeyer | PD-1 promotes immune exhaustion by inducing antiviral T cell motility paralysis[END_REF].

Similarly, in B16 melanoma and 4T1 breast tumor models, intratumoral tumor-specific T cells increased their velocity upon anti-CTLA-4 mAb injection [START_REF] Pentcheva-Hoang | Cytotoxic T Lymphocyte Antigen-4 Blockade Enhances Antitumor Immunity by Stimulating Melanoma-Specific T-cell Motility[END_REF], [START_REF] Ruocco | Suppressing T cell motility induced by anti-CTLA-4 monotherapy improves antitumor effects[END_REF]. In other contexts, ICB appeared to stabilize interactions between T cells and their targets. Indeed, in a mouse model of allogeneic hematopoietic stem cell transplantation, anti-PD-1 mAb treatment favored the graft versus leukemia effect in lymph nodes by promoting stable interactions between T cells and their targets [START_REF] Michonneau | The PD-1 Axis Enforces an Anatomical Segregation of CTL Activity that Creates Tumor Niches after Allogeneic Hematopoietic Stem Cell Transplantation[END_REF]. These effects are consistent with the idea that PD-1-PD-L1 interactions inhibit TCR induced stop signal [START_REF] Fife | Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal[END_REF]. These different studies suggest that ICB can have multiple impact on T cell dynamics highlighting the need to dissect their MOA in specific settings. The impact of anti-PD1 has also been visualized by IVM in a colon carcinoma model using cytokine reporter mice [START_REF] Garris | Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12[END_REF]. These imaging experiments revealed the induction of IFN-g and IL-12 at the tumor site reflecting the importance of cellular crosstalks between tumor infiltrating lymphocytes (TILs) and DCs essential for therapeutic activity [START_REF] Garris | Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12[END_REF]. Finally, IVM can help pinpoint in vivo bottlenecks of anti-PD-1 therapy. For example, Fc-mediated uptake of anti-PD-1 mAb by neighboring tumor-associated macrophages was found to limit the effect of antibody therapy on TILs in mice bearing MC38 tumors [START_REF] Garris | Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12[END_REF]. Notably, blocking FcRIIB improved anti-tumor T cell responses.

Importantly, removing glycans present on the anti-PD1 mAb Fc portion also improved antibody activity [START_REF] Arlauckas | In vivo imaging reveals a tumor-associated macrophage-mediated resistance pathway in anti-PD-1 therapy[END_REF]. Altogether, these imaging observations may contribute to the rational design of future therapeutics. New checkpoint inhibitors targeting other inhibitory proteins (e.g. Tim 3) and/or other immune cells have gradually emerged [START_REF] Tian | Targeting Tim-3 in Cancer With Resistance to PD-1/PD-L1 Blockade[END_REF] and should benefit from future IVM studies to detail their MOA in vivo. For instance, anti-CD47mAbs which prevent interactions between SIRPa-expressing macrophages and CD47-expressing tumor cell are thought to unleash the break on phagocytosis but characterizing the precise in vivo impact of this strategy remains to be performed [START_REF] Tseng | Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response[END_REF]- [START_REF] Advani | CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma[END_REF].

Immunomodulation by bispecific antibodies Bispecific antibodies have been recently developed with the strategy to bring effectors and target cells into contact to promote cell elimination. In particular, BiTEs (bispecific T cell engager) consist of two linked single-chain variable fragments (scFvs), recognizing a T cell-specific CD3 subunit at one end and a tumor-associated antigen (TAA) at the other end. Blinatumomab, a bispecific T cell engager targeting CD3 and CD19 is now used for the treatment of B cell acute lymphoblastic leukemia and several clinical trials are ongoing to broaden up the use of bispecific antibodies to the treatment of solid tumors [START_REF] Wu | Recent advances and challenges of bispecific antibodies in solid tumors[END_REF].

In a humanized mouse model of colon carcinoma, IVM showed that a bispecific antibody targeting the CEA antigen and the CD3 protein increased the effector to target ratio by crosslinking multiple T cells to a single tumor target [START_REF] Lehmann | In Vivo Fluorescence Imaging of the Activity of CEA TCB, a Novel T-Cell Bispecific Antibody, Reveals Highly Specific Tumor Targeting and Fast Induction of T-Cell-Mediated Tumor Killing[END_REF]. Similarly, an anti CD3xCD20 T cell bispecific antibody increased stable synapses between human T cell and tumor cells [START_REF] Cremasco | Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model[END_REF]. In the PyMT mouse model of breast cancer, tracking of a fluorescent BiTE directed against epidermal growth factor receptor (EGFR)vIII revealed that BiTE anti-tumor activity was highly dependent on antibody dose and TAA expression level. In addition, CD103 + DCs appeared necessary to maximize therapy efficacy by enhancing CTL expansion [START_REF] You | Visualizing Spatial and Stoichiometric Barriers to Bispecific T-cell Engager Efficacy[END_REF]. Thus far, only few studies have undertaken real time visualization of bispecific antibodies in vivo and future work will undoubtedly bring important insights on their MOA and possible limitations. In a model of solid tumor, CTL killing was relatively inefficient with an average of one target eliminated every 6 hr [START_REF] Breart | Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice[END_REF]. Reflecting this observation, tumor regression required a high density of intratumoral effector T cells [START_REF] Breart | Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice[END_REF]. By contrast, CAR T cells appeared more rapid at killing lymphoma B cells in the bone marrow, a process that took on average only 25 minutes [START_REF] Cazaux | Single-cell imaging of CAR T cell activity in vivo reveals extensive functional and anatomical heterogeneity[END_REF], [START_REF] Boulch | A cross-talk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity[END_REF]. Yet, CAR T cells were heterogenous in their capacity to induce cell death in part due to the differential capacity to recognize their targets as recorded by calcium (Ca 2+ ) signaling . In melanoma-bearing mice, CTL interactions with tumor cells could be prolonged when combined with an anti-CD137mAb resulting in a more efficient induction of tumor cell apoptosis [START_REF] Weigelin | Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb[END_REF].

Related to the efficiency of CTL killing is the capacity of tumor cells to resist CTL attack. In vitro studies had initially provided evidence that tumor cells were capable of avoiding CTL-mediated killing by rapidly repairing membrane damages [START_REF] Khazen | Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse[END_REF]. Such sublethal hits result in a transient increase of intracellular Ca 2+ due to pore formation and a rapid return to basal levels after repair. The use of tumor cells expressing a Ca 2+ reporter confirmed that most CTL hits delivered in vivo were indeed sublethal [START_REF] Khazen | Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo[END_REF]. Of note, sublethal hits by multiple T cells can have additive effects, favoring target cell apoptosis [START_REF] Weigelin | Cytotoxic T cells are able to efficiently eliminate cancer cells by additive cytotoxicity[END_REF], [START_REF] Halle | In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity[END_REF].

Furthermore, CTL activity can be largely modulated by the environment, as shown in a mouse model of graft versus leukemia effect, where CTL interaction with targets was specifically inhibited in lymph nodes but not in the liver due to differential PD-L1/PD-L2 expression [START_REF] Michonneau | The PD-1 Axis Enforces an Anatomical Segregation of CTL Activity that Creates Tumor Niches after Allogeneic Hematopoietic Stem Cell Transplantation[END_REF]. Beyond enhancing the rate of target cell killing by effectors, ACT can profoundly modulate the host immune response. For instance, IFNg production by CAR T cells can drive the recruitment and activation of endogenous T and NK cells and sustain CAR T cells cytotoxicity [START_REF] Boulch | A cross-talk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity[END_REF]. Mechanisms of failure of ACT include the emergence of antigennegative tumor variants. In this respect, dual expression of anti-CD19 and anti-CD123 CARs limited tumor relapse in a xenograft B-ALL tumor model and IVM revealed that CAR T cells could still eliminate CD19 negative tumor cells through CD123 recognition [START_REF] Ruella | Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies[END_REF]. Another potential limitation of CAR T cells is the severe adverse events experienced by some patients including cytokine release syndrome and neurotoxicity [START_REF] Brudno | Toxicities of chimeric antigen receptor T cells: recognition and management[END_REF]- [START_REF] Fischer | CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities[END_REF]. Yet, mechanisms leading to neuronal dysfunction remain unclear. IVM was used to visualize brain capillaries and established that mice treated with CAR T cell therapy had disrupted blood flow in the brain due to plugged capillaries [START_REF] Faulhaber | Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy[END_REF]. Mechanical obstruction of brain capillaries was associated with abnormal behavior providing a possible mechanistic basis for neurotoxicity [START_REF] Faulhaber | Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy[END_REF].

While T cells have been the focus of most ACT, other immune cells may represent an interesting alternative. Intra-lymphatic adoptive transfer of CCR7-transduced regulatory macrophages (CCR7 + Mregs) could successfully migrate to the T cell zone of the lymph node where they reduced the time of interaction between antigen-specific T cells and DCs and attenuated delayed-type hypersensitization responses [START_REF] Yu | Targeted delivery of regulatory macrophages to lymph nodes interferes with T cell priming by preventing the formation of stable immune synapses[END_REF]. Other immune cells such as gd T cells, NK cells and macrophages are attractive candidates for CAR-based strategies and in vivo imaging approaches are expected to provide valuable information into their respective activities.

Immunomodulation by cytokines

Interfering with cytokine signaling represents an important immunomodulation strategy. However, IVM analysis of such immunomodulatory approaches remains challenging as altering cytokine networks may not have an immediate impact on immune cell dynamics or may have visible consequences only after a long period of time. Nevertheless, injection of chemokines can have rapid effects on immune cell behaviors as noted with injection of CCR7 ligands that promoted intranodal T cell motility [START_REF] Worbs | CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo[END_REF]. In a few instances, cytokines were seen rapidly altering cell behaviors. For example, liver-associated NKT cells arrested in liver sinusoids upon injection of IL12 and IL18 [START_REF] Velázquez | Cutting edge: activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of liver sinusoids[END_REF].

However, it is possible to dissect the effects of cytokine signaling by IVM more readily using appropriate fluorescent biosensors. For example, the use of an IFN-g-responsive reporter or that of a STAT1-GFP probe have revealed the long-range diffusion of IFN-g within the tumor microenvironment [START_REF] Thibaut | Bystander IFN-γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment[END_REF], [START_REF] Hoekstra | Long-distance modulation of bystander tumor cells by CD8+ T-cell-secreted IFN-γ[END_REF].

Thus, longitudinal IVM through imaging windows combined with functional reporters may provide opportunities to dissect cytokine-based immunomodulation.

Optogenetics for immunomodulation with spatiotemporal control

Strategies to modulate immune responses using magnetic field, temperature or light have recently emerged. In particular, optogenetics use light sensitive protein domains to manipulate cell functions with exquisite spatiotemporal control (figure 3) [START_REF] Gautier | How to control proteins with light in living systems[END_REF]. The use of optogenetics in the context of IVM may prove to be particularly useful for fundamental analysis of immunomodulatory pathways and may even find clinical applications [START_REF] Sahel | Partial recovery of visual function in a blind patient after optogenetic therapy[END_REF]. Optogenetic control of calcium signals has been achieved in vivo and shown to promote T cell arrest in LN [START_REF] Bohineust | Optogenetic manipulation of calcium signals in single T cells in vivo[END_REF]. In this respect, manipulation of Ca 2+ signals with light in either DCs or T cells has been exploited to increase anti-tumor CTL responses [START_REF] He | Near-infrared photoactivatable control of Ca2+ signaling and optogenetic immunomodulation Aldrich R[END_REF], [START_REF] Kim | Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment[END_REF]. Optogenetics also provide opportunities to better understand and control immune cell migration. For example, a strategy to control protein secretion with light have highlighted how neutrophils respond to chemokineproducing cells in vivo [START_REF] Coombs | Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish[END_REF]. Furthermore, local photoactivation of CTLs expressing a light-sensitive CXCR4 promoted their migration in vitro and could be used to direct them towards B16 melanoma tumors in vivo [START_REF] Xu | Optogenetic control of chemokine receptor signal and T-cell migration[END_REF]. The capacity to elicit a specific cellular function in vivo and visualize its outcome at the single cell level represents a unique opportunity to dissect complex immune networks.

Limitations and future improvement

With few exceptions, in vivo imaging has been largely limited to rodent models and the mechanisms of action of certain therapies may differ in humans. Indeed, some anatomical or gene expression differences persist between mice and humans, with rodent models also lacking genetic diversity. One way to partially circumvent some of these limitations may lie in the combination of IVM with humanized mouse models, although this is often at great cost. Moreover, the rather clean environment of mice husbandry is unlikely to reflect the constant exposure of humans to infectious threats and is likely impacting the immune responses to immunomodulators [START_REF] Medetgul-Ernar | Standing on the shoulders of mice[END_REF]. Finally, experimental procedures associated with IVM are not devoid of limitations. For example, access to certain organs such as the lungs or the heart can be challenging and requires extensive technical expertise [START_REF] Li | Intravital 2-photon imaging of leukocyte trafficking in beating heart[END_REF]- [START_REF] Nicolás-Ávila | A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart[END_REF]. In addition, access to a variety of fluorescent reporter mice is often needed to identify the cellular players implicated in immunomodulation. Nevertheless, the constant development of fluorescent biosensors, optogenetic actuators and animal/diseases models (zebrafish, mouse, humanized mouse) should fuel the use of IVM for understanding the ever-growing panel of immunomodulatory treatments.

General conclusion

With the development of multiple approaches to modulate immune responses to treat a variety of diseases, it remains important to clarify the mode of action of these therapies with single cell resolution.

IVM offers such possibility and has proven to be a key approach to unravel new mechanisms of immunomodulation. In particular, comparing the behavior of immune cells in the same location before or after immune intervention can be particularly instructive for therapies with rapid effects.

  Immunomodulation by adoptive cell therapies Adoptive cell therapy (ACT) represents an extreme form of immunomodulation whereby the density of effector cells is artificially increased for therapeutic purposes. T cell-based ACT includes transfer of TILs and T cells with engineered TCRs or CARs. IVM in combination with genetically-encoded fluorescent sensors to detect signaling and cell death have been particularly useful to quantify the efficiency of T cell-mediated killing in different settings.

Figure 1 .

 1 Figure 1. IVM offers a unique opportunity to visualize the immediate effects of immunomodulators in real time (A) Intravital microscopy (IVM) allows the exact same location to be imaged before and immediately after the injection of an immunomodulatory drug. This is particularly useful to characterize the early effects of an immune intervention on cell behaviors. DC is for dendritic cells. (B) An example of images obtained by IVM of the bone marrow of a mouse with an established B cell tumor, before and the first few minutes following injection of an anti-CD20mAb. Macrophages, stained with an anti-F4/80 antibody

Figure 2 .

 2 Figure 2. Insights into the mode of action of depleting antibodies using intravital imaging (A) Multiple modes of actions have been reported in vitro for eliminating cells following antibody treatment and IVM can be used to dissect their relative contribution in vivo (B) As an example, the mode of action of anti-CD20mAb has been characterized in different anatomical sites and some limitations have been identified thanks to the use of IVM. In particular, tumor B cell elimination by anti-CD20mAb is restricted in time and space in the bone marrow. ADCP, antibody-dependent cell phagocytosis, ADCC antibody-dependent cell cytotoxicity, ADCD antibody-dependent cell death

Figure 3 .

 3 Figure 3. Optogenetic control of cellular functions to study immunomodulation through IVM The use of optogenetics in the context of IVM can refine our fundamental understanding of several immunomodulatory pathways. For example, optogenetic control can activate chemokine receptor signaling to promote cell adhesion to blood vessels (top panel), or trigger cytokine/chemokine secretion in turn favoring cell activation and recruitment (middle panel), or stimulate calcium signalling to promote cell arrest and improved cell cytotoxicity (bottom panel).

Figure 1

 1 Figure 1

Figure 2 Before

 2 Figure 2

Figure 2 Figure 3

 23 Figure 2

Figure 3 Cytokine

 3 Figure 3

Acknowledgments

The work was supported by Institut Pasteur, Inserm, La Ligue contre le Cancer (P.B.), ANR JCJC (C.L.G) and an Advanced grant (ENLIGHTEN) from the European Research Council (P.B.). Some figures have been designed using bioRender and Adobe illustrator.

Data availability statement:

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Conflict of interest:

The authors declare no commercial or financial conflict of interest.