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1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, the Netherlands; 2 GABBA PhD Program, University of Porto, Porto, Portugal five

Immuneregulatory properties of lymphoid stromal cells

AP Baptista1,2, RE Mebius1 Abstract Evolution has equipped the immune system with a vast network of specialized lymphoid tissues that, being located at strategic positions, grant permanent immunesurveillance. This network of secondary lymphoid organs (SLOs) is crucial for the homeostasis of the immune system, as evidenced by the severely immunocompromised phenotype of the animals that lack it. SLOs are supported by non-hematopoeitic stromal elements, which are becoming increasingly recognized as central players in the regulation of the overall immune system. In this introductory note, we five summarize our current understanding of lymphoid stromal cells. We discuss their origin and role in the development of SLOs and how they contribute to the homeostasis of the immune system in the steady state and during immune responses.

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Introduction Host protection against pathogenic insults depends upon the dynamic interaction between pathogen-derived and diverse hematopoietic cells specialized in processing and presentation, followed by insult clearance. As the frequency of antigen-specific lymphocytes is relatively low1, 2, generation of appropriate immune responses critically depends on the presence of specialized secondary lymphoid organs (SLOs), such as nodes, and Peyer’s patches3-5. SLOs form nuclei of immunesurveillance, where danger signals and antigens contained within lymph, blood and/or mucosa are scrutinized by both the innate and the adaptive immune five systems. Indeed, SLOs are designed as very efficient filtration systems that capture pathogens6, preventing their spreading throughout the host, and allow the presentation of their antigens in an appropriate manner to lymphocytes7, 8. Importantly, the likelihood that antigen-specific T and B cells will find their cognate antigen within SLOs is maximized by the highly sophisticated microarchitecture of these organs9-11. Typically, SLOs have separate T and compartments, whose formation and maintenance relies on the presence of specialized stromal cells of non- hematopoietic origin12. While fibroblastic reticular cells (FRCs) are present within T cells, follicular dendritic cells (FDCs) form the structural backbone of B cell follicles9-11. In addition, to these two stromal populations, other lymphoid organ CD45- populations include marginal reticular cells (MRCs)13 and blood and lymphatic endothelial cells (BECs and LECs)9-11. Importantly, further stromal cell subsets are likely to be defined as research progresses. Lymphoid stromal cells have long been viewed simply as cells that create and define the architecture of lymphoid tissues, passively facilitating the communication between immune cells. However, it is now clear that they have a more subtle role in the overall homeostasis and responsiveness of the immune system. In this introductory chapter, we summarize our current understanding of the immuneregulatory properties of lymphoid stromal cells. We will discuss, among other topics, their origin and role in the development of lymphoid tissues, their role in the homeostasis and maturation of lymphocytes, their role in the control of lymphocyte recirculation in the steady-state and during immune responses and their role as antigen-presenting cells.

Lymphoid stromal cells and lymphoid tissue development Early steps in lymphoid tissue organogenesis SLOs develop during embryogenesis or within the first weeks after birth as a result of the interaction + - + + + between lymphotoxin (LTα1β2)-expressing CD45 CD3 CD4 IL7Rα c-kit lymphoid tissue inducer (LTi) cells and lymphotoxin β receptor (LTβR)-bearing VCAM-1+ICAM-1+ lymphoid tissue organizer (LTo) cells14-16. LTi cells cluster at sites of prospective lymphoid tissue formation attracted by the 17-22 CXCL13, CCL19 and CCL21, initially expressed in a LTα1β2-independent manner . 23 LTi clustering induces LTα1β2 expression , enabling LTi cells to communicate with the surrounding mesenchymal stromal cells. LTβR triggering induces the differentiation of lymphoid stromal cells, leading to increased expression of CXCL13, CCL19 and CCL21, and additionally to the induction of the adhesion ligands intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion

111 molecule 1 (VCAM-1) and the cytokines IL7 and tumor necrosis factor-related activation induced cytokine (TRANCE)24, 25. Together these factors promote the recruitment, survival and retention of additional LTi cells into the lymphoid tissue primordia14-16. Importantly, in addition to promote

LTi cell survival, stromal cell-derived IL7 and TRANCE also induce the expression of LTα1β2 on LTi cells22, 23. Hence, lymphoid stromal cell differentiation and increased LTi cell recruitment create a positive feedback loop that allows the rapid expansion and consolidation of the developing lymphoid tissues17. Noteworthy, stromal organizer cells also produce lymphangiogenic factors upon LTβR triggering21, which are likely to contribute for the development of both blood and five lymphatic vascular systems within the lymphoid tissue anlagen.

Lymphoid tissue compartmentalization The factors responsible for LTi cell recruitment into the lymphoid tissue anlagen are also responsible for the attraction of lymphocytes17, 26-28. As B and T cells arrive, they seem to take over the role of

LTi cells as the main source of LTα1β2 and TNF, therefore contributing to the further differentiation of the different stromal cell subsets that compose the backbone of definitive lymphoid organs and are responsible for their ultimate architecture29-33. Importantly, different stromal cell subsets are present within the different microdomains of lymphoid organs (fig. 1). In a schematic way, SLOs can be divided in three different microdomains: an outer antigen- sampling zone in which pathogens present in lymph () or blood (spleen) are captured; a area, rich in T cells and dendritic cells (DCs); and B cell follicles, containing B cells. The antigen-sampling zones of lymph nodes (subcapsular sinus) and spleen (marginal zone) are -rich areas, which are becoming increasingly recognized as central to the initiation of immune responses, as evidence that antigen processing and presentation can take place in these regions is growing6, 34-38. The structural support for the antigen-sampling zones of SLOs is provided by MRCs13 (fig. 1a). These cells were recently identified by Katakaiet al. as CD45- non- hematopoietic cells expressing ICAM-1, VCAM-1, mucosal 1 (MAdCAM-1) and TRANCE13. Importantly, these cells produce the CXCL1313, which has been shown to have a non-redundant role in the development of peripheral lymph nodes20. Therefore, it was been suggested that MRCs may represent the adult counterpart of LTo cells and may be involved in the re-establishment of the steady-state lymphoid tissue architecture upon the clearance of inflammatory insults39. The T cell areas of SLOs – the periarteriolar lymphoid sheath of the spleen, the paracortex of lymph nodes and the interfollicular region of Peyer’s patches and colonic patches – are supported by a scaffold of FRCs (fig. 1c). FRCs are readily identified by their capacity to produce the chemokines CCL19 and CCL2140, which are crucial for the delineation of T cell areas41, 42. Importantly, FRCs in the T cell zones of spleen and lymph nodes surround the central arterioles and (HEVs), respectively, forming a sponge-like network that controls the interstitial migration of lymphocytes upon their arrival at SLOs41, 42. As CD4+ and CD8+ T cell move along the guidance paths laid down by FRCs41, 42, they will receive input from DCs, which are anchored to the FRC network43. B cell follicles are structured by a dense network of FDCs (fig. 1b). Similarly to T cells in the T cell area, B cells in B cell follicles are in close contact with stromal cells41, which provide

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Figure 1. Lymphoid tissue compartmentalization by lymphoid stromal cells. Lymphoid tissues, here represented by a lymph node, have a highly structured architecture including distinct antigen-sampling zones (1), B cell follicles (2) and T cell areas (3). Each one of these areas is organized by distinct lymphoid stromal cell populations. 1. Antigen-sampling zones (subcapsular sinus in lymph nodes) are supported by marginal reticular cells (MRCs). 2. B cell follicles are built by CXCL13-producing follicular dendritic cells (FDCs). CXCL13 attracts CXCR5+ B cell into the follicle. 3. T cell areas are assembled by fibroblastic reticular cells (FRCs). FRCs produce CCL19 and CCL21, which together delimit the T cell area by attracting CCR7-expressing T cells.

them essential survival and differentiation factors44-48. Noteworthy, FDCs produce CXCL1317, which is essential for the delineation of B cell follicles via the attraction of CXCR5+ B cells26. Importantly, during immune responses, germinal centres are organized in a germinal centre light zone, in which B cells interact with FDCs producing CXCL13, and a germinal centre dark zone, in which these interactions are commanded by CXCL1249. It is, however, presently unclear whether CXCL12-producing stromal cells are a specialized subset of FDCs or a yet unidentified stromal cell subset. The structural support provided by the stromal cells mentioned above is mediated to a large extent by the production of (ECM) components, such as , and laminin32. Importantly, these components do not directly contact with lymphocytes, as they are secreted throughout the stromal cell surface opposite to the lymphocytes and are

113 completely enwrapped by the stromal cells43. Directional secretion of ECM components leads to the development of conduits, which form a web of tubes that extends from the antigen- sampling zone throughout the entire T cell area and B cell follicles towards central arterioles and HEVs43, 50-52. The conduits consist of an outer tube made up of stromal cells and their basement membrane and an inner core made of parallel type I and III collagen fibers, which are physically separated by a microfibrillar zone rich in 1 and fibrillin43 2 . Notably, conduits serve as a highway that allows the rapid diffusion of small immune modulators, such as antigens, chemokines and cytokines, produced either within the SLO itself or in the peripheral tissues five drained by the SLO43, 50-52. In the T cell zone, DCs anchored to the FRC network seem to extend short protrusions through the basement membrane of conduits43. Reaching the conduit lumen, they are thought to sample its contents, which may lead to the presentation of conduit-derived antigens to T cells43. Importantly, recent data suggest that FRCs lay down the entire conduit system, which are afterwards replaced by FDCs and MRCs in the B cell follicles and antigen- sampling areas of SLOs, respectively53.

Lymphoid stromal cell development The origin and differentiation of lymphoid stromal cells is still an elusive subject. Based on cell lineage, SLO stromal cells should be classified in two main groups: whereas BECs and LECs would be grouped into the endothelial family, embryonic LTo cells as well as adult FRCs, FDCs and MRCs would constitute the mesenchymal group. Supporting this classification, we have recently found, by using Nestin-Cre x Rosa26-EGFP mice, in which activation of Cre led to the permanent labeling of the progeny of Nestin+ mesenchymal stem cells, that FRCs, FDCs and MRCs share a common progenitor (REM, unpublished observations). Similarly, Liao and Ruddle have previously unraveled the presence within adult SLOs of cells with a mixed phenotype between BECs and LECs, which may be responsible for the synchrony between blood and lymphatic vascular development observed during immune responses54. These results question, however, when stromal cell lineage commitment occurs and whether such commitment is permanent. Despite these uncertainties, it is however secure to assume that lymphoid stromal cells differentiate under the influence of immune cells (fig. 2). The embryonic development of LTo cells may start independently of immune cells, as retinoic acid-secreting cells, most likely neurons, induce the production of CXCL13 in adjacent mesenchymal cells20. Subsequently, retinoic acid- induced CXCL13 promotes the recruitment of CXCR5-expressing LTi cells into the SLO anlagen20.

LTi cells in turn interact with the primitive LTo cells, via LTα1β2-LTβR, inducing their differentiation into mature VCAM-1highICAM-1highMadCAM-1+ LTo cells55, which are now able to efficiently retain hematopoietic cells14-16. In the Peyer’s patch anlagen, this differentiation step may be additionally stimulated by lymphoid tissue initiator (LTin) cells that cluster at the prospective sites of Peyer’s patch development due to RET signaling and induce VCAM-1 expression56, 57. MRCs, similarly to their embryonic LTo cell counterparts, require continuous LTβR signalling to maintain their differentiated status13, 58. The necessary LTβR signals are likely to have origin in adult LTi-like cells produced in the , as severe combine immunodeficiency (SCID) mice, which lack both T and B cells, still have CXCL13-expressing stromal cells59.

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Figure 2. Lymphoid stromal cells differentiate in contact with immune cells. 1. Lymphoid tissue organizer (LTo) cells develop under the influence of retinoic acid, which is most likely being produced locally by neurons. Retinoic acid induces the synthesis of CXCL13, which recruits lymphoid tissue inducer (LTi) cells into the site of prospective lymphoid tissue development. LTα1β2-expressing LTi cells drive the full differentiation of LTβR+ LTo cells, which are characterized by the secretion of IL7, TRANCE, CCL19, CCL21, CXCL13 and VEGF-C. These factors drive the recruitment of additional immune cells into the developing lymphoid tissue, as well as their survival and further differentiation. 2. In the developed lymphoid tissue, LTi cells are thought to maintain marginal reticular cells (MRCs) via LTα1β2-LTβR interactions. 3. B cells induce the differentiation of follicular dendritic cells (FDCs) via LTα1β2 and TNF. 4. T cells differentiate fibroblastic reticular cells (FRCs) via LTα1β2 and TNF. 5. Dendritic cells, emigrating from peripheral tissue toward the vicinity of high endothelial venules (HEVs), maintain the mature phenotype of the HEV endothelium.

115 FRC development depends on TNF-R1 and LTβR signalling25, 32, 33. Within the spleen, these 33 60 signals seem to be delivered by B cells , as neonatal splenic LTi cells lack LTα1β2 expression . Within the lymph node, however, B cells are dispensable, as mice lacking them have been shown to develop normal FRC networks33, 53. Indeed, as in vitro ECM deposition by stromal cell lines obtained by long-term culture of lymph node stromal cells has been observed upon co- culture with CD4+ T cells32, it is more likely that, within lymph nodes, T cell recruitment may be the driving force behind FRC differentiation. Notwithstanding, B cell recruitment leads to the remodeling of the primitive FRC network into the more sparse reticular network found within five B cell follicles53. Development of FDC networks also depends on TNF-R1 and LTβR signaling. B cells (and 61 29-31, 48, 62, 63 possibly also T cells ) need to express TNF and LTα1β2 to mediate FDC differentiation . Importantly, TNF-R1 and LTβR induce distinct NF-kB signaling pathways – while TNF-R1 exclusively induces the canonical NF-kB signaling pathway, LTβR induces both the canonical and alternative signaling pathways24, 64, 65 – that differentially affect FDC development. Indeed, FDC networks seem to be normal in mice deficient in components of the classical NF-kB signaling pathway (p50-/- and c-Rel-/- mice), and largely absent in mice deficient in components of the alternative NF-kB signaling pathway (p52-/-, RelB-/- and NIK-/- mice)66-71. Both p52-/- and RelB-/- mice show a complete lack of FDC-M1+ networks in B cell follicles and a reduction in CXCL13 expression66-68, 70. These results would suggest that the alternative NF-kB signaling pathway alone is both essential and sufficient to induce the development of FDCs; however, ablation of the canonical NF-kB signaling pathway specifically on FDCs (inhibitor of IkB kinase (IKK)2fl/flx CD21-Cre mice) impairs the proper development of B cell responses72 indicating that FDCs require the classical NF-kB pathway to be fully functional. Finally, it is worth mentioning that the development and functionality of FDCs also depends on TLR signalling46, 73. The recognition of danger signals by FDCs increases the expression of ICAM1 and VCAM1 on the surface of FDCs promoting the accumulation of B cells in germinal centers46, 73. The development of the endothelial lineages within SLOs is far less understood. LECs originally arise from venous BECs that have acquired the expression of the transcription factor Prox-1 and budded from their original position within the blood vasculature74, 75. These cells migrate towards the place of prospective lymph node formation by unknown mechanisms and encapsulate the developing SLO76. For a long time, it was thought that formation of lymph sacs was the initial step in lymph node formation; however, recent research has showed that they are not required for the initial clustering of LTi cells at the lymph node anlagen76. Similarly to LECs, BECs are also likely to differentiate outside SLOs and only later migrate into their parenchyma. A special feature of the blood vasculature within lymph nodes and Peyer’s patches is the formation of HEVs that allow the continuous extravasation of naïve lymphocytes from the bloodstream77. HEVs are specialized post-capillary venous enlargements characterized by the cuboid appearance of their endothelial lining and the expression of – peripheral node addressin (PNAd) in peripheral and mesenteric lymph nodes and/or MAdCAM1 in mesenteric lymph nodes and Peyers’patches77. We have long ago showed, by surgically severing lymphatic vessels, that these particular features of HEVs within peripheral lymph nodes were dependent on factors present in the afferent lymph78. These results were recently extended by another research

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group, which showed that DCs migrating in the afferent lymph travel towards the vicinity of HEVs and engage BECs, thereby inducing LTβR signalling, which is required for the maintenance of the HEV’s functional phenotype as it induces the expression of a wide set of genes involved in addressin synthesis79.

Lymphoid stromal cells and lymphocyte homeostasis five Lymphocyte pools are kept constant by the steady release of lymphocytes from the and bone marrow, balanced by their death in the periphery80. The lifespan of T cells is controlled, on one hand, by tonic TCR stimulation as evidenced by reduced survival of CD8+ and CD4+ T cells in the absence of MHC class I and MHC class II molecules, respectively81-83; and on the other hand, by cytokine signals, such as those provided by IL784-86. Importantly, these signals are delivered to T cells within SLOs, as deprivation of SLO access causes the death of most naïve T cells84, 87. Similarly, T cells in LTα-/- mice, which lack lymph nodes and organized splenic white pulp areas, fail to undergo homeostatic proliferation and therefore fail to restore their homeostasis under lymphopenic conditions87. In this regard, it was recently shown that FRCs support the survival of naïve CD8+ and CD4+ T cells by providing both IL7 and CCL1984. Likewise, B cells also receive the necessary trophic factors within SLOs, as their exclusion from follicular niches also results in cell death44. FDCs express high levels of B cell activating factor (BAFF)45, 46 and are therefore a likely candidate to control B cell homeostasis. Lymphocyte survival factors within SLOs are limited. Such limitedness drives competition between lymphocytes, which ensures that their numbers remain constant and their repertoire diverse80. This is particularly important, as the maintenance of a restricted but diverse repertoire assures that pathogen-specific lymphocytes are likely present within the host, while avoiding potential self-reactive lymphocytes to become dominant and cause immunopathology. Importantly, T as well as B lymphocytes emerge from the primary lymphoid organs as immature cells. To become fully functional naïve lymphocytes, which are able to properly respond to antigenic stimulation, recent emigrants must undergo maturation within the periphery. This maturation occurs within SLOs, although the process by which it is achieved differs for each lymphocyte population. Maturation of recent thymic emigrants (RTEs) requires egress from the thymus and recirculation through either lymph nodes or spleen88. Without egress from the thymus (“wannabee” RTEs) and residence in SLOs (“homeless” RTEs), RTEs remain partially immature, secreting reduced levels of IL2 and proliferating less vigorously88-90. In contrast, immature recent B cell emigrants actively exclude themselves from lymph nodes via secretion of IFNγ, which prevents their -mediated adhesion to the extracellular matrix protein fibronectin91. As a consequence, their final maturation into IgM+IgDhigh cells occurs in the spleen92, 93. Importantly, it was recently shown that during this developmental stage in the spleen, B cells encounter their cognate antigens and as a consequence downmodulate the surface expression of their IgM B cell receptor, thereby increasing their activation threshold94. Therefore, it seems that splenic B cell maturation further imposes tolerance by functionally suppressing the autoreactive B cell repertoire.

117 Lymphoid stromal cells and leukocyte migration Lymphocyte recirculation in the steady-state As mentioned previously, the frequency of antigen-specific lymphocytes is relatively low – estimations ranging from 20 to 200 and 80 to 1200 naïve antigen-specific precursor cells per mouse for CD4+ and CD8+ T cells, respectively1, 2. To facilitate the recognition of cognate five antigens, naïve lymphocytes continuously recirculate through SLOs, which also continuously gather information from the periphery becoming a mirror of its health status that can be easily scrutinized95. Lymphocyte recirculation entails the recruitment of cells from the bloodstream, their migration through the lymphoid environment along its stromal cell networks and, eventually their egress through lymphatic or blood vessels and migration to other SLOs. Importantly, many of these steps are controlled by factors produced by and/or expressed on lymphoid stromal cells. Extravasation of naïve lymphocytes from the bloodstream into lymph nodes and Peyer’s patches begins with adhesive interaction between lymphocytes and the endothelial lining of HEVs96, 97 (fig. 3). This process is mediated by the expression of selectins on lymphocytes and adhesive molecules, such as PNAd and MAdCAM1, on BECs98-101. Due to the weak interaction between these molecules and the shear force caused by the blood flow, lymphocytes do not initially attach strongly and therefore start to roll along the vessel wall96, 97. During rolling, lymphocytes will be increasingly stimulated by chemokines attached to the luminal surface of BECs. Naïve CCR7- expressing T cells are stimulated by the chemokines CCL19 and CCL21102; central memory T cells by CXCL12103; and B cells by the aforementioned chemokines as well as CXCL13104. Importantly, not all these chemokines seem to be produced by BECs and therefore their expression on the luminal surface of BECs may rather rely upon their transport across endothelial cells105, 106. At the luminal side, chemokines remain attached to heparan sulphate107. Chemokine receptor triggering on the rolling lymphocytes induces many cellular events, including cytoskeletal rearrangement and integrin activation, which together allow the firm arrest and spreading of lymphocytes on the ICAM-1+VCAM-1+ HEV luminal surface100, 108. After firm arrest, lymphocytes may have to crawl to nearby cellular junctions, where they are allowed to cross into the SLO parenchyma109, 110. The process of leukocyte junctional diapedesis is still enigmatic, but it is likely to require both stromal cell-derived adhesion molecules as well as chemokines. Traditionally, it is thought that lymphocytes follow a chemotactic gradient in order to enter tissues; however, as both in vitro and in vivo evidence suggest that chemokines are chemokinetic rather chemotactic111-113, it is more conceivable that chemokines simply trigger the necessary cell movement to push lymphocytes across predefined transendothelial paths. After crossing the endothelial layer and its basement membrane, lymphocytes are transiently trapped in a perivascular space built by the FRCs surrounding HEVs, which they exit preferentially at predefined locations to enter the SLO parenchyma41, 114. Following entry into the SLO parenchyma, T and B cells come into immediate contact with FRCs41 and quickly move to their respective domains attracted by stromal cell-derived chemokines26, 41, 42. Interstitial T cell motility is promoted by the CCR7 ligands CCL19 and CCL21

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Figure 3. Lymphocyte migration into the lymphoid tissue parenchyma. Lymphocyte extravasion from the bloodstream into lymph nodes or Peyer’s patches is initiated by adhesive interactions between lymphocytes and high endothelial venules (HEVs), which are mediated by selectins on the lymphocyte surface and adhesion molecules on endothelial cells (1). Under shear forces, these interactions are labile and thus cause lymphocyte rolling. During rolling, lymphocytes are stimulated by chemokines attached to the endothelial cell surface (2). Chemokine receptor signaling induces conformational activation of allowing firm arrest and spreading of lymphocytes on the integrin ligands (ICAM-1, VCAM-1) exposed on HEVs (3). Subsequently, lymphocytes crawl to nearby cellular junctions and undergo junctional diapedesis into the lymphoid tissue parenchyma. After crossing the endothelial layer, lymphocytes are transiently trapped in the perivascular space built by the and fibroblastic reticular cells (FRCs) that surround HEVs. They migrate into the lymphoid tissue parenchyma sensu stricto via preformed portals coming into immediate contact with FRCs along which they move stimulated by chemokines (4).

immobilized on the surface of FRCs112, 113, 115. Conversely, B cell motility is mainly dictated by CXCL13 expressed on FDCs and MRCs116. CCR7 ligands, however, may also contribute to B cell migration towards B cell follicles, as B cells initially crawl on top of the FRC network41. Importantly, chemokine receptor signaling in the absence of shear stress does not induce integrin activation117 and therefore does not lead to lymphocyte firm arrest in the lymphoid environment; indeed, leukocyte migration in lymph nodes and in artificial three-dimensional matrixes has been shown to occur independently of integrins117, 118. Instead, chemokine receptor signaling promotes a seemingly random walk on top of the stromal networks41, 42, which facilitates cognate antigen search. During antigen search, T and B cell migration is restricted to their specific domains, with cells making sharp turns as they reach the boundaries of their respective areas119. The majority of lymphocytes do not succeed at finding their cognate antigen and therefore, after a searching period of half a day to a day, they will leave the SLO in which they are and return to the circulation. Lymphocyte egress occurs in a sphingosine-1-phosphate (S1P)-dependent manner120. S1P concentrations are higher in blood and lymph as compared to SLOs owing to the specific action of S1P lyase in lymphoid tissues121. Therefore, this enzyme is thought to create a S1P gradient that imprints directionality towards the blood and lymph upon S1P receptor 1 (S1P1) triggering. While plasma S1P is produced mainly erythrocytes and determines lymphocyte

119 egress from the spleen to the blood across venous sinuses, lymph S1P is produced by radio- resistent Lyve1+ LECs and drives egress from lymph nodes towards the lymph via lymphatic cortical sinus122, 123. Importantly, S1P stimulation leads to the internalization of its receptor124. Surface S1P1 expression remains low during transit in the blood and lymph, but steadily increases upon re-entry in SLOs124. This cyclical pattern of S1P1 expression is thought to control the ability of lymphocytes to overcome the retention signals present within the SLO parenchyma and therefore to determine the time spent by them within each SLO125. In this regard, it was recently shown that CCR7 and S1P1 signaling antagonize each other, as CCR7-deficient T cells are less efficiently five retained within lymph nodes upon pharmacological inhibition of S1P1 than wild-type T cells are and inactivation of Gαi-protein coupled chemokine receptors restores the egress competence of S1P1-deficient T cells125.

Leukocyte migration during immune responses An immediate consequence of infection is the transient increase in lymph node cellularity, which is accomplished by simultaneously enhancing lymphocyte homing to and blocking lymphocyte egress from SLOs126. This concerted change in the lymphocyte recirculation pattern enhances the likelihood of lymphocytes finding their cognate antigen. Enhanced lymphocyte recruitment seems to occur initially in response to the thermal stress (fever) caused by the release of inflammatory mediators, as this induces an increase in arterial vessel diameter and in the intravascular display of ICAM1 and CCL21 on HEVs127. In addition to CCL21 presentation, which is likely to account for the increased recruitment of CCR7+ naïve lymphocytes127, presentation of CCL3, CCL4, CXCL9 and CXCL10 on HEVs also takes places128-130. Presentation of CCL3 and CCL4 is likely responsible for enhanced recruitment of CCR5+ memory T cells126, 130, and therefore may account for the rapid and vigorous onset of secondary immune responses. Conversely, display of CXCL9 and CXCL10 allows the recruitment of not only natural killer (NK) cells but also of CCR7-CXCR3+ effector T cells129. NK cells provide interferon γ (IFNγ) for the polarization of Th1 responses129; CCR7-CXCR3+ effector CD8+ T cells kill DCs presenting their cognate antigen128, potentially contributing to the resolution of the immune response. Similarly to their role in the early enhancement of lymphocyte recruitment, the early release of inflammatory mediators is also responsible for the suppression of lymphocyte egress131, 132. Type I IFNs induce the rapid increase in the expression of CD69 on lymphocytes, which sequesters the egress receptor S1P1 intracellularly and thus prevents S1P signaling131. Interestingly, high levels of type I IFNs were shown to induce the selective death of non-specific T cells during the early stages of immune responses to infection133. This, presumably, ensures that sufficient room and survival factors are available for the massive expansion of antigen-specific lymphocytes that will happen. More importantly, it may also ensure that non-specific lymphocytes are not accidentally activated within the highly inflammatory environment of the reactive SLO. The source of both the inflammatory mediators and the chemokines that lead to the accumulation of lymphocytes within the reactive SLOs is presently unclear. They could be produced locally in the SLOs, in which case lymphoid stromal cells would be a likely producer, or they could be produced in the infection site and transported to the HEVs through the blood or afferent lymph and the SLO conduit system43, 50, 51.

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Finally, it must be considered that in order to fulfill their main function, i.e. facilitate the induction of immune responses, SLOs must also capture and present antigens. Antigens can reach these organs either as soluble entities or associated with antigen-presenting cells, such as DCs95. migration towards lymphoid organs is associated with their maturation program, which includes a dramatic shift in chemokine receptor expression134, 135. In particular, CCR7 upregulation makes DCs responsive to LEC-derived CCL21, thereby forcing their migration into lymphatic vessels and SLOs136, 137. Importantly, similarly as interstitial lymphocyte migration, dendritic cell intravasation into lymphatic vessels does not require integrin-mediated adhesive interactions118; nor does it require pericellular proteolysis138. Instead, DCs squeeze themselves five through the discontinuous perilymphatic basement membrane and flap valves of lymphatic vessels138. DC squeezing through these preformed portals is dependent on the production of the semaphorin Sema3a by the lymphatics139. Sema3a promotes actomyosin contraction at the trailing edge of the migrating DC, which induces a forward movement that allows its passage through the narrow gaps of the lymphatic endothelium139. Importantly, lymph node VEGFa- expressing resident dendritic cells and later on VEGFa-expressing B cells induce reactive lymph nodes to expand their lymphatic network, which in turn supports increased DC migration from the periphery140-143. Similarly, VEGF production by FRCs promotes the proliferation of BECs and the consequent growth of the blood vasculature tree144, which may enhance the mobilization of lymphocytes from the bloodstream. Although DCs accumulate close to HEVs145, the mechanisms by which lymphocytes initially find the appropriate antigen-bearing DCs within reactive SLOs are still obscure. It may be only a stochastic event, whose efficiency depends on both the frequency of antigen-specific lymphocytes and antigen-presenting DCs, or it may be that mature DCs produce factors that specifically attract lymphocytes to their vicinity. In this regard, it should be noted that DC maturation is linked to the production of three different waves of chemokines146. The third and last wave, which occurs at a time when DCs should have reached the draining SLO, is characterized by the production of CCL19 and CXCL13146, which are known attractants of naïve T and B lymphocytes, respectively. Following the initial antigen-driven contact between CD4+ T cells and DCs, it is, however, clear that these CD4+ T cell-DC conjugates start secreting CCL3 and CCL4, which attract CCR5- expressing naïve CD8+ T cells147. CCR5 becomes expressed on naïve CD8+ T cells in an antigen- independent way upon their entry in reactive SLOs147. Also soon after activation, T and B cells change their chemokine receptor profile and thus their chemokine responsiveness. Activated T cells decrease their CCR7 expression and migrate to the border of the T and B cell domains148. Likewise, B cells also migrate to the T/B cell border, but as a result of CCR7 upregulation149. This movement of antigen-engaged T and B cells towards each other facilitates the delivery of T cell help and consequently accelerates the development of T-dependent B cell response150. Importantly, strong cognate interactions between T and B cells promote the differentiation of follicular helper T (Tfh) cells148, 151. Tfh cell differentiation induces the expression of CXCR5 through Bcl6-mediated suppression of CXCR5 antagonizing miRNAs151-153. CXCR5 expression enables Tfh cells to move into the B cell follicle attracted by FDC-derived CXCL13, where they will keep providing help to B cells in the germinal center150. Importantly, as antigen-engaged B cells move from the T/B border back to the follicle, by virtue of CCR7 downregulation, they

121 initially migrate to the outer and inter follicular regions in a process regulated by the G-protein coupled receptor Epstein-Barr Virus-induced molecule 2 (EBI2)154, 155. This early movement to outer and inter follicular regions seems to favour the early plasmablast response, as adoptively transferred EBI2-/- B cells give rise to reduced IgM and IgG titers154, 155. Lymphoid stromal cells are the cellular source of the EBI2 ligand 7α,25-dihydroxycholesterol (7α25-DHC)156, 157. Subsequently, as antigen-engaged B cells differentiate into germinal center B cells, they downregulate EBI2 and move to the FDC-rich follicle center154, 155. In this location, B cells receive several signals from the FDC network, including BAFF, IL6 and Notch ligands, which together promote B cell survival, five somatic hypermutation and class switch158-161.

Homing of activated lymphocytes From the analysis of the data discussed above, it is clear that SLO stromal cells play a critical role in the regulation of immune responses. However, their role in this regulation does not end with the encounter of antigen-presenting cells with lymphocytes. Indeed, after they have promoted cognate interactions between dendritic cells and T lymphocytes and between T and B cells, they will also control the characteristics of the ensuing responses. Importantly, they will determine the tissue tropism of the effector cells generated (fig. 4). Gut tropism is dictated by the expression of the integrin α4β7 and the chemokine receptor CCR9, whose induction critically depends on the vitamin A metabolite retinoic acid (RA)162-164. The RA-producing enzyme retinaldehyde dehydrogenase type 2 (RALDH2) is expressed by the stromal cell compartment of mesenteric lymph nodes, but not of skin-draining lymph nodes, thereby conferring to the former the unique ability to imprint gut homing165, 166. T cell migration to the skin, on the other hand, requires expression of E- and P-selectin ligands and expression of CCR4 and/or CCR10167-171. The induction of these skin-homing molecules occurs preferentially in skin-draining lymph nodes172, suggesting that the stromal cells of these lymph nodes dictate their induction. Definitive proof for this is, however, lacking as the molecular mechanisms that drive the induction of skin-homing molecules remain elusive. Importantly, imprinting of tissue-specific homing receptors is not definitive and under appropriate stimulatory conditions can be reversed173, 174. In this regard, it was recently shown that, upon completion of 3 division cycles, antigen-activated CD8+ T cells leave the reactive lymph node and migrate to both the site of infection as well as to distant antigen-free lymph nodes175. In this later location, CD8+ T cells acquire additional homing-specificity, which is thought to provide systemic protection against the dissemination of the pathogen and against secondary challenges even if they occur through a distinct route175.

Contraction of immune responses It is thought that once sufficient numbers of effector cells have been generated and the infectious insult cleared, the immune response undergoes a programmed contraction. During this phase, the majority of the antigen-activated cells generated during the immune response die off176; and the surviving cells form a pool of long-lived memory cells, which populates non-lymphoid tissues177. There is little insight into which factors, if they exist, trigger the contraction phase. Badovinac et al. have elegantly showed that the onset and kinetics of CD8+ T cell contraction after two different infections were independent of the infectious dose/antigen display and the magnitude of the initial CD8+ T cell expansion178, therefore suggesting that the contraction phase

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Figure 4. Lymphoid stromal cells imprint tissue homing characteristics on activated lymphocytes. In lymphoid tissues, naïve antigen-specific lymphocytes get activated by antigen-carrying dendritic cells, proliferate and differentiate into effector lymphocytes. a. In mucosa-draining lymph nodes, effector lymphocytes acquire gut-tropism through the induced expression of CCR9 and α4β7 integrins. These molecules are induced by retinoic acid secreted from retinaldehyde dehydrogenase type 2 (RALDH2)- expressing dendritic cells and lymphoid stromal cells. b. In skin-draining lymph nodes, effector lymphocytes acquire CCR4 and/or CCR10 and E- and P-selectin expression, which direct them to the skin. The factors produced by skin-draining lymph node stromal cells that induce this differentiation program on lymphocytes are unknown, although vitamin D has been proposed to induce at least some characteristics of skin tropism.

is a hardwired pre-programmed event. What it is currently known is how antigen-activated CD8+ T cells become committed either to cell death or to cell survival. Such decision seems to be dependent on the intrinsic ability of activated T cells to use IL7179. While killer cell lectin-like receptor subfamily G member 1 (KLRG1)hiIL7Rlo CD8+ T cells cannot respond to IL7 and therefore die relatively fast, KLRG1loIL7Rhi CD8+ T cells readily use IL7 and survive for extended periods179. The IL7-dependent nature of memory T cell survival, suggest that FRCs, which are the main source of IL7 in SLOs84, may impose themselves in the decision between effector cell death and memory cell survival. In line with this, it was been previously reported that the FRC chemokines CCL19 and CCL21 promote activation-induced cell death in activated T cells180. Consequently, it may not be surprising that the release of IFNγ by activated T cells, during Th1 priming, reduces the expression of homeostatic chemokines at the peak of the immune response181. Such downregulation, on the one hand, may allow activated T cells present within reactive SLOs to freely expand; however, on the other hand, it may also induce a temporary susceptibility to secondary infections, as new naïve lymphocytes cannot be recruited into SLOs and therefore cannot be primed against a second antigen while the first response is underway181. Furthermore, IFNγ release by activated T cells (together with TNF) induces FRCs and LECs to express the enzyme inducible nitric oxide synthase (iNOS), which catalyzes the production of nitric oxide (NO)182-184. NO inhibits the proliferation of activated T cells, preferentially Th1 cells, providing protection against unrestrained T cell activation and its deleterious effects182-184.

123 Antigen-presentation by lymphoid stromal cells Although the main function of SLOs, as discussed above, may be the promotion of adequate immune responses, they have recently been shown to be crucial for the induction of as well185-191. Importantly, this function seems to be differently regulated within different SLOs as, by means of lymph node transplantation, cervical lymph nodes, but not peripheral lymph nodes, were found to induce tolerance to inhaled antigens192. Given that upon five lymph node transplantation, only the stromal cell compartment remains of donor origin165, 166, these data suggested that the stromal cells within the cervical lymph node were instrumental in the induction of tolerance. In line with this, several groups have now shown that SLO-resident stromal cells promote CD8+ T cell tolerance185-188, 190, 191. Lee et al. showed that expression of the model antigen ovalbumin (OVA) as a self antigen under the control of the intestinal fatty acid-binding protein (iFABP) promoter led to the presentation of OVA peptides in the context of MHC-I molecules and consequently to the deletion of OVA-specific OT-I CD8+ T cells190. Similarly, expression of the influenza hemagglutinin (HA) gene under the control of the promoter of the glial fibrillary acidic protein (GFAP-HA mice) was found to drive the deletion of HA-specific CD8+ T cells191. These results were further extended by showing that presentation of endogenously expressed antigens by lymph node stromal cells also leads to abortive activation and death of antigen-specific CD8+ T cells185-188 (fig. 5). Indeed, SLO-resident cells were found to constitutively express several peripheral tissue restricted antigens (PTAs)185, 186. Importantly, PTA expression is not restricted to a single stromal cell population – CD45-gp38+CD31- FRCs as well as CD45- gp38-CD31+ BECs, CD45-gp38+CD31+ LECs and CD45-gp38-CD31- double negative stromal cells all express PTAs185, 186. Notwithstanding, PTA expression in these different stromal cell subsets seems to be differentially regulated. Whereas the transcriptional regulator autoimmune regulator (AIRE) seems to control PTA expression on CD45-gp38-CD31- double negative stromal cells as well as extrathymic AIRE-expressing cells (eTACs), the evidence available so far suggests that AIRE does not play a role in the induction of PTAs in FRCs, BECs or LECs185-187. Indeed, in these latter three stromal cell subsets the expression of another transcriptional regulator, deformed epidermal autoregulatory factor 1 (DEAF-1), seems to control the expression of several potential PTAs189. Importantly, although AIRE and DEAF-1 seem to control distinct sets of PTAs, one should not forget that these two transcriptional regulators may cooperate to ensure optimal PTA expression, as evidenced by the downregulation of well know AIRE target genes in DEAF- 1-deficient pancreatic lymph nodes189. In addition, it must be acknowledged that lymphoid stromal cells also express MHC-II molecules187 and therefore are also likely to present PTAs to CD4+ T cells. To our knowledge, so far only one study assessed the consequences of lymph node stromal cell-mediated antigen- presentation on CD4+ T cell immunity. Magnusson et al. found that, in contrast to direct antigen- presentation to CD8+ T cells, direct antigen-presentation by stromal cells to CD4+ T cells did not cause their deletion nor prevented their effector function191. Finally, it would be interesting to assess whether lymphoid stromal cells can capture and present exogenous antigens. Given their physical association with the SLO conduit system,

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Figure 5. Lymphoid stromal cells enforce peripheral tolerance via auto-reactive CD8+ T cell elimination. The transcription factors autoimmuneregulator (AIRE) and deformed epidermal autoregulatory factor 1 (Deaf-1) induce peripheral tissue restricted antigen (PTA) expression on lymphoid stromal cells. PTAs are processed and presented to CD8+ T cells on MHC-I molecules, leading to abortive CD8+ T cell proliferation and deletion.

which transports low-molecular-weight antigens as well as inflammatory mediators, this seems a reasonable hypothesis.

Concluding remarks The presence of stromal cell elements within SLOs has been described a long time ago. Despite this, however, their systematic study has just started. In the past few years, SLO-resident stromal cells were found to play a crucial role in virtually all the peripheral processes that ensure the homeostasis of the immune system and consequently host protection. They contribute to the peripheral maturation of recent thymic emigrants as well as recent immature B cell bone marrow emigrants88, 92, 93; they determine the size of peripheral T and B lymphocyte pools44, 84, 87; they select the repertoire within those pools by inducing the deletion of auto-reactive CD8+ T cells185-188, 190, 191, and therefore constitute themselves as a safeguard of immune tolerance towards self; they might even have a similar tolerogenic function towards B cells by increasing the threshold for B cell activation94; they ensure that antigen-specific lymphocytes recognize their cognate antigens by providing anchorage for antigen-presenting cells and controlling lymphocyte migration41-43; they provide instructive signals to activated lymphocytes, such as imprinting of homing information165, 166, 172; and they may control memory T cell formation. As new research develops and our knowledge about these cells progresses, new functions are likely to be added to this already long list. More importantly, the future might see the emergence of lymphoid stromal cells as potential targets for immune interventions aimed either at promoting immune responses against pathogenic insults or at preventing inadequate immune responses.

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