STAT5 is a potent negative regulator of TFH cell differentiation - PubMed (original) (raw)

STAT5 is a potent negative regulator of TFH cell differentiation

Robert J Johnston et al. J Exp Med. 2012.

Abstract

Follicular helper T cells (T(FH) cells) constitute the CD4(+) T cell subset that is specialized to provide help to germinal center (GC) B cells and, consequently, mediate the development of long-lived humoral immunity. T(FH) cell differentiation is driven by the transcription factor Bcl6, and recent studies have identified cytokine and cell-cell signals that drive Bcl6 expression. However, although T(FH) dysregulation is associated with several major autoimmune diseases, the mechanisms underlying the negative regulation of T(FH) cell differentiation are poorly understood. In this study, we show that STAT5 inhibits T(FH) cell differentiation and function. Constitutive STAT5 signaling in activated CD4(+) T cells selectively blocked T(FH) cell differentiation and GCs, and IL-2 signaling was a primary inducer of this pathway. Conversely, STAT5-deficient CD4(+) T cells (mature STAT5(fl/fl) CD4(+) T cells transduced with a Cre-expressing vector) rapidly up-regulated Bcl6 expression and preferentially differentiated into T(FH) cells during T cell priming in vivo. STAT5 signaling failed to inhibit T(FH) cell differentiation in the absence of the transcription factor Blimp-1, a direct repressor of Bcl6 expression and T(FH) cell differentiation. These results demonstrate that IL-2, STAT5, and Blimp-1 collaborate to negatively regulate T(FH) cell differentiation.

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Figures

Figure 1.

Figure 1.

STAT5 signaling selectively inhibits TFH cell differentiation and function. CD45.1+ SMARTA TCR transgenic (SM) CD4+ T cells were transduced with RVs expressing GFP and WT STAT5b (STAT5-WT), GFP and a constitutively active form of STAT5b (STAT5-CA), or only GFP (GFP). (A) Representative histograms of phospho-STAT5 levels in transduced SM cells (GFP+), without stimulation (left) or after stimulation with IL-2 (right). Phospho-STAT5 MFIs are indicated. Data are representative of two independent experiments. (B–F) Transduced SM cells (those expressing GFP) were adoptively transferred into C57BL/6J mice that were subsequently infected with LCMV (see Materials and methods). Splenocytes were analyzed 8 d after infection. Data are a composite of four (B–D) or two (E and F) independent experiments and total n = 11–16/group (B–D) or 4/group (E and F). (B) Quantitation of SM cells as a percentage of all CD4+ T cells. (C) Representative FACS plots gated on SM cells (CD4+ CD45.1+), with TFH cells (SLAMlow CXCR5high) boxed. Quantitation of SM TFH cells as a percentage of all SM cells. GFP versus STAT5-WT: ***, P = 0.0002; GFP versus STAT5-CA: ***, P < 0.0001; STAT5-WT versus STAT5-CA: ***, P < 0.0001. (D) Representative FACS plots gated on B cells (B220+), with GC B cells (Fas+ GL7+) circled. Quantitation of GC B cells as a percentage of all B cells. Uninfected C57BL/6J mice (naive) are also shown. ***, P < 0.0001. (E) Representative histograms of FoxP3 expression in SM cells and in total CD4+ T cells from an uninfected C57BL/6J mouse (naive). Quantitation of FoxP3 MFI, with natural Treg cells (nTreg; CD4+ CD25+ FoxP3+) from a naive C57BL/6J mouse included as a control. (F) Quantitation of the percentage of SM cells that produced IFN-γ after PMA/ionomycin stimulation in vitro. Error bars depict the standard error of the mean.

Figure 2.

Figure 2.

Lack of STAT5 signaling enhances TFH cell differentiation. STAT5fl/fl SM cells were transduced with an RV expressing Cre recombinase (Cre) or were not transduced but treated similarly (control). (A) Representative histograms of phospho-STAT5 levels in SM cells, with and without IL-2 stimulation. The percentage of cells that was phospho-STAT5+ is indicated. (B–J) Cre+ and control SM cells were adoptively transferred into C57BL/6J mice that were subsequently infected with LCMV. Splenocytes were analyzed 8 (B–F) or 4 (G–J) d after infection. Data are a composite of two independent experiments, and n = 8/group. (B) Quantitation of SM cells as a percentage of all CD4+ T cells. (C) Representative histograms gated on SM cells or on CD4+ T cells from an uninfected C57BL/6J mouse (naive). (D) Representative FACS plots gated on SM cells, with TFH cells (SLAMlow CXCR5high) boxed. Quantitation of SM TFH cells as a percentage of total SM cells. ***, P < 0.0001. (E) Representative FACS plots gated on SM cells, with GC TFH cells (CXCR5high GL7high) boxed. Quantitation of SM GC TFH cells as a percentage of total SM cells. **, P = 0.01. (F) IL-21 production by SM cells after PMA/ionomycin stimulation in vitro. Quantitation of IL-21+ SM cells as a percentage of total SM cells. (G) Quantitation of SM cells as a percentage of total CD4+ T cells. (H) Representative histograms of Bcl6 expression, gated on SM cells or on CD4+ T cells from an uninfected C57BL/6J mouse (naive). Quantitation of SM cell Bcl6 MFI. **, P = 0.0012. (I) Representative FACS plots gated on SM cells, with TFH (Bcl6high CXCR5high) boxed. Quantitation of SM TFH cells as a percentage of all SM cells. ***, P < 0.0001. (J) Representative FACS plots gated on SM cells, with non-TFH cells (CD25high CXCR5low) boxed. Quantitation of non-TFH SM cells as a percentage of total SM cells. ***, P < 0.0001. Error bars depict the standard error of the mean.

Figure 3.

Figure 3.

IL-2 signaling inhibits TFH cell differentiation during T cell priming. (A–D) WT and CD25+/− SM CD4+ T cells were adoptively transferred into C57BL/6J mice that were infected with LCMV 1 d later. Splenocytes were analyzed 2 (A–C) or 3 (D) d after infection. Data are representative of two independent experiments, and n = 5/group. (A) Quantitation of SM cells as a percentage of total CD4+ T cells. (B) Quantitation of SM cell CD69 and CD25 MFI. ***, P < 0.0001. (C) Representative FACS plots gated on SM cells, with TFH-committed cells (CD25low Bcl6high) boxed. Quantitation of TFH-committed SM cells as a percentage of total SM cells. ***, P < 0.0001. (D) Representative FACS plots gated on SM cells, with TFH-committed cells (Bcl6high CXCR5high) boxed. Quantitation of TFH-committed SM cells as a percentage of total SM cells. **, P = 0.01. (E and F) WT SM CD4+ T cells were adoptively transferred into C57BL/6J mice. Host mice were treated with anti–IL-2 neutralizing antibodies or isotype-matched control antibodies and infected with LCMV 1 d later. Splenocytes were analyzed 2 d after infection. Data are representative of two independent experiments, and n = 5/group. (E) Quantitation of SM cells as a percentage of total CD4+ T cells. (F) Representative FACS plots gated on SM cells, with TFH-committed cells (CD25low Bcl6high) boxed. Quantitation of TFH-committed SM cells as a percentage of total SM cells. ***, P = 0.001. Error bars depict the standard error of the mean.

Figure 4.

Figure 4.

STAT5 inhibition of TFH cell differentiation is mediated by Blimp-1. Prdm1fl/fl SM cells were transduced with STAT5-CA RV and/or with a variant of Cre RV expressing the fluorescent protein mAmetrine or were not transduced but treated similarly (control). Data are a composite of three (A–C) or two (D) independent experiments, and n = 11–12/group (A–C) or 8/group (D). (A) Representative FACS plot gated on viable (7AADlow) cells, with untransduced, singly transduced, and cotransduced cells boxed. (B–D) Transduced or control SM cells were adoptively transferred into C57BL/6J mice that were infected with LCMV 3–5 d later. Splenocytes were analyzed 8 d after infection. (B) Quantitation of SM cells as a percentage of total CD4+ T cells. (C) Representative FACS plots gated on SM cells, with TFH cells (SLAMlow CXCR5high) boxed. Quantitation of SM TFH cells as a percentage of total SM cells. Data have been normalized so that the mean control SM TFH percentage for each experiment is 100%. ***, P < 0.0001. (D) Representative FACS plots gated on SM cells, with GC TFH cells (GL7high CXCR5high) boxed. Quantitation of SM GC TFH cells as a percentage of total SM cells. Data have been normalized so that the mean control SM GC TFH percentage for each experiment is 100%. *, P = 0.0330; ***, P < 0.0001. Error bars depict the standard error of the mean.

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