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A fundamental function of CD4+ helper T (TH) cells is the regulation of B cell–mediated humoral immunity. Development of T follicular helper (TFH) cells that provide help to B cells is mediated by the cytokines interleukin-6 and interleukin-21 but is independent of TH1, TH2, and TH17 effector cell lineages. Here, we characterize the function of Bcl6, a transcription factor selectively expressed in TFH cells. Bcl6 expression is regulated by interleukin-6 and interleukin-21. Bcl6 overexpression induced TFH-related gene expression and inhibited other TH lineage cell differentiation in a DNA binding–dependent manner. Moreover, Bcl6 deficiency in T cells resulted in impaired TFH cell development and germinal center reactions, and altered production of other effector T cell subsets. Our data thus illustrate that Bcl6 is required for programming of TFH cell generation.
A critical function of CD4+ helper T (TH) cells is to provide “help” to B cells, especially in the germinal center structures where activated B cells proliferate and undergo antibody affinity maturation. Recently, T follicular helper (TFH) cells have been characterized by their expression of chemokine (C-X-C motif) receptor 5 (CXCR5) (1–3).We, as well as others, recently reported that TFH cell development is mediated by interleukin (IL)–6 or IL-21 but is independent of TH1, TH2, and TH17 cells (4, 5).
The B cell lymphoma 6 (Bcl6) transcription factor is selectively expressed by TFH cells (2, 3). Bcl6 was previously shown to be inhibitory to TH2 responses by blocking signal transducer and activator of transcription 6 (STAT6) binding to DNA (6, 7), whereas Bcl6-deficient mice developed multiorgan inflammatory diseases, enhanced immunoglobulin E (IgE) production, and defective germinal center reaction (6, 8). It is not clear whether the germinal center defect in these mice is caused by lack of proper T and/or B cell function because Bcl6 is also expressed by germinal center B cells (9). To analyze the function of Bcl6 in TFH cells, we activated naïve CD4+ T cells (CD44lowCD62LhighCD25−) from C57BL/6 mice with antibodies to CD3 and CD28 in the presence or absence of various cytokines for 1 or 2 days, and Bcl6 mRNA expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis (10). Treatment with IL-6 or IL-21 significantly up-regulated Bcl6 expression, which was strongly inhibited by the addition of exogenous transforming growth factor beta (TGFβ) (Fig. 1A). These results correlate with our previous observations that IL-6 or IL-21 alone induces TFH cell development and Bcl6 expression, whereas treatment, together with TGFβ, promotes TH17 differentiation instead (4). To determine whether IL-21 is necessary for IL-6–induced Bcl6 expression, we activated naïve wild-type and IL-21– or IL-21 receptor (IL-21R)–deficient CD4+ T cells in the presence of IL-6. IL-21– and IL-21R–deficient T cells showed significantly reduced expression of Bcl6 (fig. S1).
We next assessed whether overexpression of Bcl6 promoted TFH cell development in the absence of exogenous cytokines. Bcl6 overexpression led to increased expression of endogenous Bcl6 mRNA as well as IL-21R, IL-6R, and CXCR5 mRNA, similar to cells treated with IL-6 or IL-21 (Fig. 1B and fig. S2A). Interestingly, IL-21 expression was not up-regulated by Bcl6 overexpression.
Bcl6 has multiple zinc finger (ZF) domains, and the mutation of two of these (ZF3 and ZF5) was previously shown to abolish DNA binding but not nuclear localization (11). We thus assessed the function of Bcl6 with a mutation in either domain in the induction of TFH-specific genes. ZF3 and ZF5 mutations completely abrogated the ability of Bcl6 to up-regulate endogenous Bcl6, IL-21R, and CXCR5 expression, whereas the ZF3 exhibited less efficient inhibition of IL-6R expression than ZF5 (Fig. 1C). Thus, the regulation of TFH gene expression by Bcl6 appears to depend largely on its ability to bind DNA.
We then assessed whether Bcl6 overexpression antagonizes the differentiation of other TH lineage cells. We first overexpressed Bcl6 in cells undergoing TH17 differentiation in the presence of TGFβ and IL-6. We found that Bcl6 overexpression induced endogenous Bcl6 expression and also moderately increased the expression of CXCR5, IL-21R, and IL-6R (Fig. 2A); however, the expression of these genes was significantly lower than that induced by Bcl6 under neutral conditions or in TFH cells (Fig. 1B). Bcl6 also inhibited IL-17 protein expression when measured by intracellular staining, as well as IL-17 and IL-17F mRNA expression, in a DNA binding–dependent manner (Fig. 2, A and B). In contrast, RORγt or IL-21 expression was not affected by Bcl6. We further analyzed whether Bcl6 might influence RORγt-dependent transcription using a luciferase reporter driven by the IL-17 gene promoter and the conserved noncoding sequence 2 (CNS2) element (12). Wild-type Bcl6, but not the DNA-binding mutants ZF3 and ZF5, strongly inhibited RORγt-induced luciferase activity (Fig. 2C). Thus, similar to Foxp3, Bcl6 inhibits RORγt function but not its expression. Unlike Foxp3 (13), however, Bcl6 function appears to be dependent on its binding to DNA.
In addition to TH17 cells, we also analyzed the effect of Bcl6 in developing TH1 and TH2 cells and found that overexpression of Bcl6 increased endogenous Bcl6 expression and CXCR5 (Fig. 2D and fig. S2, B and C). Bcl6, but not ZF3 and ZF5 mutants, also significantly inhibited the expression of TH1 (IFNγ and T-bet) and TH2 (IL-4 and GATA3) genes (Fig. 2, D and E, and fig. S2, B and C). Together, these data suggest that Bcl6 autoregulates its own expression. Moreover, Bcl6 not only induces TFH gene expression in neutral conditions, similar to IL-6 or IL-21, but also inhibits the differentiation of other TH lineage cells.
To understand whether Bcl6 is necessary for TFH cell development, we used a previously reported Bcl6 knockout mouse (Bcl6−/−) (14) and crossed it with OT-II T cell receptor (TCR) transgenic mice. The OT-II TCR is specific for a peptide derived from chicken ovalbumin presented by major histocompatability complex class II; thus, the majority of T cells in these mice are CD4+. Naïve CD4+ T cells were first differentiated to promote TFH cell differentiation in vitro (4). Bcl6 deficiency resulted in substantial reduction of CXCR5 and IL-6R expression, although the expression of IL-21R and IL-21 was partially reduced (Fig. 3A).
We also subjected control and Bcl6−/− naïve T cells to TH1 and TH17 differentiation. As expected, absence of Bcl6 resulted in enhanced TH1 and TH17 differentiation and increased expression of T-bet and RORγt, respectively (Fig. 3, B and C, and fig. S3). Interestingly, IL-21 mRNA expression in Bcl6−/− cells differentiated under TH17 condition was enhanced, correlating with increased expression of IL-17 and RORγt (Fig. 3C). When activated in the presence of TGFβ to induce Foxp3 expression, Bcl6−/− T cells exhibited reduced levels of Foxp3 expression but enhanced IL-4 and IFNγ expression. Moreover, Foxp3 expression was partially restored by treatment with antibodies to IFNγ and IL-4 (Fig. 3D).
To analyze the function of Bcl6 in TFH cell generation in vivo, we first analyzed germline wild-type and Bcl6−/− mice. We found that memory T cells from aged Bcl6−/− mice produced significantly elevated amounts of IL-17, but their production of IFNγ and IL-4 was comparable to that of wild-type cells (fig. S4, A and B). IL-21 expression in IL-17+ T cells was also increased in these mice (fig. S4C). We then immunized wild-type and Bcl6−/− mice with keyhole limpet hemocyanin (KLH) protein (4), which induces an immune response where B cell activation requires T cell help. Bcl6−/− mice exhibited severely reduced CXCR5 expression on T cells and numbers of germinal center B cells (fig. S5) 7 days after immunization. Immunohistochemistry analysis also revealed that the germinal center B cells were greatly reduced in the Bcl6−/− mice when compared with immunized wild-type mice (fig. S6A). The production of KLH-specific IgG, IgM, and IgG2a was also reduced in Bcl6−/− mice, whereas IgE was enhanced (fig. S7A). In contrast, we observed increased expression of TH1, TH2, and TH17 cytokines in Bcl6−/− mice, whereas IL-21 production was not affected (fig. S5). To substantiate the above results, we also purified CD4+CD44high cells from immunized mice and measured their cytokine secretion after activation ex vivo with KLH and irradiated wild-type splenic antigen-presenting cells. We observed greatly increased expression of IL-4, IFNγ, and IL-17 in Bcl6−/− T cells when compared with wild-type T cells (fig. S8A). Furthermore, Bcl6−/− T cells exhibited defective expression of TFH-related genes, but their expression of TH1, TH2, and TH17 genes was significantly enhanced (fig. S8B). Interestingly, Bcl6 deficiency did not result in defective IL-21 production, and TH17 cells are likely the source of IL-21 in knockout mice.
To specifically analyze the function of Bcl6 in T cells, we mixed wild-type or Bcl6−/− naïve CD4+ T cells with wild-type B cells and transferred them into Rag1−/− mice, which lack T and B cells, and immunized the mice with KLH. Absence of Bcl6 in T cells again resulted in greatly reduced numbers of CXCR5+ T cells (Fig. 4A). Moreover, germinal center B cells (GL7+ FAS+) were greatly reduced in these animals (Fig. 4A), which was consistent with our immunohistochemistry analysis (fig. S6B). KLH-specific IgG, IgM, and IgG2a production was also reduced (fig. S7B). We also observed that Bcl6 deficiency resulted in enhanced production of IL-4 by T cells and KLH-specific IgE by B cells, but only moderately decreased IL-17 and IL-21 expression by knockout T cells (Fig. 4A and fig. S7B). In another experiment, we transferred wild-type and Bcl6−/− total CD4+ T cells with wild-type B cells into Rag1−/− mice and immunized them with KLH. The analysis of sorted effector-phenotype CD4+CD44high cells from the immunized mice revealed that Bcl6 deficiency in T cells resulted in the reduction of mRNA expression of most of the TFH-related genes except for IL-21 (fig. S9), which was increased together with IL-17 and RORγt, suggesting that the source of IL-21 may be TH17 cells.
Because Bcl6 deficiency also caused hyper-IL-4 production in the above experiment, we examined the effect of IL-4 neutralization. Treatment with antibody to IL-4 almost completely blocked IL-4 production by T cells, whereas defective CXCR5 expression was still observed in Bcl6−/− T cells (Fig. 4B). We also observed reduced generation of germinal center B cells and KLH-specific IgG, IgM, and IgG2a production (Fig. 4B, fig. S6C, and fig. S7C). In addition, Bcl6−/− T cells also exhibited increased IL-22, IL-17, and IFNγ production (Fig. 4B). Despite the absence of TFH cells in Bcl6−/− mice, we detected IL-21 production that was comparable to wild-type mice, likely derived from TH17 cells.
To further demonstrate the role of Bcl6 in T and B cells, we generated mixed bone marrow chimeras by transferring a mixture of congenic CD45.1+ wild-type and CD45.2+ Bcl6-deficient bone marrow cells into sublethally irradiated Rag1−/− mice. Eight weeks after reconstitution, we immunized the mice with KLH. Bcl6−/− CD4+ T cells did not mature into CXCR5+ TFH cells that also express B and T lymphocyte attenuator (BTLA) (Fig. 4C), and did not exhibit germinal center B cells. CD4+CD44high Bcl6−/− cells exhibited greatly reduced TFH gene expression; however, the expression of TH1, TH2, and TH17 genes were all increased when compared with wild-type cells (fig. S10). Moreover, Bcl6-deficient B cells did not produce germinal center B cells. On the basis of the above data, we conclude that Bcl6 expression in both T and B cells is required for the germinal center reactions.
In summary, we have demonstrated that Bcl6 is regulated by IL-6 and IL-21 but that, similar to RORγt in TH17 cells, it does not regulate the expression of IL-21. Bcl6 deficiency in vivo did not inhibit IL-21 expression, likely by TH17 cells. Thus, IL-21 expression by non-TFH cells is not sufficient to induce germinal center reactions. Our data overall indicate that Bcl6, selectively induced by IL-21 or IL-6 in the absence of TGFβ signaling, serves as a master transcriptional factor in TFH cell differentiation, analogous to RORγt and RORα, both of which are induced by IL-6 or IL-21 in the presence of TGFβ and function to promote TH17 differentiation (fig. S11). IL-6 alone induces the expression of RORγt (15), whose function is inhibited by Bcl6, similar to regulatory T cells (Treg), where the function of TGFβ-induced RORγt is suppressed by Foxp3 (13, 16). The combination of TGFβ and IL-6 not only induces RORγt expression in T cells, but in the meantime, Foxp3 and Bcl6 expression is suppressed by IL-6 and TGFβ signaling, respectively, allowing RORγt to function properly in promoting TH17 cell differentiation.
Although we proposed in the past year that TFH cell differentiation mediated by IL-6 and IL-21 is a novel lineage of TH cell differentiation, subsequently several groups reported that CXCR5+ T cells also express TH2 or TH17 cytokines in vivo (17, 18). Our current study indicates that Bcl6 promotes the expression of TFH-related genes but inhibits the differentiation of TH1, TH2, and TH17 cells. Our data thus support Bcl6-dependent TFH cell generation as a pathway that is independent of other TH cell lineages.
Supporting Online Material
Materials and Methods
Figs. S1 to S11