GATA-3 controls the maintenance and proliferation of T cells downstream of TCR and cytokine signaling - PubMed (original) (raw)

GATA-3 controls the maintenance and proliferation of T cells downstream of TCR and cytokine signaling

Yunqi Wang et al. Nat Immunol. 2013 Jul.

Abstract

GATA-3 controls T helper type 2 (TH2) differentiation. However, whether GATA-3 regulates the function of mature T cells beyond TH2 determination remains poorly understood. We found that signaling via the T cell antigen receptor (TCR) and cytokine stimulation promoted GATA-3 expression in CD8(+) T cells, which controlled cell proliferation. Although GATA-3-deficient CD8(+) T cells were generated, their peripheral maintenance was impaired, with lower expression of the receptor for interleukin 7 (IL-7R). GATA-3-deficient T cells had defective responses to viral infection and alloantigen. The proto-oncoprotein c-Myc was a critical target of GATA-3 in promoting T cell proliferation. Our study thus demonstrates an essential role for GATA-3 in controlling the maintenance and proliferation of T cells and provides insight into immunoregulation.

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The authors declare no competing financial interests.

Figures

Fig. 1

Fig. 1. TCR and cytokine stimulation promoted GATA-3 expression in CD8 T cells

(a) GATA-3 expression in CD4 T cells (dashed line), CD8 T cells (solid line) and B cells (dotted line) purified from wild-type mice and CD8 T cells purified from Cd4Cre-_Gata3_fl/fl mice (KO, shaded) as assessed by intra-cellular staining and immuno-blotting. Results shown are representative of at least three experiments. (b) GATA-3 expression in naïve CD8 T cells and CD8 T cells stimulated with anti-CD3 for 72 hours as assessed by intracellular staining and immuno-blotting. Representative results of at least three experiments are shown. (c) GATA-3 expression in CD8 T cells stimulated with anti-CD3 for 72 hours, in the absence (Mock) or presence of IL-2 or IL-4 as assessed by intracellular staining and immuno-blotting. Results shown are representative of at least three experiments. (d) Flow cytometry of IL-4 production in CD8 and CD4 T cells isolated from wild-type mice and activated with anti-CD3 and anti-CD28, in the absence (Mock) or presence of IL-2 or IL-4 for 72 hours. Representative results of at least three experiments are shown. (e) Numbers of live CD8 T cells recovered after 72 hours of culture as in d. Means ± SD were shown. (* P<0.05). (f) CFSE dilution in CD8 T cells labeled with CFSE assessed 72 hours after stimulation with anti-CD3, in the absence (Mock) or presence of IL-2 or IL-4. Results shown are representative of at least three experiments.

Fig. 2

Fig. 2. CD8 T cell development in the absence of GATA-3

(a) Flow cytometry of T cell distribution in the thymus, peripheral lymph-nodes (PLN) and spleens of Cd4Cre-_Gata3_fl/fl and Cd4Cre-_Gata3_fl/+ mice (top panels). GATA-3 expression assessed by intracellular staining and qRT-PCR (lower panels). Flow-cytometric results are representative of at least three experiments. qRT-PCR results are means ± SD of triplicates done in one experiment representative of at least three. (b) Flow cytometry of CD44 and CD62L expression in CD8 T cells isolated from PLN and spleen of Cd4Cre-_Gata3_fl/fl and Cd4Cre-_Gata3_fl/+ mice. Representative results of at least three experiments are shown. (c) Flow cytometry of IFN-γ and IL-4 production in CD8 T cells isolated from PLN and spleen of Cd4Cre-_Gata3_fl/fl and Cd4Cre-_Gata3_fl/+ mice. Results are representative of at least three experiments. (d) Flow cytometry of T cell populations in the mixed bone marrow chimera containing wild-type (CD45.1) and Cd4Cre-_Gata3_fl/fl (CD45.2) T cells (top panels). Means ± SD of five mice from one experiment representative of two are shown. GATA-3 expression assessed by intracellular staining (lower panels). (e) Flow cytometry of CD44 and CD62L expression in CD8 T cells from the mixed bone marrow chimera described in d. Results are representative of at least three experiments. (f) Flow cytometry of IFN-γ and IL-4 production in CD8 T cells from the mixed bone marrow chimera described in d. Results are representative of at least three experiments.

Fig. 3

Fig. 3. Defective peripheral maintenance of GATA-3-deficient CD8 T cells

(a) Flow cytometry of CD8 T cell distribution during peripheral maintenance experiments using wild-type (CD45.1) and Cd4Cre-_Gata3_fl/fl (CD45.2) donor cells. Representative results and means ± SD of five mice from one experiment of two are shown (* P<0.05). (b) Flow cytometry of CD8 T cell distribution during peripheral maintenance experiments using wild-type (CD45.1) and ERCre-_Gata3_fl/fl (CD45.2) donor cells. Representative results and means ± SD of five mice from one experiment of two are shown (* P<0.05). GATA-3 expression was also assessed by flow-cytometry one week after tamoxifen (TMA) treatment. (c) Flow cytometry of CD127 expression in CD8 T cells of wild-type (solid line) and Cd4Cre-_Gata3_fl/fl (dashed line) origins in the recipient mice described in a. (d) Flow-cytometry of CD127 expression in CD8 T cells of wild-type (solid line) and ERCre-_Gata3_fl/fl (dashed line) origins in recipient mice described in b. (e) Flow cytometry of CD8 T cell distribution during IL-7 response experiment. Means ± SD of triplicate in one experiment representative of two are shown (* P<0.05). (f) GATA-3 binding to a conversed non-coding sequence in Il7r locus and to the CGRE site within the TH2 locus in CD8 T cells isolated from wild-type and Cd4Cre-_Gata3_fl/fl (KO) mice detected by chromatin-immunoprecipitation (ChIP). Means ± SD of triplicates done in one experiment representative of at least three are shown. (* P<0.05)

Fig. 4

Fig. 4. Activated CD8 T cell function requires GATA-3

(a) Flow cytometry of CD25, CD44 and CD69 expression on wild-type (CD45.1) and Cd4Cre-_Gata3_fl/fl (CD45.2) CD8 T cells that were co-cultured and activated for 24 hours. Results are representative of at least three experiments. (b) The proliferation of wild-type (CD45.1) and Cd4Cre-_Gata3_fl/fl (CD45.2) CD8 T cells that were co-cultured and activated for 72 hours in the absence (Mock) or presence of indicated cytokines as assessed by CFSE dilution and flow cytometry. Results representative of at least three experiments are shown. (c) Flow cytometry of IFN-γ production by CD8 T cells described in b. Means ± SD of three experiments are shown. (d) Flow cytometry of CD25, CD44 and CD69 expression on wild-type (CD45.1) and ERCre-_Gata3_fl/fl (CD45.2) CD8 T cells that were co-cultured and activated for 24 hours in the presence of 4-HT. Results are representative of at least three experiments. (e) The proliferation of wild-type (CD45.1) and ERCre-_Gata3_fl/fl (CD45.2) CD8 T cells that were co-cultured and activated in the absence (Mock) or presence of indicated cytokines for 72 hours while 4-HT was added, as assessed by CFSE dilution and flow-cytometry. Results are representative of at least three experiments. (f) Flow cytometry of IFN-γ production by CD8 T cells described in e. Means ± SD of three experiments are shown.

Fig. 5

Fig. 5. GATA-3 is important for activated CD8 T cell expansion in vivo

(a) Flow cytometry of gp33-tetramer+ wild-type (CD45.1) and Cd4Cre-_Gata3_fl/fl (CD45.2) CD8 T cells in the recipients (CD45.1xCD45.2) after LCMV infection. Representative flow-cytometric plots and combined results from multiple mice from two experiments are shown (** P<0.01). (b) Flow cytometry of IFN-γ production by wild-type (CD45.1) and Cd4Cre-_Gata3_fl/fl (CD45.2) CD8 T cells (described in a) upon gp33-41 peptide re-stimulation. Representative flow-cytometric plots and combined results from multiple mice from two experiments are shown (** P<0.01). (c) Flow cytometry of CD8 T cell distribution in _Rag2_−/−_γc_−/− recipient mice (Balb/c) where GvH response was induced by transferred splenocytes from both wild-type (CD45.1, C57BL/6) and ERCre-_Gata3_fl/fl (CD45.2, C57BL/6) mice. Tamoxifen (TMA) or vehicle (Mock) was injected at the time of cell transfer. Representative flow-cytometric plots and means ± SD of five mice from one experiment representative of two are also shown. (NS, non-significant; * P<0.05). (d) Flow cytometry of CD8 T cell distribution in _Rag2_−/−_γc_−/− recipient mice (Balb/c) where GvH response was induced by transferred splenocytes from both wild-type (CD45.1, C57BL/6) and ERCre-_Gata3_fl/fl (CD45.2, C57BL/6) mice. Tamoxifen (TMA) or vehicle (Mock) was injected two weeks after cell transfer. Representative flow-cytometric plots and means ± SD of five mice from one experiment representative of two are shown (NS, non-significant; * P<0.05). (e) Flow cytometry of Ki67 expression in wild-type and ERCre-_Gata3_fl/fl CD8 T cells during GvH response as described in c. Result is representative of at least three experiments.

Fig. 6

Fig. 6. GATA-3 controls c-Myc expression

(a) Flow cytometry of the cell size of Cd4Cre-_Gata3_fl/fl and Cd4Cre-Gata3fl/+ CD8 T cells that were sorted naïve (CD62LhiCD44lo) and stimulated with anti-CD3 and anti-CD28 in the absence (Mock) or presence of indicated cytokines for 24 hours. Results are representative of at least three experiments. (b) The protein and mRNA expression of c-Myc in wild-type and Cd4Cre-_Gata3_fl/fl (KO) CD8 T cells that were sorted naïve (CD62LhiCD44lo) and stimulated with anti-CD3 and anti-CD28 as assessed by immuno-blotting and qRT-PCR respectively. Immuno-blotting results are representative of at least three experiments. qRT-PCR results are means ± SD of triplicates done in one experiment representative of three (*P<0.05). Immune-blotting for c-Myc in wild-type CD8 T cells transduced either with MSCV-IRES-EGFP (MIG) control virus or with MSCV-Myc-IRES-EGFP (MIG-Myc) viruses. Results are representative of at least two experiments. (c) Comparison of the proliferation of MIG and MIG-Myc -transduced (GFP+) wild-type and ERCre-_Gata3_fl/fl (KO) CD8 T cells that were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 and 4-HT for 4 days, as assessed by Cell Trace Violet dye dilution with flow cytometry. Representative results of at least three experiments are shown. (d) GATA-3 binding to conversed non-coding sequences in Myc locus and the CGRE site within the Th2 locus in CD8 T cells isolated from wild-type and Cd4Cre-_Gata3_fl/fl (KO) mice detected by ChIP. Means ± SD of triplicates done in one experiment representative of at least three are shown. (* P<0.05)

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