Dr Sarah E Coupland

MICROENVIRONMENT IN B-CELL LYMPHOMAS

Keywords: B-cell lymphoma, Microenvironment, Follicular lymphoma, Diffuse large B-cell lymphoma, Marginal zone B-cell lymphoma

whether they are published or not. The documents may come

Introduction

Evidence suggests that dynamic and bidirectional feedback processes between cancer cells and a complex network of reactive cells in their microenvironment play a critical role during different developmental stages of most malignancies and their progression. This network consists of a heterogeneous group of cells, including macrophages, follicular dendritic cells (FDCs), follicular reticular cells (FRCs), fibroblasts and endothelial cells, with further complexity being added by the extracellular matrix scaffolding.

Lymphomas are malignant neoplasms composed of monoclonal proliferations of lymphocytes, which proliferate autonomously and uncontrollably, overrunning the normal lymphocytic population. Most lymphomas occur within lymphoid organs,such as lymph nodes, spleen and the mucosa associated lymphoid tissue (MALT)-, where complex cell-cell interactions normally take place during the immune response. [START_REF] Harris | New approaches to lymphoma diagnosis[END_REF] Although various reactive cell populations have long been suspected of contributing to lymphoma outcome, recent gene expression array data has emphasised the role of the various components of microenvironment. [START_REF] Dave | Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells[END_REF][START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF] This review will focus on the key roles of the microenvironment in the development and progression of B-cell non-Hodgkin lymphoma (B-NHL). Future treatment strategies must take into account the interactions of the lymphoma microenvironment with the neoplastic cells, rather than only concentrating on the latter cells.

The relationship of B-cell NHL to their normal lymphoid counterpart structures

Non-Hodgkin lymphoma is a general term given to a large heterogeneous group of malignant lymphoproliferative disorders that represent distinct disease entities, according to their clinical, morphological, immunophenotypical and genetic features. [START_REF] Swerdlow | WHO classification of Tumours of Haematopoietic and Lymphoid Tissues[END_REF] Lymphomas are derived from either B cells or T cells at various stages of their differentiation, and can be divided into aggressive and indolent types, according to the clinical course. Most B-cell neoplasms mirror the features of the different B-cell differentiation stages (Fig. 1). For an overview of the differentiation pathways of B-and T-cells and of NHL subtyping applying the WHO Lymphoma classification, which proposes putative cells of origin for each NHL, the reader is referred to other works. [START_REF] Harris | New approaches to lymphoma diagnosis[END_REF][START_REF] Jaffe | Classification of lymphoid neoplasms: the microscope as a tool for disease discovery[END_REF] This review focuses on the contribution of the microenvironment in specific B-NHL entities, including diffuse large B-cell lymphoma (DLBCL) (Fig. 2), follicular lymphoma (FL) (Fig. 3), mantle cell lymphoma (ML) (Fig. 4), and marginal zone lymphoma (MZL) (Fig. 5). Evidence suggests that DLBCL and FL appear to arise from neoplastic B-cells that are (or have been) selected through the germinal centre of secondary lymphoid follicles (Fig. 1). The germinal centre is the key zone for Bcell differentiation driven by interactions between B cells, T cells and FDCs. [START_REF] Harris | New approaches to lymphoma diagnosis[END_REF] This "germinal centre reaction", as it is termed, is normally responsible for: a) the generation of memory B cells and plasma cells; b) affinity maturation of the immunoglobulin (Ig) receptors by somatic hypermutation and antigen selection in the Ig variable regions (IgV); and c) an increase of the defense efficiency of the secreted Ig (antibodies) by changing their effector domains by means of isotype switch.

Located in the outer region of the germinal centre is the mantle zone (Fig. 1), composed of small cells, which are considered to be naïve B-cells that have not undergone the germinal centre reaction because of the absence of any Ig gene somatic mutations. ML are thought to arise from mantle lymphocytes because of a morphological, immunophenotypical and genotypic features very similar to follicle mantle cells (Fig. 4). [START_REF] Swerdlow | WHO classification of Tumours of Haematopoietic and Lymphoid Tissues[END_REF] Finally, marginal zone lymphomas resemble physiological marginal zone cells in that they preferentially expand in marginal zones and tissues containing epithelial cells such as MALT, lung, salivary glands, etc. (Fig. 5). [START_REF] Isaacson | MALT lymphoma: from morphology to molecules[END_REF] Further, they express IgM in the complete or partial absence of IgD. They typically have mutated IgV region genes without any (or little) evidence of "ongoing" mutations, indicating that they originate from cells, which have passed through and "completed" the germinal centre reaction.

The functional microenvironment of normal lymphoid organs

Central lymphoid tissue is composed of the bone marrow and the thymus. The bone marrow is where B cells primarily arise and differentiate to mature B cells, and the thymus is where premature T cells differentiate into mature T cells (Fig. 6).

Immature B-and T-lymphocytes migrate from the bone marrow and thymus, respectively, into the blood and then into the peripheral lymphoid tissue, particularly the paracortex of lymph nodes via high endothelial venules. The latter are specialized blood vessels that support the rolling-, arrest-, and diapedesis of lymphocytes across the endothelium into the surrounding parenchyma. [START_REF] Miyasaka | Lymphocyte trafficking across high endothelial venules: dogmas and enigmas[END_REF] In the spleen, high endothelial venules are absent, and lymphocytes penetrate this organ via the terminal branches of the central arterioles that deliver them into the marginal zone. [START_REF] Bajenoff | Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes[END_REF] The molecular events involved in the migration of both B-and Tlymphocytes from blood to the peripheral lymphoid tissue are well characterized, and sequentially involve selectins, chemokines, and integrins. [START_REF] Bajenoff | Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes[END_REF][START_REF] Cyster | Follicular stromal cells and lymphocyte homing to follicles[END_REF] For example, key roles are played by chemokines such as CCL21 and CXCL13 in the migration and localization of T-cells [START_REF] Nakano | A novel mutant gene involved in T-lymphocyte-specific homing into peripheral lymphoid organs on mouse chromosome 4[END_REF][START_REF] Forster | CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs[END_REF] and B-cells [START_REF] Ansel | A chemokine-driven positive feedback loop organizes lymphoid follicles[END_REF] , respectively.

The functional architecture of the spleen and lymph nodes are supported by a complex 3-dimensional structure of stromal cells and a lymph conduit system. [START_REF] Bajenoff | Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes[END_REF][START_REF] Cyster | Follicular stromal cells and lymphocyte homing to follicles[END_REF][START_REF] Bajenoff | Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone[END_REF][START_REF] Bajenoff | B-cell follicle development remodels the conduit system and allows soluble antigen delivery to follicular dendritic cells[END_REF] The stromal reticular network can be clearly visualized by fluorescence immunohistochemistry coupled with confocal imaging using a monoclonal antibody, ER-TR7, and can be subdivided into four structurally distinct regions, each of which correlates well with the location of distinct immune cell subsets. [START_REF] Katakai | A novel reticular stromal structure in lymph node cortex: an immuno-platform for interactions among dendritic cells, T cells and B cells[END_REF] In addition, "lymphoid tissue inducer cells", characterized by a CD3 -CD4 + phenotype, contribute to the development and maintenance of the lymphoid organs. These cells express lymphotoxin (LT) α/β and tumour necrosis factor (TNF) family ligands, such as CD30L (CD153), and seem to be required for the formation of the lymphoid stroma. [START_REF] Kim | Function of CD4+CD3-cells in relation to B-and T-zone stroma in spleen[END_REF] Lymphoid inducer cells have been shown to enter embryonic lymphoid tissue and signal the development of T-cell and B-cell zones, via interactions with FRCs and FDCs. [START_REF] Bajenoff | Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes[END_REF] B-lymphocytes themselves also appear to be required for the proper development of the spleen, via the expression of LTα1β2, and for the induction of FDC networks. [START_REF] Ngo | Splenic T zone development is B cell dependent[END_REF][START_REF] Murakami | Splenic CD19-CD35+B220+ cells function as an inducer of follicular dendritic cell network formation[END_REF] Such inducer cells appear to orchestrate the development and production of the chemokines CCL21 or CXCL13 by the lymphoid stromal cells resulting in the formation of the T-cell and B-cell regions. Furthermore, lymphocytes can directly induce FRCs to form complex networks [START_REF] Bajenoff | B-cell follicle development remodels the conduit system and allows soluble antigen delivery to follicular dendritic cells[END_REF][START_REF] Katakai | Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes[END_REF] , suggesting that bidirectional interactions between cells of the immune system and the lymphoid stroma are essential for the function of lymphoid organs.

The role of peripheral lymphoid stromal cells in lymphomas

As described above, there is a complex organization of normal lymphoid tissue, which is, in turn, surrounded by a complicated network of stromal cells, serving as a structural support in the migration, homeostasis, and survival of immune cells. In lymphomas, it appears that this network of stromal cells creates a microenvironment that initially attracts cancer cells, allowing their early survival, and subsequently contributes to tumour growth. [START_REF] Gascoyne | Prognostic biomarkers in malignant lymphomas[END_REF] Many cytokines and adhesion molecules are implicated in this cross talk between stromal cells and neoplastic lymphocytes. Their role has been extensively demonstrated in multiple myeloma, some forms of B-NHL, including FL and DLBCL, as well as classical Hodgkin's lymphoma (cHL). [START_REF] Gascoyne | Prognostic biomarkers in malignant lymphomas[END_REF][21][START_REF] Jundt | Jagged1-induced Notch signaling drives proliferation of multiple myeloma cells[END_REF][START_REF] Aldinucci | The classical Hodgkin's lymphoma microenvironment and its role in promoting tumour growth and immune escape[END_REF] Soluble mediators, including interleukin (IL)-6, IL-7, IL-4, IL-8, VEGF, CXCR4, MIP-1, Jagged-1, and insulin growth factor (IGF)-1, appear to play a role in these communications. [START_REF] Gascoyne | Prognostic biomarkers in malignant lymphomas[END_REF][21][START_REF] Jundt | Jagged1-induced Notch signaling drives proliferation of multiple myeloma cells[END_REF][START_REF] Calvo | IL-4 protein expression and basal activation of Erk in vivo in follicular lymphoma[END_REF] Recently, a newly-described family of proteins has taken centre stage in the dialogue between stromal and neoplastic B-lymphocytes and plasma cells: the hedgehog (Hh) proteins. [START_REF] Dierks | Essential role of stromally induced hedgehog signaling in B-cell malignancies[END_REF][START_REF] Lindemann | Stroma-initiated hedgehog signaling takes center stage in B-cell lymphoma[END_REF] Hh proteins are expressed in lymphoid cells: upon interaction with their ligands, they signal to protect those cells from apoptosis.

Dierks et al. demonstrate that Hh ligands secreted by nodal and splenic stromal cells function as survival factors for lymphoma cells in transgenic mice models and in ex-

vivo isolated human lymphoma. [START_REF] Dierks | Essential role of stromally induced hedgehog signaling in B-cell malignancies[END_REF] Similarly, Singh et al. showed that there is an autocrine Hh signaling loop between DLBCL cells and nodal stromal cells. [START_REF] Singh | Hedgehog signaling pathway is activated in diffuse large B-cell lymphoma and contributes to tumor cell survival and proliferation[END_REF] Inhibition of the Hh protein's pathway in neoplastic B-and plasma cells induces either cell-cycle arrest or apoptosis through the downregulation of BCL-2. [START_REF] Dierks | Essential role of stromally induced hedgehog signaling in B-cell malignancies[END_REF][START_REF] Singh | Hedgehog signaling pathway is activated in diffuse large B-cell lymphoma and contributes to tumor cell survival and proliferation[END_REF] that two gene signatures unrelated to the malignant cells themselves had prognostic significance. [START_REF] Lenz | Stromal gene signatures in large-B-cell lymphomas[END_REF] The first set of genes, called "stromal-1", was associated with a favourable patient outcome and was enriched in genes encoding extracellular matrix proteins (e.g. fibronectin, collagens, laminins) or enzymes involved in the synthesis of this matrix (e.g. collagen synthesis modifiers, matrix metalloproteinases, connective tissue growth factor). Genes thought to originate from the monocytic lineage were also found to be coordinately expressed in DLBCL.

The second signature, "stromal-2", which predicted a poor patient outcome, was enriched in genes attributed to endothelial cells and functions (e.g. CD31, von Willebrand factor), as well as others involved in the regulation of angiogenesis. The "stromal-2" signature significantly correlated with blood-vessel density in the tumour specimen examined in this series. These findings suggest that survival after treatment of DLBCL is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment, and that some pathways may be further considered for therapeutic interventions. As gene expression profiling techniques are not yet feasible in the clinical laboratory, several investigators have aimed to translate these findings using immunohistochemistry, flow cytometry and real-time polymerase chain reaction (PCR) to further characterize in biopsy samples received in routine clinical practice. [START_REF] Farinha | Analysis of multiple biomarkers shows that lymphoma-associated macrophage (LAM) content is an independent predictor of survival in follicular lymphoma (FL)[END_REF][START_REF] Alvaro | Immunohistochemical patterns of reactive microenvironment are associated with clinicobiologic behavior in follicular lymphoma patients[END_REF] Farinha et al. first reported the adverse prognostic value of a high content of CD68+ macrophages in a homogenous series of FL patients using immunohistochemistry. [START_REF] Farinha | Analysis of multiple biomarkers shows that lymphoma-associated macrophage (LAM) content is an independent predictor of survival in follicular lymphoma (FL)[END_REF] These results were confirmed by some authors [START_REF] Alvaro | Immunohistochemical patterns of reactive microenvironment are associated with clinicobiologic behavior in follicular lymphoma patients[END_REF][START_REF] Canioni | High numbers of tumor-associated macrophages have an adverse prognostic value that can be circumvented by rituximab in patients with follicular lymphoma enrolled onto the GELA-GOELAMS FL-2000 trial[END_REF] but not by others. [START_REF] Taskinen | A high tumorassociated macrophage content predicts favorable outcome in follicular lymphoma patients treated with rituximab and cyclophosphamide-doxorubicin-vincristine-prednisone[END_REF] The discordant results may be due to differing methodological approaches with differing "cut-offs", as well as differences in the material evaluated (e.g. tissue micro-arrays with tissue cores versus whole tissue sections). [START_REF] Lejeune | Clinicobiological, prognostic and therapeutic implications of the tumor microenvironment in follicular lymphoma[END_REF] Of note, the adverse prognostic value of CD68+ cells could no longer be confirmed, when FL patients received a chemotherapy regimen containing rituximab, suggesting that new therapeutic modalities may alleviate pathways modifying tumour aggressiveness. [START_REF] Canioni | High numbers of tumor-associated macrophages have an adverse prognostic value that can be circumvented by rituximab in patients with follicular lymphoma enrolled onto the GELA-GOELAMS FL-2000 trial[END_REF][START_REF] Taskinen | A high tumorassociated macrophage content predicts favorable outcome in follicular lymphoma patients treated with rituximab and cyclophosphamide-doxorubicin-vincristine-prednisone[END_REF] However, the CD68 staining alone may not be able to truly reflect the macrophage component identified by gene expression profiling, and indeed the CD68+ population appears to be heterogeneous in its density and distribution. Using flow cytometry, Alvaro et al. further characterized a subset of macrophages expressing "signal transducer and activator of transcription 1" (STAT1) transcription factor, identifying this subset to be predominantly associated with a poor outcome in FL patients. [START_REF] Alvaro | Immunohistochemical patterns of reactive microenvironment are associated with clinicobiologic behavior in follicular lymphoma patients[END_REF] Using real-time polymerase chain reaction, Byers et al. elegantly measured the expression levels of 35 candidate Indicator genes, selected from microarray studies, to polyA complementary DNAs prepared from human frozen lymph nodes of FL patients. This group demonstrated that high levels of the chemokine receptor CCR1 transcript, known to be an activation marker for monocytes, were found to be associated with a poor outcome in FL. [START_REF] Byers | Clinical quantitation of immune signature in follicular lymphoma by RT-PCR-based gene expression profiling[END_REF] These results correlated with high numbers of CD68+ macrophages examined using immunohistochemistry.

Macrophages in B-NHL pathogenesis

By applying a microarray approach and validated using quantitative polymerase and the transcription factor SMAD-1 (expressed in vascular endothelium). [START_REF] Nykter | Differential gene expression in nonmalignant tumour microenvironment is associated with outcome in follicular lymphoma patients treated with rituximab and CHOP[END_REF] Low SMAD-1 and MARCO expression were associated with better progression-free survival in a patient cohort 0f 24 FL patients receiving chemotherapy and rituximab.

Taken together, these data indicate that an in-depth characterization of cells from the monocyte/macrophage lineage is warranted to precisely define their contribution to the growth and progression of lymphoma cells, particularly in FL, and to determine whether they could be potential targets of chemotherapeutic drugs.

T-cell subsets and dendritic cells in B-NHL pathogenesis and progression

The most compelling evidence of T-cell involvement in lymphoma development and survival prediction has been demonstrated in FL. [START_REF] Alvaro | Immunohistochemical patterns of reactive microenvironment are associated with clinicobiologic behavior in follicular lymphoma patients[END_REF][START_REF] Zelenetz | Follicular lymphoma: a model of lymphoid tumor progression in man[END_REF][START_REF] Zelenetz | Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection[END_REF][START_REF] Carreras | High numbers of tumor-infiltrating FOXP3-positive regulatory T cells are associated with improved overall survival in follicular lymphoma[END_REF][START_REF] Lee | Number of CD4+ cells and location of forkhead box protein P3positive cells in diagnostic follicular lymphoma tissue microarrays correlates with outcome[END_REF][START_REF] Glas | Gene-expression and immunohistochemical study of specific T-cell subsets and accessory cell types in the transformation and prognosis of follicular lymphoma[END_REF][START_REF] Wahlin | CD8+ T-cell content in diagnostic lymph nodes measured by flow cytometry is a predictor of survival in follicular lymphoma[END_REF][START_REF] Carreras | High numbers of tumor-infiltrating programmed cell death 1positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma[END_REF] The follicular structure of the tumour cells in most FL results in a close proximity and collaboration of the lymphoma cells with T-helper cells and FDC. [START_REF] Manconi | Dendritic reticulum cell pattern as a microenvironmental indicator for a distinct origin of lymphoma of follicular mantle cells[END_REF] Carbone et al. noted that lymphoma cells need interactions with T cells and FDCs in the follicles in order to proliferate. [START_REF] Carbone | CD40 ligand is constitutively expressed in a subset of T cell lymphomas and on the microenvironmental reactive T cells of follicular lymphomas and Hodgkin's disease[END_REF] As mentioned above, the germinal center is the anatomic and functional site of the lymphoid follicle where T-cell-dependent antibody response is generated and T cells help to provide signals to antigen-specific naïve B cells. Importantly, CD4+ T cells are crucial for germinal center formation and are indispensable for the differentiation of antigen-selected high-affinity B cells into plasma cells or memory B cells. [START_REF] Liu | Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation[END_REF][START_REF] Klein | Somatic hypermutation in normal and transformed human B cells[END_REF][START_REF] Manz | Maintenance of serum antibody levels[END_REF] .

T-lymphocytes probably support FL proliferation by adhesion molecules and other

stimulatory surface molecules as well as by the production of growth factors and cytokines. Generally, FL cells express CD40, the co-stimulatory molecule CD86 and less often CD80. The ligands, CD28/CD152 (CTLA4) for CD80/CD86 and CD40L (CD154) for CD40, are expressed by intratumoral T cells. [START_REF] Dorfman | In vivo expression of B7-1 and B7-2 by follicular lymphoma cells can prevent induction of T-cell anergy but is insufficient to induce significant T-cell proliferation[END_REF][START_REF] Vyth-Dreese | Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma[END_REF] FDCs express ICAM1 and VCAM1, the counterstructures for LFA-1 (CD11a/ CD18) and VLA-4 (CD49d/CD29) present on FL cells. [START_REF] Koopman | Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells[END_REF] FDCs also provide stimulatory molecules such as interleukin-15, 8D6 (CD320), BAFF (B cell activating factor belonging to the TNF family ) and hepatocyte growth factor (HGF), which may all have a role in normal germinal centre B-cell expansion and may exert a similar function in FL. [START_REF] Park | How do follicular dendritic cells interact intimately with B cells in the germinal centre?[END_REF][START_REF] Li | Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth[END_REF][START_REF] Hase | BAFF/BLyS can potentiate B-cell selection with the Bcell coreceptor complex[END_REF][START_REF] Zhang | BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors[END_REF][START_REF] Van Der Voort | Paracrine regulation of germinal center B cell adhesion through the c-met-hepatocyte growth factor/scatter factor pathway[END_REF][START_REF] Tjin | Follicular dendritic cells catalyze hepatocyte growth factor (HGF) activation in the germinal center microenvironment by secreting the serine protease HGF activator[END_REF] Production of TNF by B cells is critically important for the generation of fully developed FDCs from resident stromal cells upon p55-TNF-R signaling. [START_REF] Endres | Mature follicular dendritic cell networks depend on expression of lymphotoxin beta receptor by radioresistant stromal cells and of lymphotoxin beta and tumor necrosis factor by B cells[END_REF][START_REF] Matsumoto | Distinct roles of lymphotoxin alpha and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells[END_REF][START_REF] Victoratos | FDCspecific functions of p55TNFR and IKK2 in the development of FDC networks and of antibody responses[END_REF] Moreover, sustained interaction of TNF and LTα1β2 is important for the maintenance of mature FDCs networks. [START_REF] Mackay | Turning off follicular dendritic cells[END_REF][START_REF] Gommerman | Manipulation of lymphoid microenvironments in nonhuman primates by an inhibitor of the lymphotoxin pathway[END_REF] It has been shown that sorted neoplastic B-cells in FL produce TNF, LTα and LTβ. [START_REF] Voog | Factors that predict chemotherapy-induced myelosuppression in lymphoma patients: role of the tumor necrosis factor ligand-receptor system[END_REF] The production of these molecules supposedly is crucial for the local generation of FDCs or "FDC-like" cells from stroma at extranodal sites and in the bone marrow.

Of the above-mentioned molecules, those mainly involved in B-and T-cell contact are CD40 and its ligand CD40L, respectively, which are expressed on B cells and activated T cells. CD40L can protect from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in FL. [START_REF] Travert | CD40 ligand protects from TRAIL-induced apoptosis in follicular lymphomas through NF-kappaB activation and up-regulation of c-FLIP and Bcl-xL[END_REF] One of the key features of FL is a defect in apoptosis, due to the t(14;18) translocation, resulting in overexpression of the BCL2 protein by the neoplastic B-cells. FL cells are difficult to culture, but they may survive for weeks when stimulated through the CD40 pathway, following stimulation with CD40 and cytokine signals [START_REF] Johnson | Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40/stromal cell system[END_REF] or when co-cultured with CD4 T cells recognizing lymphoma alloantigens. [START_REF] Umetsu | Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones[END_REF] It has been observed that the CD40 antiapoptotic signaling occurs quickly, before the commencement of the mitochondrial events considered to the start of apoptosis. [START_REF] Eeva | Kinetics and signaling requirements of CD40-mediated protection from B cell receptor-induced apoptosis[END_REF] Recently, an elegant animal model found that constitutive CD40 signaling was sufficient to initiate a polyclonal proliferation of B cells, ultimately leading to the development of true B-cell lymphomas. [START_REF] Homig-Holzel | Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-kappaB pathway and promotes lymphomagenesis[END_REF] Of note, CD40 is a favorite target for the development of new monoclonal antibodies in lymphoma, and humanized anti-CD40 is currently being tested for B-chronic lymphocytic leukemia and other B-NHL. [START_REF] Fanale | Monoclonal antibodies in the treatment of non-Hodgkin's lymphoma[END_REF] Among CD4+ T cells is a subpopulation of lymphocytes termed regulatory cells (Tregs), which play a crucial role in modulating the immune regulation in humans (Fig. 6). They do this by suppressing the proliferation and cytokine production of other T-cells, including CD8+ T-cells. [START_REF] Horna | In vivo disruption of tolerogenic cross-presentation mechanisms uncovers an effective T-cell activation by B-cell lymphomas leading to antitumor immunity[END_REF][START_REF] Plumas | Tumor B cells from non-Hodgkin's lymphoma are resistant to CD95 (Fas/Apo-1)-mediated apoptosis[END_REF] A subset of regulatory Tregs in the tumor [START_REF] Hori | Control of regulatory T cell development by the transcription factor Foxp3[END_REF] , appear to have a critical role modifying immune responses, particularly suppression of tumor associated antigen-reactive lymphocytes in tumours (Fig. 6). [START_REF] Wei | Antitumor immunity and autoimmunity: a balancing act of regulatory T cells[END_REF] The presence of Tregs thus contributes to cancer cell growth. [START_REF] Beyer | Regulatory T cells in cancer[END_REF] Concordantly, the presence of a high number of Tregs in a variety of malignancies has been correlated with a poor prognosis of patients. [START_REF] Curiel | Dendritic cell subsets differentially regulate angiogenesis in human ovarian cancer[END_REF][START_REF] Okita | CD4CD25 regulatory T cells in the peripheral blood of patients with breast cancer and non-small cell lung cancer[END_REF][START_REF] Nagorsen | Tumor-infiltrating macrophages and dendritic cells in human colorectal cancer: relation to local regulatory T cells, systemic T-cell response against tumor-associated antigens and survival[END_REF][START_REF] Shindo | Regulatory T Cells and Skin Tumors, Recent Pat Inflamm[END_REF] Recent evidence suggests similarly that the quantity of tumor-infiltrating Tregs, can significantly modify the clinical outcome in B-cell NHL, particularly in FL and cHL. [START_REF] Alvaro | Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells[END_REF][START_REF] Tzankov | Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin's lymphoma[END_REF] Indeed, neoplastic B cells in NHL were shown to be able to induce forkhead/winged helix transcription factor 3 (Foxp3) expression in intratumoral CD4+CD25-T cells (Fig. 6). [START_REF] Yang | CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells[END_REF] Therefore, although neoplastic human B cells could be killed by autologous infiltrating CD8+ T cells, they appear to be protected by the intra-tumoral CD4+CD25-Tregs. [START_REF] Yang | Attenuation of CD8(+) T-cell function by CD4(+)CD25(+) regulatory T cells in B-cell non-Hodgkin's lymphoma[END_REF] Yang et al. also provided evidence that the percentages of CD8+ T cells and intratumoral Tregs in specimens from patients with B-cell NHL were proportionally inverse. [START_REF] Yang | CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells[END_REF] Interactions between Tregs and neoplastic B-cells collaborate to promote tumour growth: Tregs contribute to the ineffective antitumor response in B-NHL, while the lymphoma cells increase the numbers of Tregs within the tumor.

Generally, Tregs are proposed to contribute to immune evasion by malignancies.

There are at least two forms of Treg cells: "natural" and "induced." Natural Tregs can be induced and expanded in direct contact with tumor cells. [START_REF] Mittal | Local and systemic induction of CD4+CD25+ regulatory T-cell population by non-Hodgkin lymphoma[END_REF] This interaction can be explained by the production by the tumor cell of the CCL22 chemokine.

Recently, a new IL-17-secreting CD4+ helper T cell population increased the family of T cells into three major lineages: TH1, TH2, and TH17 cells (Fig. 6). [START_REF] Betz | Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes[END_REF]

TH17 and

Tregs represent two opposing immune responses. Yang et al. [START_REF] Yang | Malignant B cells skew the balance of regulatory T cells and TH17 cells in B-cell non-Hodgkin's lymphoma[END_REF] recently confirmed that the development of Tregs and TH17 cells is reciprocally regulated and, more importantly, that lymphoma B cells generate an inhibitory environment that favours the development of Tregs rather than TH17 cells. This lack of TH17 cells may therefore contribute to the suboptimal immune response in NHL. The complexity of this microenvironment as well as the size or fixation state of a tumour specimen most likely account for difficulties in investigating T-cell subpopulations as prognostic markers in lymphoma. Based on gene expression profiling data and the association of the above-mentioned IR-1 signature (enriched in T-cell genes) with improved survival in FL 2 , several groups analyzed various T-cell markers to further define the functional T-cell subpopulation that could be associated with a favorable outcome. Several groups have identified FOXP3+ T cells as being associated with a favorable outcome. [START_REF] Carreras | High numbers of tumor-infiltrating FOXP3-positive regulatory T cells are associated with improved overall survival in follicular lymphoma[END_REF][START_REF] Lee | Number of CD4+ cells and location of forkhead box protein P3positive cells in diagnostic follicular lymphoma tissue microarrays correlates with outcome[END_REF] These findings are noteworthy given the data mentioned above and the adverse role of Treg infiltration in other tumor subtypes. [START_REF] Curiel | Dendritic cell subsets differentially regulate angiogenesis in human ovarian cancer[END_REF][START_REF] Curiel | Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[END_REF] Interestingly, recent data suggests that FOXP3+ cell distribution significantly predicts survival and the risk of high-grade transformation in FL. [START_REF] Farinha | The architectural pattern of FOXP3-positive T cells in follicular lymphoma is an independent predictor of survival and histologic transformation[END_REF] CD8+ infiltrating T cells, assessed by immunohistochemistry or flow cytometry, were identified as being associated with a favorable outcome in two independent studies. [START_REF] Alvaro | Immunohistochemical patterns of reactive microenvironment are associated with clinicobiologic behavior in follicular lymphoma patients[END_REF][START_REF] Wahlin | CD8+ T-cell content in diagnostic lymph nodes measured by flow cytometry is a predictor of survival in follicular lymphoma[END_REF] Further, a high number of cytotoxic lymphocytes positive for the programmed cell death (PD)-1 molecule was also found to be of favorable prognosis in FL patients [START_REF] Carreras | High numbers of tumor-infiltrating programmed cell death 1positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma[END_REF] , further underlining the role of different populations of cytotoxic lymphocytes in the lymphoma microenvironment. Comparative analyses using PCR-based techniques, flow cytometry and immunohistochemistry may prove to be useful in determining the most reliable method for T-cell quantification and subtyping in routine FL specimens.

The role of chemokines and their receptors in the migration of neoplastic cells.

As mentioned above, chemokines and their receptors constitute a large family of molecules that control the trafficking of immune cells during their development and in response to inflammation. The characterization of dendritic cells in NHL, the expression of adhesion molecules, costimulatory molecules, chemokine receptors, and activation markers have been analyzed in a few studies. A significantly decreased expression of CD62L and CCR7, receptors necessary for lymph node homing, was identified in dendritic cells in NHL. [START_REF] Fiore | Dendritic cells are significantly reduced in non-Hodgkin's lymphoma and express less CCR7 and CD62L[END_REF] Dendritic cells are also phenotypically altered and reduced in number in NHL, potentially contributing to the loss of tumor control in these patients. Further, chemokines, chemokine receptors, and adhesion molecules can be critical for B lymphoma cells homing toward the supporting cells, and for the dissemination of neoplastic B-cells. In ML, the neoplastic B-cells express high levels of CXCR4, CXCR5, chemokine receptors, and VLA-4 adhesion molecule. [START_REF] Kurtova | Mantle cell lymphoma cells express high levels of CXCR4, CXCR5, and VLA-4 (CD49d): importance for interactions with the stromal microenvironment and specific targeting[END_REF] Similarly, high levels of CXCR4, CXCR5 and CCR7 have been described by DLBCL of the central nervous system. 88 CXCR4 (CXCL12 receptor) and CXCR5 (CXCL13 receptor) are crucial for trafficking and homing in on the secondary lymphoid tissue. [START_REF] Bajenoff | Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes[END_REF] ML may further disseminate through the lymphoid tissue via these receptors because stromal cells constitutively secrete CXCL12 and CXCL13 molecules. In FL, CXCL13 is secreted by the neoplastic B-cells, which also express CXCR4 and CXCR5 (ligand of CXCL13). [START_REF] Husson | CXCL13 (BCA-1) is produced by follicular lymphoma cells: role in the accumulation of malignant B cells[END_REF] CXCL13 production in a particular local environment may then lead to an accumulation of neoplastic cells. [START_REF] Smith | Expression of B-cellattracting chemokine 1 (CXCL13) by malignant lymphocytes and vascular endothelium in primary central nervous system lymphoma[END_REF] passes from the blood into the peripheral lymphoid tissue. Here, the B cell migrates to the outer region of the lymph node in the "primary" follicles and, later, to the follicle mantles. This differentiation step is associated with the additional expression of IgD. These IgM+/IgD+ B cells are known as "naive mature B-cells". When these cells come into contact with antigen, which can bind to their immunoglobulin molecules, they transform into proliferating extrafollicular B blasts, from which short-lived plasma cells and "antigen-induced" or "primed" B cells are derived. These "primed" B cells initiate and maintain the germinal center reaction, during which they transform into rapidly proliferating centroblasts. During the mitotic proliferation and differentiation of the centroblasts into centrocytes, somatic mutations in the variable region of the immunoglobulin genes are inserted in a randomized manner. The centrocytes with advantageous mutations (i.e. those which lead to an increase in the affinity of the immunoglobulin receptor) differentiate further, passing out of the germinal centre into long-lived plasma cells or into "memory" B cells. The latter remain in the marginal zone.

  lymphoma treatment could involve the disruption of the Hh signaling pathway between nodal stromal cells and the malignant lymphocytes. Recent genomic and genetic studies have led to the identification-and recognition of the importance of additional signaling pathways of the stromal network in B-cell lymphomas. Using gene expression profiling, Lenz et al. demonstrated in DLBCL

  Similar to DLBCL and their stromal signatures, recent gene expression profiling data have demonstrated the importance of non-malignant cells, particularly macrophages and dendritic cells, in germinal centre-derived B-NHL (e.g. FL and cHL) and marginal zone-derived B-NHL (e.g. MZL). It appears that the clinical heterogeneity in FL is related to the gene expression signatures of these nonmalignant cells infiltrating the tumor, rather than to intrinsic differences in the lymphoma cells. Two outcome-related signatures, termed Immune response 1 (IR1) and Immune response 2 (IR2), have been identified by gene expression profiling in FL, indicating a significant role of the microenvironment in tumor development and . 2 IR1, composed of genes mostly expressed by T-cells, was found to be associated with a more favorable clinical course, and IR2, enriched for genes expressed by macrophages and FDCs, was found to be associated with an inferior clinical course.

  reaction (qPCR) and immunohistochemistry, Harjunpää et al. proposed additional prognostic markers in FL: these were a scavenger receptor on macrophages with collagenous structure (MARCO), together with EPHA-1, a tyrosine kinase involved in transepithelial migration (expressed in granulocytes),

  by a CD4+CD25+ phenotype and regulated by the FOXP3 gene

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  Indeed, CXCL13 secretion by lymphoma cells can recruit CXCR5+ cells to create a tumor microenvironment. Conclusions In B-cell lymphomas, tumor development and expansion appears to be dependent on multiple signals mediated by the microenvironment within peripheral lymphoid tissue. Cellular interactions are required not only between the neoplastic lymphocytes but also with the numerous other actors (non-malignant cells and the stromal cell components) present on the stage. Chemokines, chemokine receptors, adhesion molecules, and adhesion receptors have an important role in this network of cells within the microenvironment. Inside this complex microenvironment, neoplastic lymphocytes have adapted mechanisms whereby they have taken control of the local host immune response. And yet, the immune system may either promote or constrain tumor cell development, depending on the relative distribution and activation status of various cell subpopulations. For evaluation of the microenvironment cellular subpopulations, uniform guidelines in methodologies would increase the comparability between studies. With regard to chemotherapeutics, we need to rethink our treatment strategy and address the complex network of multiple cell types within the tumor microenvironment instead focusing on the tumor cells alone.

Figure 1

 1 Figure1schematically demonstrates the differentiation pathway of the B-cell as it passes from the blood into the peripheral lymphoid tissue. Here, the B cell migrates to the outer region of the lymph node in the "primary" follicles and, later, to the follicle mantles. This differentiation step is associated with the additional expression of IgD. These IgM+/IgD+ B cells are known as "naive mature B-cells". When these cells come into contact with antigen, which can bind to their immunoglobulin molecules, they transform into proliferating extrafollicular B blasts, from which short-lived plasma cells and "antigen-induced" or "primed" B cells are derived. These "primed" B cells initiate and maintain the germinal center reaction, during which they transform into rapidly proliferating centroblasts. During the mitotic proliferation and differentiation of the centroblasts into centrocytes, somatic mutations in the variable region of the immunoglobulin genes are inserted in a randomized manner. The centrocytes with advantageous mutations (i.e. those which lead to an increase in the affinity of the immunoglobulin receptor) differentiate further, passing out of the germinal centre into long-lived plasma cells or into "memory" B cells. The latter remain in the marginal zone.

Figure 2 .

 2 Figure 2. A: Giemsa stain of a diffuse large B-cell lymphoma (DLBCL), centroblastic morphological variant. B: DLBCL of immunoblastic morphological variant (Giemsa, objective x40).

Figure 3 .

 3 Figure 3.Histopathology with associated immunostaining of a typical follicular lymphoma (FL). The haematoxylin and eosin (H&E) stain displays the nodular infiltration of the neoplastic B-cells, which are comprised of a mixture of centroblast-and centrocyte-like cells (Giemsa stain). Although mitoses are seen, apoptotic cells are absent. The neoplastic cells are immunoreactive with CD20, BCL-2 (as a consequence of the t(14;18) chromosomal translocation resulting in overexpression of the bcl-2 gene) and demonstrate a reduced Ki-67 growth fraction (with loss of the usual light and dark proliferation zones in the normal germinal centre). The CD3 stain highlights the T-cell population in the interfollicular zone as well as scattered cells within the neoplastic germinal centres. These T-cell populations are to have significant roles in the development and progression of FL.

Figure 4 .

 4 Figure 4. Histopathology and immunostaining of a mantle cell lymphoma, whichare comprised of small cells with ovoid or coffee-bean-shaped nuclei (Giemsa). In the background are occasional paler epithelioid dendritic cells.The number of mitotic figures can be moderate. The neoplastic B-cells in mantle cell lymphoma arise from the mantle zone, which is highlighted here in the cyclin D1 stain (top right). A higher magnification demonstrates the nuclear staining of the malignant B-cells, which also aberrantly co-express the T-cell antigen, CD5.

Figure 5 .

 5 Figure 5. Hsitopathology and immunostaining of an extranodal marginal zone B-cell lymphoma. These tumours consist of a proliferation of centrocyte-like cells in the marginal zone (MZ) surrounding a normal germinal centre (GC). The marginal zone cells can show varying degrees of plasmacellular differentiation, as highlighted in the PAS stain. The neoplastic marginal zone B-cells stain for CD20 and are usually monotypical for IgM. In this case, the neoplastic B-cells are infiltrating the overlying conjunctiva, forming lymphoepithelial lesions.

Figure 6 .

 6 Figure 6. Schematic representation of T-cell development and differentiation into regulatory T-cells and a variety of subtypes with differing functions, and from which the differing T-cell lymphomas are postulated to arise. The main regulatory T-cell considered to have a major role in the development and progress in some B-NHL are the CD4+, CD25+ and FoxP3+ T-regs.

Published on behalf of the British Division of the International Academy of Pathology Histopathology