The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation

Abstract

Several subsets of Foxp3(+) regulatory T cells (T(reg) cells) work in concert to maintain immune homeostasis. However, the molecular bases underlying the phenotypic and functional diversity of T(reg) cells remain obscure. We show that in response to interferon-gamma, Foxp3(+) T(reg) cells upregulated the T helper type 1 (T(H)1)-specifying transcription factor T-bet. T-bet promoted expression of the chemokine receptor CXCR3 on T(reg) cells, and T-bet(+) T(reg) cells accumulated at sites of T(H)1 cell-mediated inflammation. Furthermore, T-bet expression was required for the homeostasis and function of T(reg) cells during type 1 inflammation. Thus, in a subset of CD4(+) T cells, the activities of the transcription factors Foxp3 and T-bet are overlaid, which results in T(reg) cells with unique homeostatic and migratory properties optimized for the suppression of T(H)1 responses in vivo.

Figure 1. CXCR3 expression on T_reg cells is T-bet-dependent

(a,b) Representative flow cytometric analysis of CXCR3 and Foxp3 expression by splenocytes isolated from wild-type (WT) mice, (a) or age-matched WT and Tbx21^–/- mice (b). Plots are gated on CD4⁺ splenocytes. rIgG2a, isotype control. Numbers display the frequency of cells expressing the indicated markers. Data are representative of greater than six mice analyzed in this fashion. (c) Migration of CD4⁺Foxp3⁺ splenocytes isolated from the indicated mice in response to media alone, 100nM CXCL10 or 100nM CCL21 in a transwell chemotaxis assay. Data are mean and s.d. of triplicate measurements. (d) CD45.2 and CXCR3 expression on CD4⁺Foxp3⁺ cells recovered from the indicated tissues of recipients of a mixture of CD45.1⁺ WT and CD45.2⁺ Tbx21^–/– BM. Numbers depict the percent of cells positive for the indicated markers. Data are representative of three independent experiments with four mice analyzed per experiment. (e) T-bet expression (open histograms) in CD4⁺Foxp3⁺CXCR3⁺ or CD4⁺Foxp3⁺CXCR3^- splenocytes. Histograms (right) correspond to indicated gates (left). Data are representative of greater than ten mice analyzed in this fashion.

Figure 2. T-bet⁺ T_reg cells upregulate T-bet in vivo following anti-CD40 treatment

(a) Analysis of T-bet and Foxp3 expression in splenocytes from mice treated with anti-CD40 (top) or rat IgG (bottom). Plots are gated on CD4⁺ cells. Numbers in plots represent the percentage of cells positive for the indicated markers. Data are representative of three independent experiments. (b) (Top) Splenocytes from TCRβδ-KO recipients of the indicated cells and subjected to the indicated treatment were analyzed by flow cytometry. Dot plots are gated on lymphocytes. Histograms display CXCR3 and T-bet expression in CD4⁺Foxp3⁺ cells isolated from anti-CD40- (open histograms) or rat IgG- (shaded histograms) treated mice as indicated. Numbers in dot plots indicate the percentage of Foxp3⁺CD4⁺ cells of total lymphocytes. Data are representative of two independent experiments with three mice per group.

Figure 3. IFN-γR and STAT1 are promote expression of T-bet and CXCR3 by T_reg cells

(a) CXCR3 expression on splenocytes from WT, Ifngr1^–/–, or Stat1^–/– mice as indicated (n≥ three per genotype). Histograms are gated on CD4⁺Foxp3⁺ cells. Numbers indicate the percent of CXCR3⁺ cells among total CD4⁺Foxp3⁺. (b) Graph depicts the ratio of WT:Ifngr1^–/–-derived lymphocytes among CXCR3⁺CD4⁺Foxp3⁺, CXCR3^-CD4⁺Foxp3⁺ or CD4^-B220⁺ peripheral blood lymphocytes of 11 mixed BM chimeras. Each point represents an individual mouse. Statistical significance was determined using a two-way repeated measures ANOVA. A Bonferroni post-test was used to obtain the P-value for the indicated pairwise comparison. (c) CXCR3 and T-bet expression on splenocytes isolated from a WT:Ifngr1^–/– BM chimera. Plots are gated on CD4⁺Foxp3⁺ cells from WT-derived (CD45.1⁺) or Ifngr1^–/– -derived (CD45.2⁺) BM as indicated. Numbers in dot plots indicate the percent of cells positive for CXCR3. Data are representative of four mice analyzed in this fashion.

Figure 4. Functional characterization of T-bet⁺ Foxp3⁺ T_reg cells

(a) IFN-γ and Foxp3 expression by T-bet⁺CD4⁺ (top right) and T-bet^-CD4⁺ (bottom right) lymphocytes isolated from WT mice following stimulation with PMA and ionomycin. Left histogram is gated on total CD4⁺ cells, and indicates gates used to define the populations depicted in the dot plots. Numbers in plot indicate the percent of cells positive for the indicated markers. Data are representative of greater than five mice analyzed in this fashion. (b) Proliferation of CFSE-labeled CD4⁺CD25^- T cells incubated with irradiated splenocytes, anti-CD3 and anti-CD28, with or without varying concentrations of CXCR3⁺ T_reg cells. Numbers above histograms indicate T_reg:T_eff cell ratio in each culture. No stimulation, control without irradiated splenocytes and stimulatory antibodies. Numbers indicate the percent of T_eff cells that are CFSE^-. (c) Expression of the indicated markers on gated CD4⁺CXCR3⁺ (top) or CD4⁺CXCR3^- (bottom) splenocytes from WT mice. Numbers represent the frequency of cells positive for each marker as a fraction of total CD4⁺Foxp3⁺ cells. Data are representative of three mice analyzed in this fashion.

Figure 5. Decreased proliferation of T-bet-deficient T_reg cells following anti-CD40 treatment

(a) Experimental design showing cell transfer and treatment schedule. Briefly, a mixture of CD45.1⁺ WT and CD45.2⁺ Tbx21^–/– T_reg cells were injected into TCRβδ-KO mice, followed by treatment with the indicated antibodies. BrdU was added to the drinking water when indicated. (b) CD45.1 expression on splenocytes of recipient mice was analyzed by flow cytometry. Histograms are gated on Foxp3⁺CD4⁺TCRβ⁺B220^- cells, and numbers indicate the percent of cells positive and negative for CD45.1. Graphs show absolute numbers of WT- and Tbx21^–/–-derived T_reg cells recovered from the spleens of recipient mice. Each point represents an individual treated mouse. (c) BrdU incorporation by splenocytes of recipient mice was analyzed by flow cytometry. Dot plots are gated on Foxp3⁺CD4⁺TCRβ⁺B220^- splenocytes. Numbers in plots indicate percentage of BrdU⁺ cells among total WT- (CD45.1⁺) or Tbx21^–/–-derived (CD45.1^-) T_reg cells. Graphs depict the frequency of BrdU⁺ cells among WT and Tbx21^–/–-derived T_reg cell populations. For b and c, statistical significance was determined using a two-way repeated measures ANOVA. Bonferroni post-tests were used to obtain the P-values for the indicated pairwise comparisons. Data are representative of three independent experiments with three or greater mice per group

Figure 6. Impaired homeostasis of T-bet-deficient T_reg cells during persistent Mtb infection

(a) T-bet and Foxp3 expression on cells isolated from the indicated tissues of an Mtb-infected mouse (left) or an uninfected age-matched control (right). Plots are gated on CD4⁺ T cells. Numbers display the percent of cells in each of the indicated quadrants. Data are representative of five independent experiments. (b) (Left) Recipients of a mixture of WT (CD45.1⁺) and Tbx21^–/– (CD45.2⁺) BM were infected with Mtb for 105 days. Cells isolated from the indicated organs were analyzed by flow cytometry. Contour plots depict gating strategy, with numbers indicating the percent of cells positive for the indicated markers. Graphs depict the ratio of WT and Tbx21^–/–-derived cells among gated CD4^-CD8^- DN, CD4⁺Foxp3⁺ T_reg and CD4⁺Foxp3^-CD44^hi T_eff populations. Each point represents a value from an individual infected BM chimera. Data are representative of two independent experiments (n=3 per experiment).

Figure 7. T-bet expression in T_reg cells is critical for control of T_H1-mediated inflammatory responses

(a) CD44 and T-bet expression (top) or IFN-γ production (bottom) by splenocytes isolated from age-matched WT or scurfy (sf) mice, as measured by flow cytometry. Plots are gated on CD4⁺Foxp3^- cells. Numbers in plots indicate the percent of T-bet⁺ (top) or IFN-γ⁺ (bottom) cells among total CD44^hi CD4⁺Foxp3^-cells. Data are representative of three independent experiments. (b) T-bet and Foxp3 expression by splenocytes isolated from age-matched WT mice or sf mice given WT T_reg cells. Plots are gated on CD4⁺CD8^- lymphocytes. Numbers in plots indicate the percentage of CD4⁺Foxp3⁺ cells expressing T-bet. Data are representative of six independent experiments. (c) Cells isolated from the spleen and peripheral lymph nodes (pLN) of sf neonate recipients of WT or Tbx21^-/- T_reg cells were analyzed by flow cytometry. Numbers in plots indicate fraction of Foxp3⁺ T cells among total CD4⁺ reg splenocytes. (d, e) Flow cytometric and quantitative analysis of splenocytes isolated from sf mice given WT or Tbx21^–/– T_reg cells, or from age-matched WT mice. Splenocytes in e were stimulated with PMA and ionomycin prior to analysis. Plots are gated on CD4⁺Foxp3^- cells. Numbers in plots display percentage of T-bet⁺ (d) and IFN-γ⁺ (e) cells among total CD4⁺Foxp3^-CD44^hi cells. (f) Photograph of representative spleen and peripheral LNs isolated from 6 week old sf mice given WT (left) or Tbx21^–/– (right) T_reg cells as neonates. Representative of six independent experiments. For c, d and e, each point represents an individual mouse, and significance was measured using two-tailed, unpaired student’s t tests.