Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells

Abstract

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Figure 1

Homeostatic expansion of T_reg cells is driven by increased production of IL-2. (a) Percentages of DTR⁺ T_reg cells Thy1.1⁺ T_reg cells and total T_reg cells in Foxp3^Thy1.1/DTR females depleted of Foxp3^DTR+ T_reg cells on day 0. Blood leukocytes were assessed on indicated days (n=4,3,7,3,4,4,4,4 mice). (b,c) Proliferation rate (b; percentage Ki67⁺) and apoptosis rate (c; percentage activated caspase 3⁺) of Thy1.1⁺ T_reg cells, after DT treatment of Foxp3^Thy1.1/DTR females on day 0. (d) Percentages of Thy1.1⁺ T_reg cells in peripheral blood after DT treatment of saline-treated (n=3,3,3 mice/group) or CTLA4-Ig–treated Foxp3^Thy1.1/DTR female mice (n=3,3,3 mice/group). (e) Plasma IL-2 (left axis, n=27,3,3,12,14 mice/group) and surface CD25 expression on Thy1.1⁺ T_reg cells (right axis, n=4,3,7,3,4,4 mice/group) from days 0 to 15 for female Foxp3^Thy1.1/DTR mice depleted of Foxp3^DTR+ T_reg cells (f) Proportion of Thy1.1⁺ (reporter for IL-2) cells within the CD4⁺Foxp3⁻ population after DT injection of Foxp3^+/DTR.IL2-Thy1.1 mice. (g) Foxp3^Thy1.1/DTR mice were depleted of DTR⁺ T_reg cells and injected daily with an IL-2 blocking antibody (a-IL2) or an immunoglobulin isotype-matched control antibody. Splenocytes were analyzed on day 6 for the percentages of DTR⁺ and Thy1.1⁺ T_reg cells, with or without T_reg cell depletion (DT treatment) and with or without anti–IL-2 treatment (n=4,4,7,7 mice/group). (h,i) Proliferation rate (h; percentage Ki67⁺) and apoptosis rate (i; activated caspase-3⁺) of Thy1.1⁺ regulatory T cells, with or without DT treatment and with or without anti-IL-2 treatment (n=4). (a–i) Mean ± s.d., *P < 0.05, t-test. Data are representative of 3(a–f) and 2 (g–i) independent experiments.

Figure 2

The intrinsic apoptosis pathway is required to restrain T_reg cell numbers to homeostatic levels. (a) Representative flow profiles (TCRβ versus Foxp3 gated on CD4⁺ cells, Ki67 histograms gated on Foxp3⁺ CD4⁺ cells) for wild-type, Foxp3^CreBak^−/−Bax^fl/fl mice and control littermates at 6–8 weeks of age. Numbers in plots indicate (top) the percentage of Foxp3⁺TCRβ⁺ regulatory T cells and (bottom) the fraction of proliferating Foxp3⁺CD4⁺ cells (Ki67⁺). (b) Average percentages and absolute numbers (mean ± s.d.) of splenic Foxp3⁺ T_reg cells in wt, Foxp3^CreBak^−/−Bax^fl/fl mice and control littermates at 6–8 weeks of age (n=3,3,5,3 mice/group). (a–b) Data from one experiment representative of three are shown. Mean ± s.d., *P < 0.05, t-test.

Figure 3

Regulatory T cell survival is independent of Bcl-2 and Bcl-x_L. (a) C57BL/6.Ly5.1 (Ly5.1) chimeras reconstituted with a 50:50 mixture of hematopoietic precursors from wild-type Ly5.1 and either Bcl2^+/+ or Bcl2^−/− mice, analyzed 8–12 weeks later. Gates show the percentage of Ly5.2⁺CD4⁺Foxp3⁺ cells recovered from the thymus or spleen. (b) Average percentages (mean ± s.d.) of Ly5.2⁺CD4⁺Foxp3⁺ cells recovered from the thymus and spleen of the same mixed hematopoietic chimeras reconstituted with precursors from either wild-type (Bcl2^+/+) or Bcl2^−/− mice (n= 4,6 mice/group). (a,b) Data from one experiment representative of three are shown. (c) Representative flow profiles of CD4 versus Foxp3 gated on CD4⁺ cells from Foxp3^CreBcl2l1^wt/wt and Foxp3^CreBcl2l1^fl/fl mice. Gates show the percentage of CD4⁺Foxp3⁺ cells recovered from the thymus or spleen. (d) Average percentages (mean ± s.d.) of CD4⁺Foxp3⁺ T_reg cells in the thymus and spleen of Foxp3^CreBcl2l1^wt/wt and Foxp3^CreBcl2l1^fl/fl siblings at 6–8 weeks of age (n = 6,9 respectively). (c,d) Data pooled from three independent experiments are shown.

Figure 4

Spontaneous fatal immunopathology after T_reg cell–specific deletion of Mcl-1. (a) HuCD4 reporter for Mcl-1 expression was measured in lymphocyte subsets in Cd127^CreMcl1^wt/fl-huCD4 female mice, 6–8 weeks of age. Average huCD4 reporter MFI in CD4⁻CD8⁻ (DN), CD4⁺CD8⁺ (DP) and single positive (SP) thymocytes, the latter subdivided into CD4⁻CD8⁺ SP, conventional CD4⁺CD8⁻ SP and CD4⁺CD8⁻ Foxp3⁺ SP (n = 3 mice/group). (b) Average huCD4 reporter MFI in splenic CD19⁺ B cells, naïve conventional CD4⁺ (CD4⁺ nTc), activated conventional CD4⁺ (CD4⁺ actTc), Foxp3⁺ T_reg cells, naive conventional CD8⁺ (CD8⁺ nTc) and activated conventional CD8⁺ T cells (CD8⁺ actTc) (n = 3 mice/group). (c) Representative histogram of huCD4 reporter MFI in naive conventional CD4⁺ (CD4⁺ nTc), Foxp3⁺ T_reg cells and naive conventional CD8⁺ (CD8⁺ nTc), with control huCD4 staining in wild-type Foxp3⁺ T_reg cells. (a–c) Data from one experiment representative of three. (d) Weights of male Foxp3^CreMcl1^wt/wt, Foxp3^CreMcl1^wt/fl and Foxp3^CreMcl1^fl/fl littermates at 6–8 weeks of age (n=11,20,16 mice/group). (e) Survival curve for male Foxp3^CreMcl1^wt/wt, Foxp3^CreMcl1^wt/fl and Foxp3^CreMcl1^fl/fl littermates (n=16,18,18 mice/group)). (f) Plasma IgE levels in male Foxp3^CreMcl1^wt/wt and Foxp3^CreMcl1^fl/fl littermates at 4–8 weeks of age (n=6,12 mice/group). (g,h) Average disease score (g) and representative histology (h; scale bar, 200 μm) of the lungs and small intestine of male Foxp3^CreMcl1^wt/wt and Foxp3^CreMcl1^fl/fl littermates at 4–8 weeks of age (n=11, 9 mice/group). (i) Average percentage of CD44⁺CD62L^low activated cells within CD4⁺ and CD8⁺ splenic T cells in Foxp3^CreMcl1^wt/wt, Foxp3^CreMcl1^wt/fl and Foxp3^CreMcl1^fl/fl littermates at 6–8 weeks of age (n=12,9,11,10,4,d8 mice/group). (d–i) Data pooled from 3 experiments. Mean ± s.d., * P < 0.05, t-test.

Figure 5

Mcl-1 is required for T_reg cell survival. (a) Average percentages of Foxp3⁺ T_reg cells within splenic CD4⁺ T cells in male Foxp3^CreMcl1^wt/wt, Foxp3^CreMcl1^wt/fl and Foxp3^CreMcl1^fl/fl littermates at 6–8 weeks of age (n=14,12,11 mice/group). (b) Average percentages of Ki67⁺ cells within splenic Foxp3⁺ T_reg cells in Foxp3^CreMcl1^wt/wt, Foxp3^CreMcl1^wt/fl and Foxp3^CreMcl1^fl/fl littermates at 6–8 weeks of age (n=14,12,11 mice/group). (a,b) Data pooled from 3 experiments. c) Expression of the huCD4 reporter for excision of Mcl1 in Foxp3⁺ and Foxp3⁻ cells in male Foxp3^CreMcl1^fl/fl mice (n=3,4,3 mice/group). Data from one experiment representative of three. (d) Ly5.1 versus Foxp3 expression on lymph node cells from mixed chimeras with Mcl1^fl/fl or Bcl2l1^fl/fl Ly5.2 donor compartments analyzed 3 d after treatment with tamoxifen or vehicle control. Gates display the fraction of T_reg cells arising from (top) wildtype Ly5.1⁺ and (bottom) Mcl1^fl/fl or Bcl2l1^fl/fl Ly5.1⁻ cells. Plots are representative of 3 experiments, each with n = 3 mice/group. (e) Ratios of Ly5.1⁺ to Ly5.1⁻ CD4⁺Foxp3⁺ lymph node cells in mixed chimeras with the indicated Ly5.2⁺ donor compartment 3 d after treatment with tamoxifen or vehicle (n=3,3,4,6 mice/group). Data from one experiment representative of three is shown. Mean ± s.d., *P < 0.05, t-test.

Figure 6

Regulation of Mcl-1 in T_reg cells by Bim and IL-2. (a) Representative flow cytometric profiles for CD4 vs Foxp3 gated on CD4⁺ cells (gates indicate fraction of T_reg cells within CD4⁺ lymph node cells) and Ki67 histograms gated on Foxp3⁺CD4⁺ cells (gate indicates the fraction of proliferating T_reg cells) for Foxp3^wtBcl2l11^fl/fl and Foxp3^CreBcl2l11^fl/fl littermates at 6–8 weeks of age. (b) Average percentages and absolute number of CD4⁺Foxp3⁺ T_reg cells from pooled lymph nodes of Foxp3^wtBcl2l11^fl/fl and Foxp3^CreBcl2l11^fl/fl littermates (n=8,5 mice/group). Data from one experiment representative of three are shown. (c) Immunoblot analysis of Mcl-1 expression in ex vivo isolated conventional T cells (T_conv) or T_reg cells, or T_reg cells cultured overnight with or without IL-2, in the absence or presence of QVD-OPH (a broad spectrum caspase inhibitor used to prevent apoptosis in the absence of IL-2). Data from one experiment representative of three are shown. (d) Cd127^CreMcl1^wt/fl-huCD4Foxp3^wt/DTR females were depleted of Foxp3^DTR+ T_reg cells on day 0 and huCD4 (Mcl1 reporter) expression was measured in Foxp3⁺ T_reg cells during the expansion phase. MFI was normalized to T_reg cells from Cd127^CreMcl1^wt/fl-huCD4Foxp3^wt mice (n=4 mice/group). Data from one experiment representative of two are shown. (e) Cd127^CreMcl1^wt/fl-huCD4 mice were injected with IL-2-anti-IL-2 antibody complexes or saline and on day 2 were measured for CD4⁺Foxp3⁺ T_reg cell expansion in the peripheral blood and f, expression of huCD4 reporter for Mcl1 expression (n=5,4 mice/group). Mean ± s.d., * P < 0.05, t-test.