STAT5 Protein Negatively Regulates T Follicular Helper (Tfh) Cell Generation and Function*

Background: Tfh cells regulate B cell-mediated humoral immunity. Results: STAT5 regulated Blimp-1 expression, and STAT5 deficiency in CD4+ T cells resulted in an increase of Tfh generation and an impairment of B cell tolerance. Conclusion: STAT5 negatively regulates Tfh development by up-regulating Blimp-1 and thus controls the humoral immunity and B cell tolerance. Significance: These findings may help to find new ways to treat antibody-mediated autoimmune diseases. Recent work has identified a new subset of CD4+ T cells named as Tfh cells that are localized in germinal centers and critical in germinal center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and B cell lymphoma 6 (Bcl6) is specifically expressed in Tfh cells and required for their lineage specification. In the current study, we characterized the role of STAT5 in Tfh cell development. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors (CXC motif) receptor 5 (CXCR5), musculoaponeurotic fibrosarcoma (c-Maf), Bcl6, basic leucine zipper transcription factor ATF-like (Batf), and IL-21, and STAT5 deficiency greatly enhanced Tfh gene expression. Importantly, STAT5 regulated the expression of Tfh cell suppressor factor B lymphocyte-induced maturation protein 1 (Blimp-1); STAT5 deficiency impaired Blimp-1 expression and resulted in elevated expression of Tfh-specific genes. Similarly, inhibition of IL-2 potentiated Tfh generation, associated with dampened Blimp-1 expression; Blimp-1 overexpression inhibited Tfh gene expression in Stat5-deficient T cells, suggesting that the IL-2/STAT5 axis functions to regulate Blimp-1 expression. In vivo, deletion of STAT5 in CD4+ T cells resulted in enhanced development of Tfh cells and germinal center B cells and led to an impairment of B cell tolerance in a well defined mouse tolerance model. Taken together, this study demonstrates that STAT5 controls Tfh differentiation.


Recent work has identified a new subset of CD4 ؉ T cells named as Tfh cells that are localized in germinal centers and critical in germinal center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and B cell lymphoma 6 (Bcl6) is specifically expressed in Tfh cells and required for their lineage specification.
In the current study, we characterized the role of STAT5 in Tfh cell development. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors (CXC motif) receptor 5 (CXCR5), musculoaponeurotic fibrosarcoma (c-Maf), Bcl6, basic leucine zipper transcription factor ATF-like (Batf), and IL-21, and STAT5 deficiency greatly enhanced Tfh gene expression. Importantly, STAT5 regulated the expression of Tfh cell suppressor factor B lymphocyte-induced maturation protein 1 (Blimp-1); STAT5 deficiency impaired Blimp-1 expression and resulted in elevated expression of Tfh-specific genes. Similarly, inhibition of IL-2 potentiated Tfh generation, associated with dampened Blimp-1 expression; Blimp- 1

overexpression inhibited Tfh gene expression in Stat5-deficient T cells, suggesting that the IL-2/STAT5 axis functions to regulate Blimp-1 expression. In vivo, deletion of STAT5 in CD4 ؉ T cells resulted in enhanced development of Tfh cells and germinal center B cells and led to an impair-ment of B cell tolerance in a well defined mouse tolerance model. Taken together, this study demonstrates that STAT5 controls Tfh differentiation.
Recently, a new subset of CD4 ϩ T cells, named Tfh 4 cells, has emerged as a major player in B cell-mediated humoral immunity, especially in the germinal center reactions (1)(2)(3)(4). Tfh cells have been characterized by their expression of chemokine CXCR5, which is induced in T cells following activation and dependent on costimulatory signals, such as CD28, inducible costimulatory molecule, and OX40. Although activated T cells may transiently express CXCR5, Tfh cells exhibit more stable expression of this chemokine receptor (5). In addition to CXCR5, additional Tfh cell markers have also been reported, such as inducible costimulatory molecule, IL-21, and the transcription factors c-Maf, Batf, and Bcl6 (1,(5)(6)(7)(8)(9)(10)(11)(12). Moreover, the correlation between an increased number of Tfh and autoimmunity has been described in lupus model, suggesting that aberrant Tfh cell expansion can lead to the formation of autoantibodies and a lupus-like phenotype (13,14). For example, mice homologous for the sanroque allele of Roquin, which encodes the RING-type ubiquitin ligase, developed spontaneous autoantibody production and lupus-like autoimmunity, associated with spontaneous development of germinal centers and excessive number of Tfh cells (13,15).
Tfh cell development is regulated by cytokines IL-6 and IL-21 and signaling molecule STAT3, but is independent of Th1, Th2, and Th17 effector cell lineages (6). We, as well as others, recently reported that Bcl6, a transcriptional factor selectively * This work was supported, in whole or in part, by National Institutes of Health Grants R01 AI079087 and PO1 HL44612 (to D. W.), supplement R56 AI071239 (to R. W.), and Grants AI073587 and AR059010 (to S. S. W.). This work was also supported by a Scholar Award from the Leukemia and Lymphoma Society (to D. W.). 1  expressed in Tfh cells, promotes the development of Tfh cells, but inhibits the differentiation of Th1, Th2, and Th17 cells (8 -10). In contrast to Bcl6, the transcription factor Blimp-1 negatively regulates the development of Tfh cells and germinal center B cells (9). Moreover, in contrast to IL-21/STAT3-mediated Bcl6 expression, Blimp-1 induction is mediated by IL-2 through interleukin-2 receptor ␣ (IL-2R␣)-dependent mechanism (16). In the current study, we have analyzed the function of STAT5 and show that it is a negative regulator of Tfh differentiation and functions through up-regulation of Blimp-1.
Retroviral Transduction-Naive CD4 ϩ CD25 Ϫ CD62L hi CD44 lo T cells from OT-II mice were FACS-sorted and activated with Ova peptide and irradiated with wild-type splenic antigen-presenting cells under neutral (anti-IFN␥, anti-IL-4, and anti-TGF␤) conditions in the presence or absence of IL-6. Twentyfour hours after activation, cells were infected by retroviruses expressing constitutively active STAT5 or control empty vector (containing IRES-GFP). Four days after infection, FACS-sorted GFP ϩ cells were restimulated with anti-CD3 for 4 h, and gene expression was determined by real-time RT-PCR. In Fig. 1B, naive CD4 ϩ CD25 Ϫ CD62L hi CD44 lo T cells from Stat5 fl/ϩ and Stat5 fl/Ϫ mice were infected with a GFP-containing bicistronic retrovirus expressing Cre or a vector control virus and activated in the presence of the indicated cytokines. FACS-sorted GFP ϩ cells were restimulated for 4 h with anti-CD3 for real-time RT-PCR analysis. In Fig. 1D, naive CD4 ϩ T cells from Stat5 fl/ϩ and Stat5 fl/Ϫ mice coinfected with two bicistronic retroviruses expressing Cre-GFP or GFP vector and Blimp-1-hCD2 or hCD2 were further activated under neutral conditions (anti-IL-4, anti-IFN␥, and anti-TGF␤) in the presence or absence of IL-6. GFP ϩ hCD2 ϩ cells were sorted and restimulated for 4 h with anti-CD3 for real-time RT-PCR analysis.
Quantitative Real-time RT-PCR-Total RNA was prepared from T cells using TRIzol regent (Invitrogen). cDNA were synthesized using the SuperScript reverse transcriptase and oligo(dT) primers (Invitrogen), and gene expression was examined with a Bio-Rad iCycler optical system using a iQ TM SYBR Green real-time PCR kit (Bio-Rad Laboratories). The data were normalized to ␤-actin reference. The following primer pair for c-Maf was used: forward, GCAGAGACACGTCCTGGAG-TCG, and reverse, CGAGCTTGGCCCTGCAACTAGC. The primers for IL-21, CXCR5, Bcl6, Batf, c-Maf, Blimp-1, and ␤-actin were previously described (8,18).

RESULTS AND DISCUSSION
STAT5 Inhibits Tfh Differentiation-We first examined the role of STAT5 in Tfh generation in vitro. Previously, we have shown that T cells activated in vitro in the presence of IL-6 or IL-21 but without TGF␤, IL-4, and IFN-␥ signaling preferentially acquire Tfh gene expression and function to promote humoral immunity in vivo (6). To determine the role of STAT5 in Tfh generation, naive CD4 ϩ T cells from OT-II mice activated with Ova peptide and irradiated antigen-presenting cells under neutral (anti-TGF␤, anti-IL-4, and anti-IFN-␥) or IL-6 treatment (IL-6, anti-TGF␤, anti-IL-4, and anti-IFN-␥) conditions were infected with a constitutively active form of STAT5 or a vector control retrovirus that contains an IRES-GFP. Four days after infection, we sorted the retrovirus-transduced cells based on GFP expression and analyzed for their gene expression by real-time RT-PCR. Expression of constitutively active STAT5 dramatically decreased the expression of Tfh-specific genes, such as CXCR5, Bcl6, c-Maf, Batf, and IL-21 (Fig. 1A). Interestingly, we found that the expression of transcription factor Blimp-1 (encoded by Prdm1) was strongly enhanced by constitutively active STAT5 under IL-6 treatment condition. This finding correlates with the previously published observation that IL-2R␣ hi cells exhibit strong Blimp-1 expression, which represses Bcl6 (16). Thus, constitutively active STAT5 inhibits Tfh differentiation that is sufficient to suppress most of the Tfh-specific genes through induction of Blimp-1.

STAT5 Deficiency Enhances
Tfh Development-Our data indicated a potential role of STAT5 in regulation of Tfh development. We further examined the role of STAT5 in Tfh development by using Stat5-deficient CD4 ϩ T cells. C57BL/6 Stat5deficient mice with deletion of the entire Stat5A/5B locus (Stat5 Ϫ/Ϫ ) and mice with the entire Stat5A/5B locus gene flanked with loxP sites (Stat5 fl/fl ) were previously described (17). The Stat5 fl/Ϫ mice were generated by breeding Stat5 ϩ/Ϫ and Stat5 fl/ϩ mice. We used a GFP-containing bicistronic retrovirus expressing Cre to delete STAT5 in CD4 ϩ T cells derived from Stat5 fl/Ϫ mice in vitro. Naive CD4 ϩ T cells from Stat5 fl/ϩ and Stat5 fl/Ϫ mice were differentiated in vitro with plate-bound anti-CD3 and anti-CD28, infected on day 2, and subsequently further differentiated under IL-6 treatment, Th17 (IL-6 and TGF␤), and regulatory T cell (TGF␤) conditions. Four days after infection, GFP-positive cells were analyzed for their gene expression by real-time RT-PCR. Deletion of STAT5 under IL-6 treatment condition significantly increased the expression of most Tfh-specific genes (CXCR5, Bcl6, c-Maf, and Batf) except IL-21, which were strongly inhibited by exogenous TGF␤ (Fig. 1B). In addition, in the absence of STAT5, we detected enhanced mRNA expression of Batf, but not CXCR5 and Bcl6, under Th17 condition, when we administrated TGF␤ together with IL-6 ( Fig. 1B). Interestingly, deletion of Stat5 in IL-6-treated cells dramatically decreased the mRNA level of Bcl6 repressor, Blimp-1 (Fig. 1B). These results suggest that STAT5 controls Tfh development through Blimp-1.
Because IL-2 is important for Blimp-1 expression (16) and largely functions through STAT5, we next activated naive CD4 ϩ T cells under neutral or IL-6/IL-21 treatment conditions in the presence or absence of IL-2-neutralizing antibodies (Fig.  1C). Activation of cells under IL-6/IL-21 treatment condition significantly increased the mRNA expression of Tfh-specific genes and up-regulated CXCR5 and Bcl6 protein expression (Fig. 1C), which were further enhanced by blocking of IL-2. In addition, blockade of IL-2 resulted in suppression of Blimp-1 expression. Thus, our data indicate that IL-2 signaling controls Blimp-1 expression and Tfh differentiation.
To further determine whether STAT5 negatively regulates Tfh differentiation through Blimp-1, we overexpressed Blimp-1 in Stat5-deficient CD4 ϩ T cells (Fig. 1D). Naive CD4 ϩ T cells from Stat5 fl/ϩ and Stat5 fl/Ϫ mice were differentiated in vitro with plate-bound anti-CD3 and anti-CD28, infected on day 2 with two viruses expressing Cre-GFP or GFP vector and Blimp-1-hCD2 or hCD2 vector, and subsequently further differentiated under neutral or IL-6 treatment conditions. Four  A and B, sorted CD4 ϩ T cells from STAT5 fl/ϩ or STAT5 fl/Ϫ Mx1Cre/YFP (CD45.2 ϩ ) mice were adoptively transferred into CD45.1 ϩ congenic mice (n ϭ 3) before the recipient mice were subcutaneously immunized with KLH in complete Freund's adjuvant. A, 7 days after immunization, lymphoid cells from the draining lymph nodes of recipient mice were isolated, and Tfh cells in CD45.2 ϩ YFP ϩ population and germinal center B cells were analyzed. The numbers in the boxes represent the percentages. B, the sera from the recipient mice were subject to a 3-fold serial dilution, and the concentrations of KLH-specific IgM, IgG, IgG1, and IgG2a were analyzed by ELISA and averaged for each group. OD, optical density. C, 7 days later, CD4 ϩ CD45.2 ϩ CD44 hi YFP ϩ cells were sorted, and following anti-CD3 restimulation, mRNA expression of Tfh-specific genes was analyzed by real-time RT-PCR. The data shown were normalized by the expression of a reference gene Actb. The experiments were performed two times with consistent results. Error bars in panels B and C indicate S.D.
days after infection, we sorted GFP ϩ hCD2 ϩ T cells and analyzed for their gene expression by real-time RT-PCR. Under IL-6 treatment condition, Blimp-1 overexpression significantly decreased the expression of Tfh-associated genes such as CXCR5, Bcl6, c-Maf, Batf, and IL-21 in the presence of STAT5 or even in the absence of STAT5 (Fig. 1D). Thus, altogether our in vitro data suggest that STAT5-mediated Blimp-1 expression is sufficient to antagonize Tfh program.
Next, we examined the effect of STAT5 deficiency on Tfh generation and function in vivo. Stat5 fl/ϩ and Stat5 fl/Ϫ Mx1Cre/YFP mice were treated with poly(I:C), which can induce Cre expression, resulting in deletion of a "floxed" transcriptional stop cassette preceding YFP cDNA at the rosa26 locus and subsequent YFP expression to track floxed Stat5 deletion (19). Three weeks after treatment, YFP ϩ CD4 ϩ (CD45.2) cells were FACS-sorted and then adoptively transferred into C57BL/6 (CD45.1 ϩ ) mice (3 ϫ 10 6 cells/mouse) (two groups; three mice per group) followed by immunization with KLH protein emulsified in complete Freund's adjuvant. Seven days following immunization, the expression of CXCR5 and BTLA on CD45.2 ϩ CD4 ϩ YFP ϩ T cells was examined. CXCR5 and B and T lymphocyte attenuator expression was greatly increased on CD45.2 ϩ CD4 ϩ YFP ϩ T cells derived from CD45.1 recipients that received T cells from Stat5 fl/Ϫ relative to Stat5 fl/ϩ Mx1Cre/YFP mice ( Fig.  2A). Interestingly, there were approximately two times more GL7 ϩ Fas ϩ host germinal center B cells in recipients that received T cells from Stat5 fl/Ϫ relative to Stat5 fl/ϩ Mx1Cre/ YFP mice (Fig. 2A). The transferred Stat5-deficient cells also enhanced KLH-specific antibody production (Fig. 2B). In addition, analysis of sorted CD4 ϩ YFP ϩ CD44 hi cells revealed that STAT5 deficiency in T cells resulted in increased expression of Tfh-related genes (CXCR5, IL-21R, Batf, c-Maf, and IL-21) and suppression of Blimp-1, an antagonistic regulator of Tfh development (Fig. 2C). Based on the above data, we conclude that STAT5 expression in T cells is required to control development of Tfh cells and the germinal center reaction.
Fas ϩ GL7 ϩ germinal center B cells was also greatly increased in Stat5 fl/Ϫ relative to Stat5 fl/ϩ CD4Cre/YFP/Ig HEL sHEL mice (Fig. 3B, lower). Importantly, serum levels of HEL-specific IgM were markedly increased in Stat5 fl/Ϫ relative to control CD4Cre/YFP/Ig HEL sHEL mice (Fig. 3C). These data demonstrate that STAT5 deficiency in CD4 ϩ T cells causes an increase of Tfh cells and germinal center B cells in Ig HEL sHEL transgenic mice, resulting in an impairment of B cell tolerance.
Significant advances in the understanding of Tfh cell differentiation and involvement in immune responses have been made over the past few years. However, the signaling events required for commitment of CD4 ϩ Th cells to the Tfh subset are only beginning to be elucidated. IL-21/IL-6, STAT3, and Bcl6 are required for Tfh cell generation, although the precise mechanisms controlling the expression of these major Tfh factors have yet to be resolved. We have analyzed the role of STAT5 in developmental regulation of Tfh cells. We found that STAT5, which is known as a critical regulator of regulatory T cell and Th17 reciprocal development, also played a critical role in Tfh development and in B cell-mediated humoral immune responses. IL-2/STAT5 promotes the expression of Bcl6 repressor Blimp-1 and inhibits the differentiation of Tfh cells. Mice with STAT5 deficiency in CD4 ϩ T cells exhibited an increase of Tfh and germinal center B cells and impairment of B cell tolerance, suggesting that STAT5 signaling controls the Tfh generation and humoral immunity. This finding highlights a negative cross-talk between the IL-2/STAT5/Blimp-1 and the IL-6/IL-21/Bcl6 pathways in Tfh development and may help us to find ways to treat antibody-mediated autoimmune diseases associated with expansion of Tfh cells.