TGF-beta1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells

Abstract

Transforming growth factor (TGF)-beta1 is a major pluripotential cytokine with a pronounced immunosuppressive effect and its deficiency results in lethal autoimmunity in mice. However, mechanisms of its immunosuppressive action are not completely understood. Here, we report that TGF-beta1 supports the maintenance of Foxp3 expression, regulatory function, and homeostasis in peripheral CD4(+)CD25(+) regulatory T (T reg) cells, but is not required for their thymic development. We found that in 8-10-d-old TGF-beta1-deficient mice, peripheral, but not thymic, T reg cells are significantly reduced in numbers. Moreover, our experiments suggest that a defect in TGF-beta-mediated signaling in T reg cells is associated with a decrease in Foxp3 expression and suppressor activity. Thus, our results establish an essential link between TGF-beta1 signaling in peripheral T reg cells and T reg cell maintenance in vivo.

Figure 1.

Decrease in peripheral CD4⁺CD25⁺ T cells in Tgf-β1 ^−/− mice. Thymocytes (a) and splenocytes (b and c) from 8–10-d-old Tgf-β1 ^{− / −} mice or littermate controls were counted and stained for CD4 and CD8 and analyzed by flow cytometry. Peripheral CD4 T cells from Tgf-β1 ^−/− mice (gray line) or littermate controls (black line) were stained for CD44 and CD62L. Thymocytes and splenocytes from 8–10-d-old Tgf-β1 ^{− / −} mice or littermate controls were stained for CD4 and CD25 and analyzed by flow cytometry (d). Proportion of CD4⁺CD25⁺ cells among CD4 T cells (e, n = 20).

Figure 2.

Decrease in Foxp3 level in Tgf-β1 ^−/− CD4⁺CD25⁺ T cells. Thymocytes and splenocytes from 8–10-d-old Tgf-β1 ^{− / −} mice or littermate controls were stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry. Foxp3 staining in CD4⁺CD25⁻ T cells (black line) and in CD4⁺CD25⁺ (gray line) are shown. Isotype control staining is shown (dashed line). These results are representative of three different experiments.

Figure 3.

TGF-β1 is required to maintain Foxp3 in CD4⁺CD25⁺ T cells. T reg cells purified from spleen of 8–10-d-old Tgf-β1 ^{− / −} mice or littermate controls were transferred into TCRβ/δ-deficient mice injected every other day with anti–TGF-β1 antibody or isotype control IgG. 4 d after transfer, cells from spleen and lymph nodes were stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry (a). Results are representative of three different experiments. Serum TGF-β1 in treated animals was measured by ELISA (b). Western blot analysis of Smad2 phosphorylation and Foxp3 expression in purified wild-type T reg cells either freshly isolated or cultured for 24 h in the presence or absence of 100 pg/ml TGF-β1 (c).

Figure 4.

TGF-β signaling is required to maintain regulatory function T reg cells. (a) Analysis of suppressor activity of T reg cells isolated from Tgf-β1 ^{− / −} (gray symbols) or WT littermates (Lit, black symbols) in cocultures with freshly isolated B6 CD4⁺CD25⁻ responder T cells in the presence of either Tgf-β1 ^{− / −} or littermate control APCs and Con A. Proliferation of CD4⁺CD25⁻ T cells was determined after 72 h of culture by [³H]thymidine incorporation. Proliferation of CD4⁺CD25⁺ from either control or Tgf-β1 ^{− / −} mice in response to Con A was ∼300–500 cpm. The results are shown as mean cpm of [³H]thymidine incorporation in triplicate cultures ± SD. (b) T reg cells transduced with DN–TGF-βRII–MigR2 (MigR2 DN) or empty vector control (MigR2 Ev) were purified and cocultured with freshly isolated B6 CD4⁺CD25⁻ responder T cells in the presence of irradiated T cell–depleted splenic APCs and Con A. Proliferation was measured as described before. Results are representative of two or three different experiments.