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Foxp3+ follicular regulatory T cells control T follicular helper cells and the germinal center response

Foxp3+ follicular regulatory T cells control T follicular helper cells and the germinal center... Follicular helper (T ) cells provide crucial signals to germinal center B cells undergoing somatic FH hypermutation and selection that results in affinity maturation. Tight control of T numbers FH + + + maintains self-tolerance. We describe a population of Foxp3 Blimp-1 CD4 T cells constituting high high + 10-25% of the CXCR5 PD-1 CD4 T cells found in germinal center after immunization. These follicular regulatory T cells (T ) share phenotypic characteristics with T and FR FH conventional Foxp3 regulatory T cells (T ) yet are distinct from either. Similar to T cells, reg FH T development depends on Bcl-6, SAP, CD28 and B cells; however T originate from thymic- FR FR derived Foxp3 precursors, not naïve or T cells. T are suppressive in vitro and limit T and FH FR FH germinal center B cell numbers in vivo. In the absence of T , an outgrowth of non-antigen- FR specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, T cells use the reg T differentiation pathway to produce specialized suppressor cells that control the germinal FH center response. Germinal centers are clusters of rapidly-dividing B cells formed in secondary lymphoid tissues in response to T-dependent antigens. Within germinal centers, mutation of the B cell receptor V-region genes together with subsequent selection results in the production of high affinity plasma cells and memory B cells . Defective selection can result in the production 2, 3 of autoantibodies and a break in self-tolerance . Germinal center B cell selection can in high high part be mediated by a specialized helper T cell subset, CXCR5 PD-1 T follicular helper (T ) cells . T cells develop in a Bcl-6-dependent manner and provide germinal FH FH Corresponding authors: [email protected], [email protected], [email protected]. Contributed equally The authors have no conflicting financial interests. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 2 center B cells with survival and selection signals. Limiting the numbers of T cells within FH germinal centers has been shown to be critical to prevent the emergence of 5,6 autoantibodies . Little is known about T control; in mice, Qa-1-restricted CD8 T cells FH 7 + + − can regulate the T compartment and in humans, CD4 CD25 CD69 T cells with a FH 8, 9 suppressive function in vitro have been found in germinal centers . Regulatory T Cells (T ) have also been shown enter the primary B cell follicle in mice, but their phenotype, reg ontogeny and ability to control T cells remain unknown . FH T that develop in a Foxp3-dependent manner repress the growth and function of CD4 reg effector T cells. Humans and mice lacking Foxp3 cannot form T and develop fatal reg 11-15 autoimmunity . In order to repress T 1-, T 2- and T 17- mediated immune responses, H H H T have been shown to co-opt selective aspects of the differentiation programs required for reg 16-18 these T subsets: Tbet/Stat1, IRF-4 or Rorγt signaling respectively . Here we show that Foxp3 T can be diverted to become T repressors via expression of Bcl6 and SAP- reg FH mediated interaction with B cells. The resulting follicular regulatory T cells (T ) share FR features of both T and T cells, localize to germinal centers, and regulate the size of the FH reg T cell population and germinal centers in vivo. FH Foxp3 follicular regulatory T cells are distinct from T and T FH reg After immunization with a T-dependent antigen we observed that ~10-25% of + high high CD4 CXCR5 PD-1 ‘T ’ cells expressed the transcriptional regulator of the T FH reg lineage, Foxp3 (Figure 1a). These cells followed the same formation and resolution kinetics as conventional T cells (Figure 1b). Foxp3 cells could be visualized within germinal FH centers identified by immunofluorescence staining of frozen spleen sections from immunized mice (Fig. 1c); 17% ± 8% of germinal center CD3 cells also expressed Foxp3. + high high + To obtain information about the identity and function of CD4 CXCR5 PD-1 Foxp3 cells - designated T , we performed microarray expression profiling on sorted populations FR GFP 19 from Foxp3 mice seven days after SRBC immunization. T , T , non-T effector/ reg FH FH memory cells (T ) and naïve (T ) T cells were also included (sorting strategy is depicted EM N in Supplementary Fig. 1). T more closely resembled T than T , T or T (Fig. 1d FR reg FH EM N and Supplementary Table 1), with elevated expression of many T associated genes reg including Foxp3, Ctla4, Gitr, Klrg1 and Prdm1. Nevertheless, T also expressed high FR amounts of the prototypic T genes Cxcr5, Pdcd1, Bcl6, Cxcl13, and Icos. T did not FH FR express the helper cytokines IL-21 or IL-4 (Fig. 1e) or the costimulatory ligand CD40L (Fig. 1f), but expressed comparably high levels of the ligand for CXCR5, CXCL13 , as T cells FH (Figure 1f). Differential expression of T or T associated molecules was confirmed by FH reg flow cytometry and/or real time PCR (Fig. 1g-i and Supplementary Fig. 2). T expressed numerous molecules characteristically expressed by T , such as GITR and FR reg CTLA-4, but at higher levels than T , consistent with an activated T phenotype reg reg (Figure 1g). Gzma expression was comparable to T (Figure 1h) but Gzmb, a reported reg target of Bcl-6 repression was barely detectable. T also expressed high levels of Il10 FR mRNA and surface ICOS protein, which are common to both T and T cells (Figure 1j FH reg and Supplementary Fig. 2). The elevated levels of GITR, IL-10 and ICOS on T compared FR with the rest of the T pool is consistent with an effector T phenotype , which suggests reg reg T have a regulatory function. The phenotypic features shared by T cells and T may FR FH FR account for their common germinal center localization. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 3 T and T cells require similar differentiation cues for their formation and FR FH maintenance As both T cells and T co-localize in germinal centers, we sought to determine whether FH FR T formation was dependent on similar developmental cues. T cell priming through CD28 FR 24, 25 is one of the first signals required for T development . Enumeration of T and T FH FH FR −/− +/+ in mixed Cd28 CD45.2: CD45.1 Cd28 bone marrow chimeras immunized seven days previously with SRBC revealed a complete absence of both T and T cells in the FH FR 26, 27 absence of CD28 signaling (Fig. 2a-c). Consistent with previous reports , CD28- deficiency moderately reduced peripheral T numbers (Supplementary Fig. 3a). reg SAP-dependent interactions of T precursors with B cells are required for T formation FH FH 5, 28-30 and/or maintenance . We therefore investigated whether interactions with B cells and/ or SAP-mediated signals are essential for T formation. Neither T nor T cells formed FR FH FR after SRBC immunization of B cell deficient μMT mice (Fig. 2d-f) whereas T formed reg −/− normally (Supplementary Fig. 3b). In addition, immunization of Sap-deficient (Sh2d1a ) mice revealed that, similar to T cells (Figure 3g, h), T cells are dependent on SAP for FH FR their formation (Figure 3i). T were only slightly reduced in the absence of SAP reg (Supplementary Fig. 3c). These data demonstrate that the developmental requirements of T are similar to those that govern T formation and dispensable for the generation of FR FH T . reg Coordinated Bcl-6 and Blimp-1 expression in T cells FR Bcl-6, the transcriptional regulator of the T subset, regulates key molecules required for FH 31-33 follicular localization and function in a process thought to be counteracted by the transcriptional repressor Blimp-1 ; Bcl-6 and Blimp-1 also mutually repress each other 34, 35 during B cell differentiation . We asked whether, similar to T cells, T cells also FH FR expressed Bcl-6 and would be devoid of Blimp-1 expression. Quantitative RT-PCR revealed that Bcl6 was expressed in T cells (Fig. 3a). Of note, T co-expressed Prdm1, the gene FR FR encoding Blimp-1, and its expression on T was higher than in any other CD4 T cell subset FR (Fig. 3a). Expression of Bcl-6 and Blimp-1 protein in Foxp3 cells within germinal center identified was also confirmed by immunofluorescence staining of spleen sections from + + SRBC-immunized mice. All Foxp3 T within AID germinal center expressed Bcl-6, Fr albeit at low levels (Fig. 3b) and 75% stained positive for Blimp-1 7 days after immunization; this proportion was reduced to 50% by day 14. Bcl-6 is required for T formation and Blimp-1 regulates T homeostasis FR FR To determine whether Blimp-1 and/or Bcl-6 play a role in T cell formation or FR homeostasis, we reconstituted sub-lethally irradiated CD45.1 mice with a 1:1 ratio of fetal +/+ gfp/gfp −/− liver cells from congenically-marked Prdm1 and Prdm1 embryos, Bcl6 and +/+ +/+ gfp/+ Bcl6 embryos or control Prdm1 and Prdm1 embryos. Eight weeks after reconstitution the mice were infected intranasally with influenza virus (HKx31), and 10 days later T formation was assessed in the mediastinal lymph node. In contrast to published FR data , loss of Blimp-1 did not alter the proportion of T cells but caused T to double FH FR (Fig. 3c), suggesting that Blimp-1 limits the size of the T population. This is consistent FR with a recent report showing Blimp-1 limits the numbers of effector T through a Bcl-2- reg dependent mechanism . −/− +/+ Mixed Bcl6 : Bcl6 chimeras confirmed previous reports that T cells do not form in FH the absence of Bcl-6 (Fig. 3c, upper panel). Cells lacking Bcl-6 expression did not give rise to T cells despite the presence of germinal centers in the mice (Fig. 3c, lower panel). FR Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 4 Similar results demonstrating the requirement for Bcl-6 in splenic T generation were FR obtained after SRBC immunization (Supplementary fig. 4a-c). As reported previously , Bcl-6 was dispensable for T formation (Supplementary fig. 4d). Together, this suggests reg that Bcl-6 is essential for T formation and Blimp-1 expression regulates the size of the FR T population. FR Although co-expression of Bcl-6 and Blimp-1 seems paradoxical, there are precedents in which both Prdm1 and Bcl6 are co-regulated; for example in both effector and memory + 36 CD8 subsets . Blimp-1 has been recently shown to influence T function inducing an reg 23,37 effector phenotype . It is expressed by T at mucosal sites and by a small (8-12%) reg 37 + subset of splenic T , which produce IL-10 in a Blimp-1-dependent manner . Blimp-1 reg T and T cells also share expression of high amounts of IL-10, GITR and ICOS . T reg FR FR + + are thus likely to be the follicular counterparts of the Blimp-1 IL-10 effector T found at reg mucosal surfaces. T cells derive from T precursors FR reg The observation that T require the same cues as T for their differentiation raised a FR FH critical question: do T represent induced T that arise from T cells that switch on FR reg FH Foxp3 in the germinal center, or do they derive from Foxp3 T ? Plasticity of CD4 helper reg T cell subsets is well documented , as is the adoption of T transcriptional programs by 16-18 T . reg In order to test whether T derive from T cells that turn on Foxp3 expression we FR FH 5 + low − transferred 1×10 naïve cells (CD4 CD44 CD25 ) from CD45.1 mice expressing the 3A9 HEL TCR transgene (TCR ), which recognizes hen egg lysozyme (HEL) peptide presented by I-A , into congenic CD45.2 B10.BR mice. Seven days after immunization with HEL in alum, 6-10% of T cells derived from the donor HEL-TCR T cells, but no donor-origin FH T could be identified; all T derived exclusively from the recipients’ cells (Fig. 4a). FR FR HEL Between 1-2% of the transferred naïve TCR donor population developed into inducible T (Fig. 4b). To exclude that an idiosyncratic effect of the 3A9 transgene had precluded reg development of T cells, this experiment was repeated with OT-II transgenic T cells: FR Again, OT-II T cells could form T but not T (Fig. 4c, d), suggesting that T do not FH FR FR derive from T cells. FH + 6 + low − To test whether T derive from Foxp3 precursors, 1×10 naïve CD4 CD44 Foxp3 T FR int + GFP cells or CD44 Foxp3 T from unimmunized Foxp3 mice were adoptively transferred reg into congenically-marked mice. Seven days after immunization with KLH in Ribi, ~1-2% of both donor-origin T and donor-origin naïve cells had upregulated CXCR5 and PD-1 to reg high high high levels (Figure 4e). More than 90% of donor-origin CXCR5 PD-1 T cells reg retained Foxp3 expression, but none of the transferred naïve T cells that became high high CXCR5 PD-1 after immunization switched on Foxp3 to become T cells (Fig. 4e). FR + SP We then asked whether thymic T (nT ) could become T cells. Thymic Foxp3 CD4 reg reg FR − SP GFP or Foxp3 CD4 from Foxp3 CD45.2 mice were adoptively transferred into CD45.1 mice. Seven days after SRBC immunization only Foxp3 CD45.2 cells had become T ; FR Foxp3 CD45.2 cells had become T but not T (Supplementary Fig. 5). Furthermore, FH FR ~97% of T cells expressed Helios (Supplementary Fig. 5), a transcription factor which has FR been reported to be expressed by thymic-derived nT but not T induced in the ref ref periphery . In an alternative strategy to confirm that T derive from Foxp3 precursors we used mice FR in which the gene encoding for the diphtheria toxin receptor (DTR) has been inserted in the DTR Foxp3 locus (Foxp3 ), so that treatment with diphtheria toxin (DT) selectively ablates all Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 5 40 DTR T within 48 hours . Foxp3 mice were immunized with SRBC and treated with either reg DT or saline immediately afterwards. Six days after immunization, T cells had formed FR normally in mice that did not receive DT but were absent in DT-treated mice (Fig. 4f, g) demonstrating T cannot form if Foxp3 cells are absent at the time of immunization. FR Within Peyer’s patches, but not in the spleen, it has been demonstrated that a proportion of − + 41 Foxp3 T cells derive from Foxp3 precursors . To investigate whether splenic T cells FR FR stably express Foxp3, we immunized Foxp3-Cre x ROSA-Stop-flox-YFP mice, in which any cell that has expressed Foxp3 is permanently marked. Seven days after SRBC + + high high immunization, the majority (97%) of YFP CD4 CXCR5 PD-1 T cells coexpressed FR high high − + Foxp3 and less than ~0.6% of CXCR5 PD-1 cells were Foxp3 YFP (Fig. 4h), consistent with the observation that Foxp3 expression is stable in the natural T reg 43 + population . These data suggest that T cells derive from Foxp3 thymic T that co-opt FR reg the T cell differentiation program to migrate to germinal center, where they maintain FH Foxp3 expression. T cells suppress T cell proliferation in vitro and repress T cells in vivo FR FH Expression of Foxp3 by CD4 T cells initiates a transcriptional program that confers 11-13 GFP suppressor function . T and T sorted from immunized Foxp3 mice displayed FR reg comparable suppressive ability in vitro (Supplementary Fig. 6a). To determine whether T FR cells are suppressive in vivo, we sought to ablate T cells after the germinal center FR DTR response had been established. For this, DT was first administered to Foxp3 mice 5d after SRBC immunization, when T and T cells have already formed, but the response FH FR has not yet reached its peak . After 3 days of DT treatment – 8 days after immunization – only 1% of T cells and 5% of T cells were present compared with vehicle-only-treated FR reg mice (Supplementary Fig. 7), and DT-treated mice displayed a significant increase in both the proportion and total number of T cells compared to controls (Fig. 5a). At this time FH point, germinal center B cell numbers were comparable between DT- and vehicle-treated groups (Figure 5b). To confirm the in vivo regulatory role of T , it is necessary to deplete them while leaving FR the T and T compartments intact. To achieve this we generated mixed bone marrow reg FH −/− DTR chimeras with a 1:1 ratio of either congenically marked Sh2d1a :Foxp3 or +/+ DTR + −/− Sh2d1a :Foxp3 marrow. After immunization and DT treatment, CD45.2 Sh2d1a cells should not able to form T or T cells, but should form T cells normally, whereas FH FR reg DTR + Foxp3 cells should form T cells, but lack all Foxp3 cells. Thus, T and T FH FH reg populations should still be present in both chimeras, but T cells will be selectively FR −/− DTR reduced in Sh2d1a : Foxp3 mice (Supplementary Table 2). Such chimeric mice were generated and treated with DT 1 day prior to, and 2 and 5 days after SRBC immunization. At day 8 after immunization >90% of T cells in FH −/− DTR DTR Sh2d1a :Foxp3 chimeras derived from Foxp3 cells compared to ~50% in the +/+ DTR control Sh2d1a :Foxp3 mice (Supplementary Fig. 8a). The size of the T population reg was comparable in both groups of mice (Fig. 5c) with the majority (97%) of T being reg CD45.2 (Supplementary Fig. 8b). Critically, the number of T was reduced by 5 times in FR −/− DTR Sh2d1a : Foxp3 mice compared with the control chimeras (Fig. 5d, Supplementary Fig. 7c). There was also an increase in both the number and proportion of T cells (Fig. 5e) FH and of germinal center B cells (Fig. 5f), indicating that T limit the germinal center FR response during a T-dependent immune reaction in vivo. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 6 T limit the outgrowth of non-antigen-specific germinal center B cells FR To further characterize the role of T during the germinal center response, T were FR FR DTR transiently depleted at the peak of the germinal center response. For this, Foxp3 and WT control Foxp3 mice were immunized with NP-KLH in alum, and treated with DT six days later. Ten days after immunization, the fraction of germinal center B cells was not significantly different between the two groups (Fig. 6a, b) but combined T and T reg FR depletion led to a reduction in the proportion of germinal center B cells specific for the dominant epitope of the immunizing antigen, the hapten NP (Fig. 6a, c). In order to determine whether this had any long-term impact on the antigen-specific DTR antibody response, Foxp3 mice were immunized with NP-KLH in alum and treated with either DT or saline at 7, 10 and 13 days after immunization, then boosted with NP-KLH in alum 24 days after primary immunization (Fig. 6d). High (anti-NP2) & low (anti-NP12) affinity anti-NP antibody titers were assessed by ELISA prior to and d10, 15, 10 and d28 after immunization. Titers of high and low affinity antibodies were comparable until d20, when both were reduced in DT-treated mice compared with controls (Fig 6e). Four days after secondary immunization (day 28 after primary challenge), high affinity antibodies remained lower in DT-treated mice (Fig 6e). This suggests that depletion of T and T FR reg during the germinal center response does not increase antigen-specific antibody production. To test whether the reduction in the antigen-specific germinal center response was a consequence of T depletion rather than general T depletion, we generated FR reg −/− −/− Sh2d1a :Foxp3 mixed fetal liver chimeras that selectively lack T cells. Three groups FR +/+ +/+ +/+ −/− of control chimeras were generated in parallel: Sh2d1a :Foxp3 , Sh2d1a :Foxp3 −/− +/+ and Sh2d1a :Foxp3 . Eight weeks after reconstitution, chimeric mice were immunized with alum-precipitated NP-chicken gammaglobulin (NP-CGG). 21 days post-immunization, −/− −/− Sh2d1a :Foxp3 chimeras had a reduction in T cells compared to all control groups FR (Supplementary Fig. 9a and b) and an expanded T population (Supplementary Fig. 9c). FH The proportion of Foxp3 T was comparable amongst all 4 groups (Supplementary Fig. reg 9d). Consistent with a selective defect in T and not T , circulating T 1 and T 2 cells FR reg H H −/− −/− were not expanded in Sh2d1a :Foxp3 mice 14 days post-immunization (Supplementary Fig. 10a and b) nor in the spleen 21 days after immunization (Supplementary Fig. 10c and d). This also confirms that T cells are specialized in the regulation of follicular responses, FR while other T effectors control T 1 and T 2 cells. reg H H DTR As observed in Foxp3 mice, the proportion and absolute number of antigen-specific + −/− −/− (NP ) germinal center B cells was reduced in Sh2d1a :Foxp3 mice compared with control chimeras (Fig. 6f), despite formation of abundant germinal centers (Fig. 6g). NP- specific splenic memory B cells and NP-specific bone marrow plasma cells appeared −/− −/− reduced in Sh2d1a :Foxp3 chimeras compared with controls 21 days after immunization (Figure 6h and i) and the differences were statistically-significant against all −/− +/+ control groups except for the Sh2d1a :Foxp3 group, in which there was greater variability. At this time point there was no difference in anti-NP antibody titers between −/− −/− Sh2d1a :Foxp3 mice compared with control chimeras (Supplementary Fig. 11), probably due to a large component being of extrafollicular origin given that abundant NP plasma cells were still detectable in the spleen (Supplementary Fig. 11). Together, this suggests that T act to limit the outgrowth of non-antigen specific clones in the germinal FR center. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 7 Discussion We have shown here that in response to T-dependent antigens a proportion of naïve T can reg turn on Bcl-6, which allows them to adopt the T differentiation program and express the FH follicular homing receptor CXCR5 to localize to the germinal center. Here they exert suppressive functions on T cells and the germinal center response. Unlike T cells, T FH FH FR cells express Blimp-1, which is required to control their numbers in the germinal center. In response to other extracellular stimuli, naïve Foxp3 cells can turn on Tbet or increase activity of IRF4 or STAT3, required for T 1, T 2 and T 17 cell formation H H H 16-18 respectively . The specialized regulatory program determined by each of these transcription factors is likely to operate via modification of the T chemokine receptor reg profile in order to allow migration into an anatomical location where T are poised to reg regulate specific T cell responses to prevent autoimmunity or inflammation-associated tissue damage. Our data suggests T cells are specialized in controlling the germinal center reaction FR through limiting the numbers of T cells and inhibiting selection of non-antigen-specific B FH cells including those carrying self-reactive receptors. Furthermore, the ability of T cells to FR ensure dominance of antigen-specific clones over the germinal center response appears essential for formation of normal numbers of long-lived plasma cells and memory B cells. Although it is likely that T control germinal center B cells indirectly, through their ability FR to limit T numbers, it is also possible that T also negatively regulate germinal center B FH FR cells directly. This would be akin to the description of direct regulation of antigen- 45, 46 9 presenting cells by T and reports that T can directly regulate B cell function . reg reg Germinal center B cells are the predominant APCs within the germinal center microenvironment, and this makes them attractive candidates for T -mediated inhibition. FR Selection of cognate germinal center B cells by T cells is one of the key mechanisms by FH which germinal center tolerance is regulated . Dysregulation of the T population has FH 3, 5, 7, 48 been previously demonstrated to result in autoimmunity , highlighting the need to tightly control positive selection in germinal centers. Understanding the mechanisms by which T are regulated and their TCR specificity will be important for dissecting the FR pathogenesis of the increasing number of pathologies in which T cells appear to play a FH role, including disease-associated ectopic germinal center formation seen in many 3 49 50 autoimmune diseases , atherosclerosis , and chronic allograft rejection . We therefore postulate that T cells may represent a critical peripheral tolerance mechanism, essential FR for preventing germinal center-derived autoimmunity. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments We thank Xin Hu and Jessica Fitch for technical assistance, Dimitra Zotos for help with experiments not included GFP in this manuscript, Alexander Rudensky for kind provision of Foxp3 mice and Marion Espeli for helpful discussions. This work was funded by NHMRC program and project grants to CGV, a VIB PI grant to AL and a Wellcome Trust Programme Grant (083650/Z/07/Z) to KGCS. MAL is supported by an EMBO Post-doctoral Long-term Fellowship (ALTF 1041-2009) and a Raymond and Beverly Sackler Junior Research Fellowship, Churchill College, Cambridge; CGV by a Viertel Senior Medical Research Fellowship; KGCS by a Lister Prize Fellowship and TFR by the National Institute of Health Research Cambridge Biomedical Research Centre; and AL by a JDRF Career Development Fellowship and a Marie Curie Reintegration Grant Fellowship. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. 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Nature Reviews. 2005; 5:853–865. 48. Hsu HC, et al. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice. Nature Immunology. 2008; 9:166–175. [PubMed: 18157131] 49. Grabner R, et al. Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice. The Journal of Experimental Medicine. 2009; 206:233–248. [PubMed: 19139167] 50. Wehner JR, et al. B cells and plasma cells in coronaries of chronically rejected cardiac transplants. Transplantation. 2010; 89:1141–1148. [PubMed: 20386145] Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 11 high high + Figure 1. A proportion of CXCR5 PD-1 CD4 cells express the transcription factor Foxp3 (a,b) After SRBC immunization Foxp3 cells were identified in the high high + CXCR5 PD-1 CD4 ‘T ’ compartment, these cells follow the same kinetics as FH + + classic T cells. (c) Foxp3 cells (red) are present within the Bcl6 germinal center area FH (green) following SRBC immunization. Scale bar represents 100μm. (d) Heat map + GFP comparing the gene expression profiles of different CD4 T cell subsets from Foxp3 mice seven days after immunization. Red: high gene expression; blue: low gene expression. The cells were sorted using the following markers and for simplicity will be referred by the + low − abbreviations in parentheses throughout: CD4 CD44 Foxp3 naïve (T ) cells, + high int/low int/low − CD4 CD44 CXCR5 PD-1 Foxp3 effector/memory (T ) cells, EM + int int/low int/low + CD4 CD44 CXCR5 PD-1 Foxp3 regulatory T cells (T ), reg + high high − CD4 CXCR5 PD-1 Foxp3 T follicular helper (T ) cells and FH + high high + CD4 CXCR5 PD-1 Foxp3 follicular regulatory (T ) cells. (e) Il21 and Il4 mRNA FR measured by quantitative PCR from sorted cells using the strategy described in (d) normalized to Gapdh. Heights of the bars represent the mean and error bars represent the range of expression from 3 biological replicates. nd: gene expression not detected. (f) Left: Intracellular expression of CD40L as determined by flow cytometry in T (blue), T reg FH (green) and T (red) cell populations; the grey histogram represents a staining control from FR an immunized CD40L-deficient mouse. Right: Cxcl13 mRNA measured by quantitative RT- PCR as described in (e). (g) Cell surface expression of GITR, CD25 and intracellular CTLA4 in T (blue), T (green) and T (red) cell populations; grey histograms represent reg FH FR the isotype control. (h) Relative Gzmb and Gzma mRNA determined by quantitative RT- PCR as described in (e). (i) Percentage of CD103 cells within the T , T & T reg FH FR populations, each symbol represents one mouse. (j) Left: Cell surface expression of ICOS as Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 12 determined by flow cytometry in T (blue), T (green) and T (red) cell populations; the reg FH FR grey histogram represents staining level of an isotype control. Right: Il10 mRNA detected by quantitative RT-PCR of as described in (e). Flow cytometric and RT-PCR data are representative of at least three independent experiments. In (e)-(i): Statistical significance was determined using a one-way ANOVA analysis with Bonferroni’s multiple testing correction; * P<0.05; **P<0.01; ***P<0.001. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 13 Figure 2. T require the same differentiation cues as T cells for their development FR FH Flow cytometric contour plots (a, d, g) and dot plots of T (b, e, h) and T (c, f, i) cells in FH FR the groups of mice described below, seven days after SRBC immunization. (a-c) Mixed −/− bone marrow chimeras generated by sub-lethally irradiating Rag2 mice and reconstituting +/+ their immune system with a 1:1 ratio of bone marrow cells from CD45.1 Cd28 and −/− +/+ +/+ CD45.2 Cd28 mice or control CD45.1 Cd28 and CD45.2 Cd28 . (d-f) C57BL/6 (BL/ +/+ −/− 6) and B-cell deficient μMT mice. (g-i) Sh2d1a and Sh2d1a mice. Each symbol represents one mouse and horizontal bars represent median values. Figures represent one of 3 independent experiments with similar results. Statistical significance was determined using a Mann-Whitney Test: *P<0.05, **P<0.01. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 14 Figure 3. T cells express Bcl-6 and Blimp-1 FR (a) Bcl6 and Prdm1 mRNA normalized to Gapdh determined by quantitative RT-PCR from sorted cells using the strategy described in Fig. 1d and Supplementary Fig. 1. Heights of the bars represent the mean and error bars represent the range of expression from 3 biological replicates. Statistical significance was determined using a one-way ANOVA analysis with Bonferroni’s multiple testing correction; * P<0.05; **P<0.01; ***P<0.001. Bar graphs are representative of 3 experiments. (b) Immunofluorescence of frozen spleen sections from mice immunized seven days previously with SRBC. The germinal center is demarcated by the white dotted line in the three consecutive sections. Upper panel: AID (red) and CD3 (green); middle panel: Foxp3 (red) and Bcl-6 (green); lower panel: Foxp3 (red) and Blimp1 (green). Scale bar represents 100μm. (c) Flow cytometric contour plots of T (upper FH panels) & T (lower panels) formation in the draining (mediastinal) lymph node ten days FR after intranasal influenza infection of mixed fetal liver chimeras reconstituted with a 1:1 gfp/gfp +/+ ratio of fetal liver cells from E14.5 CD45.2 Prdm1 : CD45.1 Prdm1 embryos, E14.5 −/− +/+ gfp/+ CD45.2 Bcl6 : CD45.1 Bcl6 embryos or control E14.5 CD45.2 Prdm1 : CD45.1 +/+ Prdm1 embryos. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 15 Figure 4. T derive from Foxp3 precursors FR + high high + Flow cytometric contour plots of splenic CD4 CXCR5 PD-1 cells (a) or CD4 cells 5 3A9 (b) seven days after 1×10 transferred transgenic TCR HEL-specific CD45.1 T cells were adoptively transferred into congenically distinct CD45.2 B10.Br mice and immunized + high high with HEL in alum. Flow cytometric contour plots of splenic CD4 CXCR5 PD-1 cells + 5 (c) or CD4 cells (d) seven days after adoptive transfer of 1×10 OT-II OVA-specific Thy1.2 T cells into congenically distinct Thy1.1 C57BL6 mice and immunization with OVA in alum. (e) Flow cytometric contour plots of splenic CD4 T cells from CD45.1 C57BL/6 6 + int + mice seven days after adoptive transfer of 1×10 sorted naïve CD4 CD44 Foxp3 T (top reg + low − panel) or CD4 CD44 Foxp3 naïve T cells (lower panel) from unimmunized CD45.2 GFP Foxp3 mice and KLH in Ribi immunization. Transferred CD45.2 cells are shown in red, the endogenous CD45.1 cells are represented by the grey contour plots. Histograms showing + + high high Foxp3-GFP expression in transferred CD45.2 CD4 CXCR5 PD-1 cells. (f) Contour + DTR plots of splenic CD4 T cells and quantification of T cells (g) from Foxp3 mice six FR days after SRBC immunization and administration of either 0.9% saline (top panel) or DT + + high high (lower panel). Histograms show Foxp3 cells within the CD4 CXCR5 PD-1 compartment. (h) Flow cytometric contour plots of splenic CD4 cells from Foxp3-cre x ROSA-Stop-flox-YFP mice immunized seven days previously with SRBC (left panel). + high high Enumeration of the proportion of CD4 CXCR5 PD-1 cells that expressed YFP and/or Foxp3 (right panel). Each symbol represents one mouse and horizontal bars represent median values. Figures are representative of 2-4 independent experiments. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 16 Figure 5. T regulate the size of the T population FR FH Flow cytometric contour plots and graphs of T cells (a) and germinal center B cells (b) FH DTR from the spleens of Foxp3 mice immunized eight days previously (d0) with SRBC. Five days after immunization the mice were treated with either DT or saline. (c-f) Analysis of −/− mixed bone marrow chimeras generated by sub-lethally irradiating Rag2 mice and −/− DTR reconstituting their immune system with either a 1:1 ratio of Sh2d1a CD45.2 : Foxp3 +/+ DTR CD45.1 bone marrow or control Sh2d1a CD45.2 : Foxp3 CD45.1 bone marrow. Eight weeks after reconstitution chimeric mice were immunized with SRBC and treated with 50μg/Kg of DT on one day prior to immunization and d2 and d5 thereafter. Splenocytes were analyzed on d8 for the proportion and total number of + high high + + + CD4 CXCR5 PD-1 Foxp3 T cells (c), CD4 Foxp3 T (d), FR reg + high high + high high CD4 CXCR5 PD-1 T cells (e) and B220 GL-7 CD95 germinal center B FH cells (f). Each symbol represents one mouse and horizontal bars represent median values. Statistical significance was determined using a Mann-Whitney Test: *P<0.05, **P<0.01. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 17 Figure 6. T restrict the outgrowth of non-antigen specific clones in the germinal center FR + + Flow cytometric contour plots (a) and graphs (b) of total GL-7 CD95 germinal center B + WT cells and (c) NP germinal center B cells ten days after immunization of Foxp3 and DTR Foxp3 mice that have been treated with DT 6 days after NP-KLH immunization. Statistical analyses performed using Mann Whitney U-test. Experimental outline (d) of DTR immunization and DT or saline treatment scheme of Foxp3 mice (n=8 per group) to examine the antigen specific immunoglobulin response over time, mice were bled prior to, and d10, d15, d20 and d28 after primary immunization. Mice were given a booster immunization 24 days after the primary immunization. (e) ELISA analysis of NP12 and NP2 antibodies in the experiment outlined in (d). Error bars represent the standard error of the mean from eight individual mice from one experiment, representative of two experiments. Statistical analyses in (e) were performed using a two-way ANOVA with Bonferroni post test to compare differences at each time point. Graphs and flow cytometric contour plots of + + + + NP germinal center B cells (f), total GL-7 CD95 germinal center B cells (g) and NP bone marrow plasma and memory cells (h, i) 21 days after NP-CGG immunization of −/− chimeric mice generated by reconstituting Rag2 mice with a 1:1 mix of −/− −/− +/+ +/+ +/+ −/− −/− +/+ Sh2d1a :Foxp3 , Sh2d1a :Foxp3 , Sh2d1a :Foxp3 and Sh2d1a :Foxp3 fetal liver. Statistical analyses in (f, g, h and i) were performed using a one-way ANOVA with Bonferroni post test correction. Each symbol represents one mouse and horizontal bars represent median values. *P<0.05, **P<0.01, ***P<0.001. Nat Med. Author manuscript; available in PMC 2012 January 24. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature medicine Pubmed Central

Foxp3+ follicular regulatory T cells control T follicular helper cells and the germinal center response

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1078-8956
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1546-170X
DOI
10.1038/nm.2425
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Abstract

Follicular helper (T ) cells provide crucial signals to germinal center B cells undergoing somatic FH hypermutation and selection that results in affinity maturation. Tight control of T numbers FH + + + maintains self-tolerance. We describe a population of Foxp3 Blimp-1 CD4 T cells constituting high high + 10-25% of the CXCR5 PD-1 CD4 T cells found in germinal center after immunization. These follicular regulatory T cells (T ) share phenotypic characteristics with T and FR FH conventional Foxp3 regulatory T cells (T ) yet are distinct from either. Similar to T cells, reg FH T development depends on Bcl-6, SAP, CD28 and B cells; however T originate from thymic- FR FR derived Foxp3 precursors, not naïve or T cells. T are suppressive in vitro and limit T and FH FR FH germinal center B cell numbers in vivo. In the absence of T , an outgrowth of non-antigen- FR specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, T cells use the reg T differentiation pathway to produce specialized suppressor cells that control the germinal FH center response. Germinal centers are clusters of rapidly-dividing B cells formed in secondary lymphoid tissues in response to T-dependent antigens. Within germinal centers, mutation of the B cell receptor V-region genes together with subsequent selection results in the production of high affinity plasma cells and memory B cells . Defective selection can result in the production 2, 3 of autoantibodies and a break in self-tolerance . Germinal center B cell selection can in high high part be mediated by a specialized helper T cell subset, CXCR5 PD-1 T follicular helper (T ) cells . T cells develop in a Bcl-6-dependent manner and provide germinal FH FH Corresponding authors: [email protected], [email protected], [email protected]. Contributed equally The authors have no conflicting financial interests. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 2 center B cells with survival and selection signals. Limiting the numbers of T cells within FH germinal centers has been shown to be critical to prevent the emergence of 5,6 autoantibodies . Little is known about T control; in mice, Qa-1-restricted CD8 T cells FH 7 + + − can regulate the T compartment and in humans, CD4 CD25 CD69 T cells with a FH 8, 9 suppressive function in vitro have been found in germinal centers . Regulatory T Cells (T ) have also been shown enter the primary B cell follicle in mice, but their phenotype, reg ontogeny and ability to control T cells remain unknown . FH T that develop in a Foxp3-dependent manner repress the growth and function of CD4 reg effector T cells. Humans and mice lacking Foxp3 cannot form T and develop fatal reg 11-15 autoimmunity . In order to repress T 1-, T 2- and T 17- mediated immune responses, H H H T have been shown to co-opt selective aspects of the differentiation programs required for reg 16-18 these T subsets: Tbet/Stat1, IRF-4 or Rorγt signaling respectively . Here we show that Foxp3 T can be diverted to become T repressors via expression of Bcl6 and SAP- reg FH mediated interaction with B cells. The resulting follicular regulatory T cells (T ) share FR features of both T and T cells, localize to germinal centers, and regulate the size of the FH reg T cell population and germinal centers in vivo. FH Foxp3 follicular regulatory T cells are distinct from T and T FH reg After immunization with a T-dependent antigen we observed that ~10-25% of + high high CD4 CXCR5 PD-1 ‘T ’ cells expressed the transcriptional regulator of the T FH reg lineage, Foxp3 (Figure 1a). These cells followed the same formation and resolution kinetics as conventional T cells (Figure 1b). Foxp3 cells could be visualized within germinal FH centers identified by immunofluorescence staining of frozen spleen sections from immunized mice (Fig. 1c); 17% ± 8% of germinal center CD3 cells also expressed Foxp3. + high high + To obtain information about the identity and function of CD4 CXCR5 PD-1 Foxp3 cells - designated T , we performed microarray expression profiling on sorted populations FR GFP 19 from Foxp3 mice seven days after SRBC immunization. T , T , non-T effector/ reg FH FH memory cells (T ) and naïve (T ) T cells were also included (sorting strategy is depicted EM N in Supplementary Fig. 1). T more closely resembled T than T , T or T (Fig. 1d FR reg FH EM N and Supplementary Table 1), with elevated expression of many T associated genes reg including Foxp3, Ctla4, Gitr, Klrg1 and Prdm1. Nevertheless, T also expressed high FR amounts of the prototypic T genes Cxcr5, Pdcd1, Bcl6, Cxcl13, and Icos. T did not FH FR express the helper cytokines IL-21 or IL-4 (Fig. 1e) or the costimulatory ligand CD40L (Fig. 1f), but expressed comparably high levels of the ligand for CXCR5, CXCL13 , as T cells FH (Figure 1f). Differential expression of T or T associated molecules was confirmed by FH reg flow cytometry and/or real time PCR (Fig. 1g-i and Supplementary Fig. 2). T expressed numerous molecules characteristically expressed by T , such as GITR and FR reg CTLA-4, but at higher levels than T , consistent with an activated T phenotype reg reg (Figure 1g). Gzma expression was comparable to T (Figure 1h) but Gzmb, a reported reg target of Bcl-6 repression was barely detectable. T also expressed high levels of Il10 FR mRNA and surface ICOS protein, which are common to both T and T cells (Figure 1j FH reg and Supplementary Fig. 2). The elevated levels of GITR, IL-10 and ICOS on T compared FR with the rest of the T pool is consistent with an effector T phenotype , which suggests reg reg T have a regulatory function. The phenotypic features shared by T cells and T may FR FH FR account for their common germinal center localization. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 3 T and T cells require similar differentiation cues for their formation and FR FH maintenance As both T cells and T co-localize in germinal centers, we sought to determine whether FH FR T formation was dependent on similar developmental cues. T cell priming through CD28 FR 24, 25 is one of the first signals required for T development . Enumeration of T and T FH FH FR −/− +/+ in mixed Cd28 CD45.2: CD45.1 Cd28 bone marrow chimeras immunized seven days previously with SRBC revealed a complete absence of both T and T cells in the FH FR 26, 27 absence of CD28 signaling (Fig. 2a-c). Consistent with previous reports , CD28- deficiency moderately reduced peripheral T numbers (Supplementary Fig. 3a). reg SAP-dependent interactions of T precursors with B cells are required for T formation FH FH 5, 28-30 and/or maintenance . We therefore investigated whether interactions with B cells and/ or SAP-mediated signals are essential for T formation. Neither T nor T cells formed FR FH FR after SRBC immunization of B cell deficient μMT mice (Fig. 2d-f) whereas T formed reg −/− normally (Supplementary Fig. 3b). In addition, immunization of Sap-deficient (Sh2d1a ) mice revealed that, similar to T cells (Figure 3g, h), T cells are dependent on SAP for FH FR their formation (Figure 3i). T were only slightly reduced in the absence of SAP reg (Supplementary Fig. 3c). These data demonstrate that the developmental requirements of T are similar to those that govern T formation and dispensable for the generation of FR FH T . reg Coordinated Bcl-6 and Blimp-1 expression in T cells FR Bcl-6, the transcriptional regulator of the T subset, regulates key molecules required for FH 31-33 follicular localization and function in a process thought to be counteracted by the transcriptional repressor Blimp-1 ; Bcl-6 and Blimp-1 also mutually repress each other 34, 35 during B cell differentiation . We asked whether, similar to T cells, T cells also FH FR expressed Bcl-6 and would be devoid of Blimp-1 expression. Quantitative RT-PCR revealed that Bcl6 was expressed in T cells (Fig. 3a). Of note, T co-expressed Prdm1, the gene FR FR encoding Blimp-1, and its expression on T was higher than in any other CD4 T cell subset FR (Fig. 3a). Expression of Bcl-6 and Blimp-1 protein in Foxp3 cells within germinal center identified was also confirmed by immunofluorescence staining of spleen sections from + + SRBC-immunized mice. All Foxp3 T within AID germinal center expressed Bcl-6, Fr albeit at low levels (Fig. 3b) and 75% stained positive for Blimp-1 7 days after immunization; this proportion was reduced to 50% by day 14. Bcl-6 is required for T formation and Blimp-1 regulates T homeostasis FR FR To determine whether Blimp-1 and/or Bcl-6 play a role in T cell formation or FR homeostasis, we reconstituted sub-lethally irradiated CD45.1 mice with a 1:1 ratio of fetal +/+ gfp/gfp −/− liver cells from congenically-marked Prdm1 and Prdm1 embryos, Bcl6 and +/+ +/+ gfp/+ Bcl6 embryos or control Prdm1 and Prdm1 embryos. Eight weeks after reconstitution the mice were infected intranasally with influenza virus (HKx31), and 10 days later T formation was assessed in the mediastinal lymph node. In contrast to published FR data , loss of Blimp-1 did not alter the proportion of T cells but caused T to double FH FR (Fig. 3c), suggesting that Blimp-1 limits the size of the T population. This is consistent FR with a recent report showing Blimp-1 limits the numbers of effector T through a Bcl-2- reg dependent mechanism . −/− +/+ Mixed Bcl6 : Bcl6 chimeras confirmed previous reports that T cells do not form in FH the absence of Bcl-6 (Fig. 3c, upper panel). Cells lacking Bcl-6 expression did not give rise to T cells despite the presence of germinal centers in the mice (Fig. 3c, lower panel). FR Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 4 Similar results demonstrating the requirement for Bcl-6 in splenic T generation were FR obtained after SRBC immunization (Supplementary fig. 4a-c). As reported previously , Bcl-6 was dispensable for T formation (Supplementary fig. 4d). Together, this suggests reg that Bcl-6 is essential for T formation and Blimp-1 expression regulates the size of the FR T population. FR Although co-expression of Bcl-6 and Blimp-1 seems paradoxical, there are precedents in which both Prdm1 and Bcl6 are co-regulated; for example in both effector and memory + 36 CD8 subsets . Blimp-1 has been recently shown to influence T function inducing an reg 23,37 effector phenotype . It is expressed by T at mucosal sites and by a small (8-12%) reg 37 + subset of splenic T , which produce IL-10 in a Blimp-1-dependent manner . Blimp-1 reg T and T cells also share expression of high amounts of IL-10, GITR and ICOS . T reg FR FR + + are thus likely to be the follicular counterparts of the Blimp-1 IL-10 effector T found at reg mucosal surfaces. T cells derive from T precursors FR reg The observation that T require the same cues as T for their differentiation raised a FR FH critical question: do T represent induced T that arise from T cells that switch on FR reg FH Foxp3 in the germinal center, or do they derive from Foxp3 T ? Plasticity of CD4 helper reg T cell subsets is well documented , as is the adoption of T transcriptional programs by 16-18 T . reg In order to test whether T derive from T cells that turn on Foxp3 expression we FR FH 5 + low − transferred 1×10 naïve cells (CD4 CD44 CD25 ) from CD45.1 mice expressing the 3A9 HEL TCR transgene (TCR ), which recognizes hen egg lysozyme (HEL) peptide presented by I-A , into congenic CD45.2 B10.BR mice. Seven days after immunization with HEL in alum, 6-10% of T cells derived from the donor HEL-TCR T cells, but no donor-origin FH T could be identified; all T derived exclusively from the recipients’ cells (Fig. 4a). FR FR HEL Between 1-2% of the transferred naïve TCR donor population developed into inducible T (Fig. 4b). To exclude that an idiosyncratic effect of the 3A9 transgene had precluded reg development of T cells, this experiment was repeated with OT-II transgenic T cells: FR Again, OT-II T cells could form T but not T (Fig. 4c, d), suggesting that T do not FH FR FR derive from T cells. FH + 6 + low − To test whether T derive from Foxp3 precursors, 1×10 naïve CD4 CD44 Foxp3 T FR int + GFP cells or CD44 Foxp3 T from unimmunized Foxp3 mice were adoptively transferred reg into congenically-marked mice. Seven days after immunization with KLH in Ribi, ~1-2% of both donor-origin T and donor-origin naïve cells had upregulated CXCR5 and PD-1 to reg high high high levels (Figure 4e). More than 90% of donor-origin CXCR5 PD-1 T cells reg retained Foxp3 expression, but none of the transferred naïve T cells that became high high CXCR5 PD-1 after immunization switched on Foxp3 to become T cells (Fig. 4e). FR + SP We then asked whether thymic T (nT ) could become T cells. Thymic Foxp3 CD4 reg reg FR − SP GFP or Foxp3 CD4 from Foxp3 CD45.2 mice were adoptively transferred into CD45.1 mice. Seven days after SRBC immunization only Foxp3 CD45.2 cells had become T ; FR Foxp3 CD45.2 cells had become T but not T (Supplementary Fig. 5). Furthermore, FH FR ~97% of T cells expressed Helios (Supplementary Fig. 5), a transcription factor which has FR been reported to be expressed by thymic-derived nT but not T induced in the ref ref periphery . In an alternative strategy to confirm that T derive from Foxp3 precursors we used mice FR in which the gene encoding for the diphtheria toxin receptor (DTR) has been inserted in the DTR Foxp3 locus (Foxp3 ), so that treatment with diphtheria toxin (DT) selectively ablates all Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 5 40 DTR T within 48 hours . Foxp3 mice were immunized with SRBC and treated with either reg DT or saline immediately afterwards. Six days after immunization, T cells had formed FR normally in mice that did not receive DT but were absent in DT-treated mice (Fig. 4f, g) demonstrating T cannot form if Foxp3 cells are absent at the time of immunization. FR Within Peyer’s patches, but not in the spleen, it has been demonstrated that a proportion of − + 41 Foxp3 T cells derive from Foxp3 precursors . To investigate whether splenic T cells FR FR stably express Foxp3, we immunized Foxp3-Cre x ROSA-Stop-flox-YFP mice, in which any cell that has expressed Foxp3 is permanently marked. Seven days after SRBC + + high high immunization, the majority (97%) of YFP CD4 CXCR5 PD-1 T cells coexpressed FR high high − + Foxp3 and less than ~0.6% of CXCR5 PD-1 cells were Foxp3 YFP (Fig. 4h), consistent with the observation that Foxp3 expression is stable in the natural T reg 43 + population . These data suggest that T cells derive from Foxp3 thymic T that co-opt FR reg the T cell differentiation program to migrate to germinal center, where they maintain FH Foxp3 expression. T cells suppress T cell proliferation in vitro and repress T cells in vivo FR FH Expression of Foxp3 by CD4 T cells initiates a transcriptional program that confers 11-13 GFP suppressor function . T and T sorted from immunized Foxp3 mice displayed FR reg comparable suppressive ability in vitro (Supplementary Fig. 6a). To determine whether T FR cells are suppressive in vivo, we sought to ablate T cells after the germinal center FR DTR response had been established. For this, DT was first administered to Foxp3 mice 5d after SRBC immunization, when T and T cells have already formed, but the response FH FR has not yet reached its peak . After 3 days of DT treatment – 8 days after immunization – only 1% of T cells and 5% of T cells were present compared with vehicle-only-treated FR reg mice (Supplementary Fig. 7), and DT-treated mice displayed a significant increase in both the proportion and total number of T cells compared to controls (Fig. 5a). At this time FH point, germinal center B cell numbers were comparable between DT- and vehicle-treated groups (Figure 5b). To confirm the in vivo regulatory role of T , it is necessary to deplete them while leaving FR the T and T compartments intact. To achieve this we generated mixed bone marrow reg FH −/− DTR chimeras with a 1:1 ratio of either congenically marked Sh2d1a :Foxp3 or +/+ DTR + −/− Sh2d1a :Foxp3 marrow. After immunization and DT treatment, CD45.2 Sh2d1a cells should not able to form T or T cells, but should form T cells normally, whereas FH FR reg DTR + Foxp3 cells should form T cells, but lack all Foxp3 cells. Thus, T and T FH FH reg populations should still be present in both chimeras, but T cells will be selectively FR −/− DTR reduced in Sh2d1a : Foxp3 mice (Supplementary Table 2). Such chimeric mice were generated and treated with DT 1 day prior to, and 2 and 5 days after SRBC immunization. At day 8 after immunization >90% of T cells in FH −/− DTR DTR Sh2d1a :Foxp3 chimeras derived from Foxp3 cells compared to ~50% in the +/+ DTR control Sh2d1a :Foxp3 mice (Supplementary Fig. 8a). The size of the T population reg was comparable in both groups of mice (Fig. 5c) with the majority (97%) of T being reg CD45.2 (Supplementary Fig. 8b). Critically, the number of T was reduced by 5 times in FR −/− DTR Sh2d1a : Foxp3 mice compared with the control chimeras (Fig. 5d, Supplementary Fig. 7c). There was also an increase in both the number and proportion of T cells (Fig. 5e) FH and of germinal center B cells (Fig. 5f), indicating that T limit the germinal center FR response during a T-dependent immune reaction in vivo. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 6 T limit the outgrowth of non-antigen-specific germinal center B cells FR To further characterize the role of T during the germinal center response, T were FR FR DTR transiently depleted at the peak of the germinal center response. For this, Foxp3 and WT control Foxp3 mice were immunized with NP-KLH in alum, and treated with DT six days later. Ten days after immunization, the fraction of germinal center B cells was not significantly different between the two groups (Fig. 6a, b) but combined T and T reg FR depletion led to a reduction in the proportion of germinal center B cells specific for the dominant epitope of the immunizing antigen, the hapten NP (Fig. 6a, c). In order to determine whether this had any long-term impact on the antigen-specific DTR antibody response, Foxp3 mice were immunized with NP-KLH in alum and treated with either DT or saline at 7, 10 and 13 days after immunization, then boosted with NP-KLH in alum 24 days after primary immunization (Fig. 6d). High (anti-NP2) & low (anti-NP12) affinity anti-NP antibody titers were assessed by ELISA prior to and d10, 15, 10 and d28 after immunization. Titers of high and low affinity antibodies were comparable until d20, when both were reduced in DT-treated mice compared with controls (Fig 6e). Four days after secondary immunization (day 28 after primary challenge), high affinity antibodies remained lower in DT-treated mice (Fig 6e). This suggests that depletion of T and T FR reg during the germinal center response does not increase antigen-specific antibody production. To test whether the reduction in the antigen-specific germinal center response was a consequence of T depletion rather than general T depletion, we generated FR reg −/− −/− Sh2d1a :Foxp3 mixed fetal liver chimeras that selectively lack T cells. Three groups FR +/+ +/+ +/+ −/− of control chimeras were generated in parallel: Sh2d1a :Foxp3 , Sh2d1a :Foxp3 −/− +/+ and Sh2d1a :Foxp3 . Eight weeks after reconstitution, chimeric mice were immunized with alum-precipitated NP-chicken gammaglobulin (NP-CGG). 21 days post-immunization, −/− −/− Sh2d1a :Foxp3 chimeras had a reduction in T cells compared to all control groups FR (Supplementary Fig. 9a and b) and an expanded T population (Supplementary Fig. 9c). FH The proportion of Foxp3 T was comparable amongst all 4 groups (Supplementary Fig. reg 9d). Consistent with a selective defect in T and not T , circulating T 1 and T 2 cells FR reg H H −/− −/− were not expanded in Sh2d1a :Foxp3 mice 14 days post-immunization (Supplementary Fig. 10a and b) nor in the spleen 21 days after immunization (Supplementary Fig. 10c and d). This also confirms that T cells are specialized in the regulation of follicular responses, FR while other T effectors control T 1 and T 2 cells. reg H H DTR As observed in Foxp3 mice, the proportion and absolute number of antigen-specific + −/− −/− (NP ) germinal center B cells was reduced in Sh2d1a :Foxp3 mice compared with control chimeras (Fig. 6f), despite formation of abundant germinal centers (Fig. 6g). NP- specific splenic memory B cells and NP-specific bone marrow plasma cells appeared −/− −/− reduced in Sh2d1a :Foxp3 chimeras compared with controls 21 days after immunization (Figure 6h and i) and the differences were statistically-significant against all −/− +/+ control groups except for the Sh2d1a :Foxp3 group, in which there was greater variability. At this time point there was no difference in anti-NP antibody titers between −/− −/− Sh2d1a :Foxp3 mice compared with control chimeras (Supplementary Fig. 11), probably due to a large component being of extrafollicular origin given that abundant NP plasma cells were still detectable in the spleen (Supplementary Fig. 11). Together, this suggests that T act to limit the outgrowth of non-antigen specific clones in the germinal FR center. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 7 Discussion We have shown here that in response to T-dependent antigens a proportion of naïve T can reg turn on Bcl-6, which allows them to adopt the T differentiation program and express the FH follicular homing receptor CXCR5 to localize to the germinal center. Here they exert suppressive functions on T cells and the germinal center response. Unlike T cells, T FH FH FR cells express Blimp-1, which is required to control their numbers in the germinal center. In response to other extracellular stimuli, naïve Foxp3 cells can turn on Tbet or increase activity of IRF4 or STAT3, required for T 1, T 2 and T 17 cell formation H H H 16-18 respectively . The specialized regulatory program determined by each of these transcription factors is likely to operate via modification of the T chemokine receptor reg profile in order to allow migration into an anatomical location where T are poised to reg regulate specific T cell responses to prevent autoimmunity or inflammation-associated tissue damage. Our data suggests T cells are specialized in controlling the germinal center reaction FR through limiting the numbers of T cells and inhibiting selection of non-antigen-specific B FH cells including those carrying self-reactive receptors. Furthermore, the ability of T cells to FR ensure dominance of antigen-specific clones over the germinal center response appears essential for formation of normal numbers of long-lived plasma cells and memory B cells. Although it is likely that T control germinal center B cells indirectly, through their ability FR to limit T numbers, it is also possible that T also negatively regulate germinal center B FH FR cells directly. This would be akin to the description of direct regulation of antigen- 45, 46 9 presenting cells by T and reports that T can directly regulate B cell function . reg reg Germinal center B cells are the predominant APCs within the germinal center microenvironment, and this makes them attractive candidates for T -mediated inhibition. FR Selection of cognate germinal center B cells by T cells is one of the key mechanisms by FH which germinal center tolerance is regulated . Dysregulation of the T population has FH 3, 5, 7, 48 been previously demonstrated to result in autoimmunity , highlighting the need to tightly control positive selection in germinal centers. Understanding the mechanisms by which T are regulated and their TCR specificity will be important for dissecting the FR pathogenesis of the increasing number of pathologies in which T cells appear to play a FH role, including disease-associated ectopic germinal center formation seen in many 3 49 50 autoimmune diseases , atherosclerosis , and chronic allograft rejection . We therefore postulate that T cells may represent a critical peripheral tolerance mechanism, essential FR for preventing germinal center-derived autoimmunity. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments We thank Xin Hu and Jessica Fitch for technical assistance, Dimitra Zotos for help with experiments not included GFP in this manuscript, Alexander Rudensky for kind provision of Foxp3 mice and Marion Espeli for helpful discussions. This work was funded by NHMRC program and project grants to CGV, a VIB PI grant to AL and a Wellcome Trust Programme Grant (083650/Z/07/Z) to KGCS. MAL is supported by an EMBO Post-doctoral Long-term Fellowship (ALTF 1041-2009) and a Raymond and Beverly Sackler Junior Research Fellowship, Churchill College, Cambridge; CGV by a Viertel Senior Medical Research Fellowship; KGCS by a Lister Prize Fellowship and TFR by the National Institute of Health Research Cambridge Biomedical Research Centre; and AL by a JDRF Career Development Fellowship and a Marie Curie Reintegration Grant Fellowship. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. 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A proportion of CXCR5 PD-1 CD4 cells express the transcription factor Foxp3 (a,b) After SRBC immunization Foxp3 cells were identified in the high high + CXCR5 PD-1 CD4 ‘T ’ compartment, these cells follow the same kinetics as FH + + classic T cells. (c) Foxp3 cells (red) are present within the Bcl6 germinal center area FH (green) following SRBC immunization. Scale bar represents 100μm. (d) Heat map + GFP comparing the gene expression profiles of different CD4 T cell subsets from Foxp3 mice seven days after immunization. Red: high gene expression; blue: low gene expression. The cells were sorted using the following markers and for simplicity will be referred by the + low − abbreviations in parentheses throughout: CD4 CD44 Foxp3 naïve (T ) cells, + high int/low int/low − CD4 CD44 CXCR5 PD-1 Foxp3 effector/memory (T ) cells, EM + int int/low int/low + CD4 CD44 CXCR5 PD-1 Foxp3 regulatory T cells (T ), reg + high high − CD4 CXCR5 PD-1 Foxp3 T follicular helper (T ) cells and FH + high high + CD4 CXCR5 PD-1 Foxp3 follicular regulatory (T ) cells. (e) Il21 and Il4 mRNA FR measured by quantitative PCR from sorted cells using the strategy described in (d) normalized to Gapdh. Heights of the bars represent the mean and error bars represent the range of expression from 3 biological replicates. nd: gene expression not detected. (f) Left: Intracellular expression of CD40L as determined by flow cytometry in T (blue), T reg FH (green) and T (red) cell populations; the grey histogram represents a staining control from FR an immunized CD40L-deficient mouse. Right: Cxcl13 mRNA measured by quantitative RT- PCR as described in (e). (g) Cell surface expression of GITR, CD25 and intracellular CTLA4 in T (blue), T (green) and T (red) cell populations; grey histograms represent reg FH FR the isotype control. (h) Relative Gzmb and Gzma mRNA determined by quantitative RT- PCR as described in (e). (i) Percentage of CD103 cells within the T , T & T reg FH FR populations, each symbol represents one mouse. (j) Left: Cell surface expression of ICOS as Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 12 determined by flow cytometry in T (blue), T (green) and T (red) cell populations; the reg FH FR grey histogram represents staining level of an isotype control. Right: Il10 mRNA detected by quantitative RT-PCR of as described in (e). Flow cytometric and RT-PCR data are representative of at least three independent experiments. In (e)-(i): Statistical significance was determined using a one-way ANOVA analysis with Bonferroni’s multiple testing correction; * P<0.05; **P<0.01; ***P<0.001. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 13 Figure 2. T require the same differentiation cues as T cells for their development FR FH Flow cytometric contour plots (a, d, g) and dot plots of T (b, e, h) and T (c, f, i) cells in FH FR the groups of mice described below, seven days after SRBC immunization. (a-c) Mixed −/− bone marrow chimeras generated by sub-lethally irradiating Rag2 mice and reconstituting +/+ their immune system with a 1:1 ratio of bone marrow cells from CD45.1 Cd28 and −/− +/+ +/+ CD45.2 Cd28 mice or control CD45.1 Cd28 and CD45.2 Cd28 . (d-f) C57BL/6 (BL/ +/+ −/− 6) and B-cell deficient μMT mice. (g-i) Sh2d1a and Sh2d1a mice. Each symbol represents one mouse and horizontal bars represent median values. Figures represent one of 3 independent experiments with similar results. Statistical significance was determined using a Mann-Whitney Test: *P<0.05, **P<0.01. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 14 Figure 3. T cells express Bcl-6 and Blimp-1 FR (a) Bcl6 and Prdm1 mRNA normalized to Gapdh determined by quantitative RT-PCR from sorted cells using the strategy described in Fig. 1d and Supplementary Fig. 1. Heights of the bars represent the mean and error bars represent the range of expression from 3 biological replicates. Statistical significance was determined using a one-way ANOVA analysis with Bonferroni’s multiple testing correction; * P<0.05; **P<0.01; ***P<0.001. Bar graphs are representative of 3 experiments. (b) Immunofluorescence of frozen spleen sections from mice immunized seven days previously with SRBC. The germinal center is demarcated by the white dotted line in the three consecutive sections. Upper panel: AID (red) and CD3 (green); middle panel: Foxp3 (red) and Bcl-6 (green); lower panel: Foxp3 (red) and Blimp1 (green). Scale bar represents 100μm. (c) Flow cytometric contour plots of T (upper FH panels) & T (lower panels) formation in the draining (mediastinal) lymph node ten days FR after intranasal influenza infection of mixed fetal liver chimeras reconstituted with a 1:1 gfp/gfp +/+ ratio of fetal liver cells from E14.5 CD45.2 Prdm1 : CD45.1 Prdm1 embryos, E14.5 −/− +/+ gfp/+ CD45.2 Bcl6 : CD45.1 Bcl6 embryos or control E14.5 CD45.2 Prdm1 : CD45.1 +/+ Prdm1 embryos. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 15 Figure 4. T derive from Foxp3 precursors FR + high high + Flow cytometric contour plots of splenic CD4 CXCR5 PD-1 cells (a) or CD4 cells 5 3A9 (b) seven days after 1×10 transferred transgenic TCR HEL-specific CD45.1 T cells were adoptively transferred into congenically distinct CD45.2 B10.Br mice and immunized + high high with HEL in alum. Flow cytometric contour plots of splenic CD4 CXCR5 PD-1 cells + 5 (c) or CD4 cells (d) seven days after adoptive transfer of 1×10 OT-II OVA-specific Thy1.2 T cells into congenically distinct Thy1.1 C57BL6 mice and immunization with OVA in alum. (e) Flow cytometric contour plots of splenic CD4 T cells from CD45.1 C57BL/6 6 + int + mice seven days after adoptive transfer of 1×10 sorted naïve CD4 CD44 Foxp3 T (top reg + low − panel) or CD4 CD44 Foxp3 naïve T cells (lower panel) from unimmunized CD45.2 GFP Foxp3 mice and KLH in Ribi immunization. Transferred CD45.2 cells are shown in red, the endogenous CD45.1 cells are represented by the grey contour plots. Histograms showing + + high high Foxp3-GFP expression in transferred CD45.2 CD4 CXCR5 PD-1 cells. (f) Contour + DTR plots of splenic CD4 T cells and quantification of T cells (g) from Foxp3 mice six FR days after SRBC immunization and administration of either 0.9% saline (top panel) or DT + + high high (lower panel). Histograms show Foxp3 cells within the CD4 CXCR5 PD-1 compartment. (h) Flow cytometric contour plots of splenic CD4 cells from Foxp3-cre x ROSA-Stop-flox-YFP mice immunized seven days previously with SRBC (left panel). + high high Enumeration of the proportion of CD4 CXCR5 PD-1 cells that expressed YFP and/or Foxp3 (right panel). Each symbol represents one mouse and horizontal bars represent median values. Figures are representative of 2-4 independent experiments. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 16 Figure 5. T regulate the size of the T population FR FH Flow cytometric contour plots and graphs of T cells (a) and germinal center B cells (b) FH DTR from the spleens of Foxp3 mice immunized eight days previously (d0) with SRBC. Five days after immunization the mice were treated with either DT or saline. (c-f) Analysis of −/− mixed bone marrow chimeras generated by sub-lethally irradiating Rag2 mice and −/− DTR reconstituting their immune system with either a 1:1 ratio of Sh2d1a CD45.2 : Foxp3 +/+ DTR CD45.1 bone marrow or control Sh2d1a CD45.2 : Foxp3 CD45.1 bone marrow. Eight weeks after reconstitution chimeric mice were immunized with SRBC and treated with 50μg/Kg of DT on one day prior to immunization and d2 and d5 thereafter. Splenocytes were analyzed on d8 for the proportion and total number of + high high + + + CD4 CXCR5 PD-1 Foxp3 T cells (c), CD4 Foxp3 T (d), FR reg + high high + high high CD4 CXCR5 PD-1 T cells (e) and B220 GL-7 CD95 germinal center B FH cells (f). Each symbol represents one mouse and horizontal bars represent median values. Statistical significance was determined using a Mann-Whitney Test: *P<0.05, **P<0.01. Nat Med. Author manuscript; available in PMC 2012 January 24. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Linterman et al. Page 17 Figure 6. T restrict the outgrowth of non-antigen specific clones in the germinal center FR + + Flow cytometric contour plots (a) and graphs (b) of total GL-7 CD95 germinal center B + WT cells and (c) NP germinal center B cells ten days after immunization of Foxp3 and DTR Foxp3 mice that have been treated with DT 6 days after NP-KLH immunization. Statistical analyses performed using Mann Whitney U-test. Experimental outline (d) of DTR immunization and DT or saline treatment scheme of Foxp3 mice (n=8 per group) to examine the antigen specific immunoglobulin response over time, mice were bled prior to, and d10, d15, d20 and d28 after primary immunization. Mice were given a booster immunization 24 days after the primary immunization. (e) ELISA analysis of NP12 and NP2 antibodies in the experiment outlined in (d). Error bars represent the standard error of the mean from eight individual mice from one experiment, representative of two experiments. Statistical analyses in (e) were performed using a two-way ANOVA with Bonferroni post test to compare differences at each time point. Graphs and flow cytometric contour plots of + + + + NP germinal center B cells (f), total GL-7 CD95 germinal center B cells (g) and NP bone marrow plasma and memory cells (h, i) 21 days after NP-CGG immunization of −/− chimeric mice generated by reconstituting Rag2 mice with a 1:1 mix of −/− −/− +/+ +/+ +/+ −/− −/− +/+ Sh2d1a :Foxp3 , Sh2d1a :Foxp3 , Sh2d1a :Foxp3 and Sh2d1a :Foxp3 fetal liver. Statistical analyses in (f, g, h and i) were performed using a one-way ANOVA with Bonferroni post test correction. Each symbol represents one mouse and horizontal bars represent median values. *P<0.05, **P<0.01, ***P<0.001. Nat Med. Author manuscript; available in PMC 2012 January 24.

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Nature medicine – Pubmed Central

Published: Jul 24, 2011

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