STAT5 protein negatively regulates T follicular helper (Tfh) cell generation and function

Abstract

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.

FIGURE 1.

Stat5 negatively regulates Tfh-specific gene expression. A, naive OT-II T cells were activated under neutral conditions (in the presence of antibodies against IL-4, IFNγ, and TGFβ) in the presence or absence of IL-6 and infected with a GFP-containing bicistronic retrovirus expressing constitutively active STAT5 (STAT5) or a vector control virus (C). GFP⁺ cells were sorted and restimulated for 4 h with anti-CD3 for real-time RT-PCR analysis. The data shown were normalized by the expression of a reference gene Actb. B, naive CD4⁺ T cells from Stat5^fl/+ and Stat5^fl/− mice were infected with a GFP-containing bicistronic retrovirus expressing Cre or a vector control virus and activated in the presence or absence (none) of the indicated cytokines. The data shown were normalized by the expression of a reference gene Actb. C, naive OT-II cells were activated under neutral conditions in the presence or absence (none) of IL-6/IL-21 and neutralizing antibodies to IL-2 (αIL-2) for 4 days. mRNA expression of Tfh-related genes was analyzed by real-time RT-PCR analysis (upper). The data shown were normalized by the expression of a reference gene Actb. CXCR5- and Bcl6-expressing cells were also assessed by FACS staining (lower). The numbers in the dot plot quadrants represent the percentages. D, naive CD4⁺ T cells from Stat5^fl/+ and Stat5^fl/− mice were activated with plate-bound anti-CD3 and anti-CD28, coinfected with two bicistronic retroviruses expressing Cre-GFP or GFP vector and Blimp-hCD2 or hCD2, and further activated under neutral conditions (anti-IL-4, anti-IFNγ, and anti-TGFβ) in the presence or absence (none) of IL-6. GFP⁺hCD2⁺ cells were sorted and restimulated for 4 h with anti-CD3 for real-time RT-PCR analysis. 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 A–D indicate S.D.

FIGURE 2.

Stat5 negatively regulates development of Tfh cells. A and B, sorted CD4⁺ T cells from STAT5^fl/+ or STAT5^fl/− Mx1Cre/YFP (CD45.2⁺) mice were adoptively transferred into CD45.1⁺ congenic mice (n = 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⁺YFP⁺ 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⁺CD45.2⁺CD44^hiYFP⁺ 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.

FIGURE 3.

Increase of Tfh and germinal center B cells and impairment of B cell tolerance in mice lacking STAT5 specifically in T cells. A, increase of Tfh and germinal center B cells in Stat5^fl/− CD4Cre/YFP mice. Splenocytes from 2–3-month-old Stat5^+/+ and Stat5^fl/− CD4Cre/YFP mice were stained with antibodies to CD4 and CXCR5 or B220, Fas, and GL7. Percentages indicate CD4⁺CXCR5⁺ cells in the gated CD4⁺YFP⁺ (upper) or CD4⁺YFP⁻ (middle) cells and Fas⁺GL7⁺ cells in the gated B220⁺ cells (lower). B, increase of Tfh and germinal center B cells in Stat5^fl/− CD4Cre/YFP/Ig^HELsHEL mice. Splenocytes from Stat5^fl/+ and Stat5^fl/− CD4Cre/YFP/Ig^HELsHEL mice were stained with antibodies to CD4 and CXCR5 or B220, Fas and GL7. Percentages indicate CD4⁺CXCR5⁺ cells in the gated CD4⁺YFP⁺ (upper) or CD4⁺YFP⁻ (middle) cells and Fas⁺GL7⁺ cells in the gated B220⁺ cells (lower). C, increased levels of HEL-specific antibodies in Stat5^fl/− CD4Cre/YFP/Ig^HELsHEL mice. Sera from 2–3-month-old control (Stat5^+/+, Stat5^+/−, Stat5^fl/+) and Stat5^fl/− CD4Cre/YFP/Ig^HELsHEL mice were obtained. HEL-specific antibody levels in the serum of each individual mouse were determined by ELISA. Data shown were obtained from five mice of each genotype. Error bars indicate S.D.