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Fibroblastic Reticular Cells: Organization and Regulation of the T Life Cycle

This information is current as Flavian D. Brown and Shannon J. Turley of September 29, 2021. J Immunol 2015; 194:1389-1394; ; doi: 10.4049/jimmunol.1402520 http://www.jimmunol.org/content/194/4/1389 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Brief Reviews Immunology

Fibroblastic Reticular Cells: Organization and Regulation of the T Lymphocyte Life Cycle Flavian D. Brown*,† and Shannon J. Turley‡ The of any in the body is gener- tissues (3). As such, the LN serves as a central site for every stage ally referred to as stroma. This complex network is of the T life cycle by recruiting naive T cells from the , commonly composed of leukocytes, promoting naive survival, providing an environment for components, mesenchymal cells, and a collection of T cell differentiation or tolerance, and influencing the homing , blood, and lymphoid vessels. Once viewed pri- properties of memory T cells. marily as a structural entity, stromal cells of mesenchy- In addition to hematopoietic cells, the LN contains specialized mal origin are now being intensely examined for their stromal cells, including blood endothelial cells, lymphatic en- ability to directly regulate various components of im- dothelial cells, follicular dendritic cells (DCs), marginal reticular Downloaded from a + mune cell function. There is particular interest in the cells, integrin 7 pericytes, and fibroblastic reticular cells (FRCs) (4–6). LN-resident stromal cells were long viewed simply as ability of stromal cells to influence the homeostasis, ac- structural determinants, uninvolved in immune cell homeostasis tivation, and proliferation of T . One exam- or ongoing immune responses. However, a series of publications ple of this regulation occurs in the node, where over the past decade uncovered several fascinating immunoreg-

fibroblastic reticular cells support the maintenance of ulatory properties of LN stromal cells. In particular, FRCs are http://www.jimmunol.org/ naive T cells, induce Ag-specific tolerance, and restrict concentrated in the paracortical region (T cell zone) of the LN the expansion of newly activated T cells. In an effort to and are endowed with several functions that regulate the activity highlight the varied immunoregulatory properties of of T lymphocytes. fibroblastic reticular cells, we reviewed the most recent FRCs are thought to originate from mesenchymal pre- advances in this field and provide some insights into po- precursors in the microenvironment of the LN tential future directions. The Journal of Immunology, anlagen during ontogeny (7). Engagement of the lympho- 2015, 194: 1389–1394. toxin-b receptor on these precursors drives their differentia-

tion into lymphoid tissue–organizing cells, which ultimately by guest on September 29, 2021 leads to the development of myofibroblastic precursors that he life cycle of T lymphocytes begins in the give rise to mature FRCs in the postnatal LN (7–11). The as immature precursor T cells undergo positive and + + T cell zone of the adult LN is especially enriched with the T negative selection to mature into CD4 and CD8 presence of mature FRCs characterized by the expression of single-positive cells (1). Following migration from the thymus, podoplanin (gp38) and extracellular matrix proteins, such as T cells recirculate from the blood through lymph nodes (LNs) ERTR-7 and (6). We now know that into lymphatics and back into the blood, searching for the recruitment to and survival within LNs are maintained by presence of their target Ag (2). When a naive T cell becomes FRC-derived and (12, 13). FRCs also activated in the LN by a professional APC presenting its cognate directly induce deletional T cell tolerance and can restrict the Ag, the T cell will either mount an effector response or will expansion of newly activated T cells (14–19). In this article, become tolerant to avoid . In the presence of we review the immunoregulatory characteristics of LN FRCs, appropriate costimulation, activated T cells undergo rapid clonal with particular emphasis on how these cells organize and expansion in the LN, acquire effector functions, and gain the regulate several phases of the T lymphocyte life cycle. ability to migrate to their Ag source in peripheral tissues. The vast majority of effector T cells die during the contraction phase FRCs facilitate lymphocyte arrival and organization in the LN of an immune response, but a small fraction remain as circu- The random joining of TCR regions during T cell develop- lating long-lived effector or central memory cells, poised to ment produces a naive T cell repertoire with only a few cells mount a robust recall response in nonlymphoid and lymphoid with high affinity for any individual peptide–MHC (20, 21).

*Division of Medical Sciences, Harvard Medical School, Boston, MA 02115; Abbreviations used in this article: DC, ; FRC, fibroblastic reticular cell; †Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; HEV, high endothelial ; iFABP, intestinal fatty acid–binding protein; iNOS, and ‡Department of , Genentech, South San Francisco, CA 94080 inducible NO synthase; LN, ; MHC II, MHC class II; MLN, mesenteric LN; MPEC, memory precursor effector cell; tOVA, truncated form of OVA; Treg, Received for publication October 2, 2014. Accepted for publication November 13, . 2014. This work was supported by a Howard Hughes Medical Institute Gilliam Fellowship for Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 Advanced Study (to F.D.B.). Address correspondence and reprint requests to Dr. Shannon J. Turley, Department of Cancer Immunology, Genentech, One DNA Way, South San Francisco, CA 94080. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402520 1390 BRIEF REVIEWS: REGULATION OF T LYMPHOCYTES BY FRCs

To trigger an effective immune response, this rare population of ligation between the CCR7 receptor on DCs and FRC- T cells must initially engage an APC presenting its cognate Ag. derived chemokines CCL19 and CCL21 (41, 42) (Fig. 1C). To increase the likelihood of encountering its target Ag, naive Upon arrival in the LN, migratory DCs cross the floor of T cells continuously circulate between lymphoid organs, such the subcapsular sinus and infiltrate the parenchyma. Similar as the Peyer’s patches, , and LNs (22). Circulating to naive T cells, DCs also use the reticular FRC network as T lymphocytes enter the LN through specialized blood vessels a scaffold to navigate within the T cell zone (26), which named high endothelial (HEVs) (2, 23). FRCs sur- increases potential interactions between Ag-bearing DCs and round HEVs and interact with extravasated in the naive T cells. In addition to gradients (41), DC perivenular space to maintain HEV integrity during lymphocyte migration along FRC networks depends on signaling between trafficking (24). This regulation requires the ligation of FRC- CLEC-2 and its ligand gp38 (43). Engagement of DC- bound gp38 to the C-type lectin receptor CLEC-2 on platelets. expressed CLEC-2 to gp38 on FRCs promotes actin poly- Activated platelets then release sphingosine-1-phosphate in the merization in DCs, which facilitates spreading, protrusion perivenular space, which maintains adherens junctions between extension, and migration along FRC networks (43). Disrup- HEVs (24). Loss of FRC-bound gp38 or CLEC-2 expression tion of this signaling pathway causes impaired DC trafficking on platelets compromises LN vascular integrity at steady-state to and within the LN, ultimately leading to reduced T cell and during immune responses (24). The absence of CLEC-2 (43). In addition to modulating DC motility, the on platelets also leads to a defect in T and recirculation CLEC-2–gp38 axis influences the contractility of FRCs (44, through the LNs after repeated immunizations (25). 45). At steady-state, gp38 maintains FRCs in a highly tense Downloaded from Lymphocyte chemoattractants CCL19, CCL21, and CXCL12 and contracted state within the LN reticular network. CLEC- are expressed by LN FRCs and function to support naive T cell 2 ligation inhibits gp38 signaling, which causes FRCs to trafficking across HEVs and retain T cells in the LN paracortex stretch and expand as a result of the relaxation of their ac- through their ligation to CCR7 and CXCR4 (6, 12, 26–28) tomyosin cytoskeleton (44, 45). Effectively, the same Ag- (Fig. 1A). After exiting the HEVs, naive T cells use the FRC bearing DCs that initiate T cell priming during an immune network as a defined structural path to migrate within the response also cause relaxation of the FRC network, which http://www.jimmunol.org/ paracortex, according to the chemokine gradients. The sur- allows space for T cell influx and clonal expansion (44, 45). vival and homeostasis of naive T cells that reach the T cell zone are also supported by FRC-derived IL-7 (13) (Fig. 1B). Ag presentation by FRCs induces Accordingly, disruption of the FRC network during HIV- The priming instructions received by naive T cells in the LN associated LN fibrosis significantly correlates with a reduced either trigger effector T cell differentiation or produce func- + number of naive CD4 T cells in the LN (29). Indeed, LN tionally inert clones that remain in a hyporesponsive state or fibrosis in models of SIV in rhesus macaques restricts T cell eventually become deleted. The has evolved access to FRC-derived IL-7, which drives apoptosis in both such that this priming fate depends primarily on whether naive by guest on September 29, 2021 + + naive CD4 and CD8 T cell populations (30). Treatment of T cells harbor a TCR specific for a potentially dangerous SIV-infected macaques with agents that reduce LN fibrosis foreign Ag or contain receptors that recognize self-peptides. + preserves the FRC network and is associated with larger CD4 Despite the immune system’s design to eliminate self- T cell populations in the LNs compared with untreated reactive clones in the thymus during negative selection, a rel- controls (31, 32). Genetic ablation of LN FRCs in mouse atively high frequency of T cells with autoreactive potential models alters T cell localization in the paracortex, decreases escape into the periphery (46). Therefore, mechanisms to T cell survival, and impairs Ag-specific T cell priming (33). enforce peripheral tolerance are critical for the prevention By maintaining HEV integrity and secreting soluble media- of autoinflammation and tissue destruction. Until recently, tors to facilitate migration and survival, FRCs place T cells in steady-state trafficking of Ag-loaded immature DCs was position to locate their cognate Ag. widely accepted as the primary mechanism of peripheral deletional tolerance (47, 48). However, a report from Lee FRCs support the interactions between Ag-presenting DCs and T cells et al. (14) in 2007 identified an important role for LN stromal After arrival in the LN via HEVs, naive T cells spend ∼8–12 h cells in inducing CD8+ T cell tolerance (Fig. 1D). A trans- exploring the LN parenchyma for their cognate Ag (34). genic model system was used for this study, whereby a trun- FRCs directly facilitate Ag availability to T cells by creating cated form of OVA (tOVA) was expressed as a self-Ag under a conduit system that extends deep into the LN parenchyma, the control of the intestinal fatty acid–binding protein as well as by supporting migratory DC entry, maturation, and (iFABP) promoter (49). As expected, adoptive transfer of trafficking into the LN from peripheral tissues. FRCs secrete OVA-specific CD8+ T cells (OTI) led to proliferation in gut- and ensheath extracellular matrix components to form a re- draining tissues, such as the mesenteric LNs (MLNs) and ticular conduit network within the T cell zone of the LN and Peyer’s patches. Surprisingly, however, transferred cells also spleen (6, 35–38). This system functions as a molecular sieve, proliferated in the nondraining LNs. This proliferation even allowing expedited delivery of chemokines and small soluble occurred when DCs and other marrow–derived APCs Ags from upstream tissues into the parenchyma of draining were prevented from presenting Ag (14). These data led to the 2 LNs (37, 39) (Fig. 1). Small lymph-borne Ags from the discovery that CD45 gp38+ nonhematopoietic stromal cells conduit are sampled by LN-resident DCs and subsequently were responsible for the presentation of tOVA and subsequent presented to T cells (37, 40). Under inflammatory conditions, activation of OTI cells (14). Most notably, after some initial a second flux of Ag-loaded migratory DCs arrives in the LN proliferation, the adoptively transferred T cells were subse- and presents Ag to primed T cells (40). This trafficking and quently lost from the T cell pool, highlighting the tolerogenic upregulation of costimulatory molecules are induced by the capacity of LN stroma (14). The Journal of Immunology 1391

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FIGURE 1. Lymph node paracortex. LN FRCs regulate several aspects of the T cell life cycle. (A) FRCs facilitate lymphocyte arrival and organization in the LN. FRC-derived chemokines CCL19 and CCL21 support naive T cell trafficking across HEVs and retain T cells in the LN paracortex through their ligation to CCR7. (B) T cell survival is maintained by FRCs. FRC-derived IL-7 supports the survival and homeostasis of naive T cells that reach the T cell zone. (C) FRCs facilitate the interactions between Ag-presenting DCs and T cells. The trafficking of migratory DCs to the LN is induced by the ligation between the CCR7 receptor on DCs and FRC-derived chemokines CCL19 and CCL21. Upon arrival in the LN, DC migration along the FRC network requires the engagement of DC-expressed CLEC-2 to gp38 on FRCs. Disruption of this signaling axis ultimately leads to reduced T cell priming. (D) FRCs induce T cell tolerance via the expression of peripheral tissue–restricted Ags. Upon Ag presentation, FRCs induce deletional tolerance of MHC class I–restricted CD8+ T cells and hyporesponsiveness of MHC II–restricted CD4+ T cells. (E) FRCs restrict the expansion of newly activated T cells. Activated T cells release IFN-g and TNF-a, which act synergistically to endow FRCs with suppressive capabilities, mediated through the activity of NO. This suppression occurs in an Ag-independent fashion, which may ultimately be required to protect the organ from excessive swelling and damage during ongoing immune responses. (F) FRCs influence the maintenance of memory precursor effector cells. Ablation of FRCs during the late phase of an immune response leads to a modest reduction in the percentage of memory precursor effector cells. The mechanism controlling this reduction has not been determined, although FRC-derived IL-7 and IL-15 are hypothesized to be involved.

Advances in purification techniques for LN stromal cells pression. The study also provided evidence that MHC class II (50) led to the identification of FRCs as the specific stromal on FRCs also can be acquired from DCs via a contact- cell population responsible for the ectopic expression of dependent mechanism involving the transfer of DC-derived tOVA in the iFABP-tOVA mouse (15). These techniques MHC II+ exosomes (16). Accordingly, DCs pulsed with also allowed Malhotra et al. (6) to conduct a comprehensive FITC-labeled OVA also were able to transfer peptide-loaded transcriptomic analysis of different LN stroma cell subsets. MHC II complexes to FRCs (16). To assess the influence of Pairwise analyses of ligands and cognate receptors across this peptide-loaded MHC II transfer on CD4+ T cells in vivo, stroma and hematopoietic cells suggested a number of po- the investigators used the CD11cDOG mouse model in tentially interesting interactions. Notably, upregulation of which OVA protein is exclusively expressed by DCs (51). MHC class II (MHC II) by FRCs in response to inflamma- OVA-specific CD4+ T cells (OTII) precultured with FRCs tory stimuli suggests that FRCs might also tolerize class II– from these mice exhibited a delayed proliferative response restricted CD4+ T cells (6). Dubrot et al. (16) substantiated upon restimulation with anti-CD3/CD28 Abs (16). Based this notion with a recent study showing that FRCs express low on these findings, it was proposed that FRCs acquired OVA- levels of endogenous MHC II through the PIV promoter MHC II complexes from DCs in vivo, which endowed them region of CIITA, a known master regulator of class II ex- with the ability to induce Ag-specific CD4+ T cell hypores- 1392 BRIEF REVIEWS: REGULATION OF T LYMPHOCYTES BY FRCs ponsiveness (16) (Fig. 1D). Altogether, these studies eluci- (54, 55). As such, this system has provided a useful tool for dated a role for FRCs in Ag presentation and T cell anergy. investigating location-specific properties that are intrinsic to stroma. The capacity of LN stroma to induce the generation FRCs restrict the expansion of newly activated T cells of de novo regulatory T cells (Tregs) was explored in a recent In addition to tolerizing T cells through direct Ag presentation, study (56) in which -draining celiac LNs and gut- FRCs can limit T cell responses by curtailing the expansion draining MLNs were transplanted into the popliteal fossa of newly activated T cell pools. This proliferative restriction after excision of the endogenous popliteal LN. Compared applies to CD4+ and CD8+ T cells and, surprisingly, occurs with transplanted popliteal LN controls, celiac and MLNs represented a significantly superior environment for de novo independently of Ag presentation (17–19). According to 2 several reports, T cells activated by either anti-CD3/CD28 Treg differentiation from adoptively transferred naive Foxp3 Abs or peptide-pulsed DCs experienced delayed division ki- CD4+ OTII T cells (56). Interestingly, the ability to induce netics when cocultured with FRCs (17–19). Additional FRC: Tregs was not recapitulated when LNs were transplanted from T cell coculture experiments from mice deficient for candidate germ-free mice or mice with vitamin A deficiency (56). The mediators revealed a molecular cross-talk whereby T cell– phenomenon also required the presence of DCs, because Treg derived IFN-g and TNF-a act synergistically to endow FRCs generation was abolished when DCs were depleted (56). with suppressive capabilities that are mediated through the Overall, these data suggest a model whereby the intestinal activity of inducible NO synthase (iNOS) (17–19) (Fig. 1E). microenvironment imprints LN stroma with Treg-inducing

In Transwell assays, the suppressive affects of FRCs were properties that are ultimately fulfilled via a synergistic rela- Downloaded from significantly diminished, indicating contact dependency or a tionship with DCs. Given their interactions with DCs (43) requirement for close cellular proximity (17–19). The exis- and their density within the T cell zone (4), FRCs may be the tence of FRC-mediated T cell suppression was corroborated main LN stromal population responsible for Treg induction in vivo. Compared with wild-type iFABP-tOVA mice, in this model. It is also possible that FRC-derived NO might Lukacs-Kornek et al. (17) showed enhanced proliferation of act directly on newly activated T cells to induce Tregs. In line

OTI T cells transferred into iFABP-tOVA mice lacking iNOS with this hypothesis, work by Liew and colleagues (57, 58) http://www.jimmunol.org/ expression. Similar proliferation trends were observed using showed that NO can promote the generation of CD4+ 2 2 2 iNOS / mice infected with OVA-expressing vesicular sto- CD25+ Foxp3 Tregs with both in vitro and in vivo sup- matitis (19) or immunized with OVA-loaded bone pressive functions. marrow DCs (18). FRCs also may regulate T cell differentiation by supporting Given the current data, we can only speculate on the reasons the maintenance of memory precursor effector cells (MPECs) for this suppressive relationship. One possible clue may come and long-lived memory populations. A recent report by from the in vivo expression profile of iNOS in FRCs. As Denton et al. (59) provided some validation to this hypothesis

reported by immunostaining of skin-draining LNs and MLNs, by showing a modest reduction in MPEC percentages fol- by guest on September 29, 2021 OTI T cells transferred into iFABP-tOVA mice induced iNOS lowing FRC depletion during the late phase of an ongoing expression in only a proportion of FRCs in vivo (17). This influenza virus infection. Interestingly, FRC ablation reduced result, coupled with the fact that FRCs in vitro attenuated the percentage of MPECs without negatively impacting the T cell proliferation without complete abrogation (17–19), abundance of short-lived effector cells (59). Selective reduc- suggests that FRCs may exist as a heterogeneous population tion of MPECs likely occurred in response to decreased FRC- with regard to their suppressive capacity. Indeed, functional derived IL-7, which is known to support MPEC formation heterogeneity of FRCs was described recently for their B cell (60) (Fig. 1F). IL-7 and IL-15 are prosurvival factors for both homeostatic potential (33). Therefore, it is possible that FRCs CD4+ and CD8+ LN-homing central memory T cells (61– reside in discrete microdomains that promote T cell prolif- 65). Therefore, it is not surprising to find that resting LN eration in a spatially and temporally restricted fashion, while CD8+ central memory T cells closely mirror the microana- limiting uncontrolled T cell expansion. Ultimately, this tomical distribution of naive CD8+ T cells (66), whose sur- mechanism may be required to protect lymphoid organs from vival is also supported by FRC-derived IL-7 (13). Although excessive swelling and damage during ongoing immune CD4+ memory T cells are already known to associate with responses. Although we know that FRCs proliferate and ex- IL-7–expressing VCAM-1+ stroma cells in the pand to accommodate LN enlargement during immune (67), additional studies are needed to formally test whether responses(52,53),whetherand how these cells regulate CD4+ memory T cells preferentially reside on IL-7–produc- changes in LN structure during inflammation remain com- ing LN FRCs. Additionally, FRCs were recently shown to pletely unknown. More in vivo studies are needed to test the express IL-15 in vivo (68), adding to their potential role as validity of this hypothesis. supporters of maintenance (Fig. 1F). Can FRCs influence the differentiation of T cells? Conclusions Differentiation of naive T cells into regulatory cells, specific Over the past decade, our understanding of the immunolog- helper subsets, or long-lived memory cells represents the final ical relevance of the stroma has grown significantly. Based on stages of the T lymphocyte life cycle. The involvement of LN recent and emerging evidence, the role of stromal cells within stroma in this final stage can be investigated using a variety of the LN is now appreciated to be more complex than their techniques, including LN transplantation models. Upon LN previous categorization as a mere structural entity. We now transplantation, graft-derived LN stroma are largely retained in know that FRCs govern lymphocyte recruitment and orga- the transplant, whereas all hematopoietic cells migrate out of nization in the LN and support encounters between Ag- the LN and are replaced by host-derived hematopoietic cells presenting DCs and T cells. FRCs also induce deletional The Journal of Immunology 1393 tolerance via Ag presentation and are expected to participate in 13. Link, A., T. K. Vogt, S. Favre, M. R. Britschgi, H. Acha-Orbea, B. Hinz, J. G. Cyster, and S. A. Luther. 2007. 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