The receptor S1P1 overrides regulatory T cell-mediated immune suppression through Akt-mTOR

Abstract

Regulatory T cells (T(reg) cells) are critically involved in maintaining immunological tolerance, but this potent suppression must be 'quenched' to allow the generation of adaptive immune responses. Here we report that sphingosine 1-phosphate (S1P) receptor type 1 (S1P1) delivers an intrinsic negative signal to restrain the thymic generation, peripheral maintenance and suppressive activity of T(reg) cells. Combining loss- and gain-of-function genetic approaches, we found that S1P1 blocked the differentiation of thymic T(reg) precursors and function of mature T(reg) cells and affected T(reg) cell-mediated immune tolerance. S1P1 induced selective activation of the Akt-mTOR kinase pathway to impede the development and function of T(reg) cells. Dynamic regulation of S1P1 contributed to lymphocyte priming and immune homeostasis. Thus, by antagonizing T(reg) cell-mediated immune suppression, the lipid-activated S1P1-Akt-mTOR pathway orchestrates adaptive immune responses.

Figure 1. S1P₁ negatively regulates thymic Foxp3⁺ T_reg population

(a,b) Flow cytometry of total and gated CD4SP thymocytes isolated from wild-type (WT) control, S1P₁-KO (a) and S1P₁-Tg mice (b). Panels on the right show the proportions and absolute numbers of Foxp3⁺ CD4SP T_reg cells. Data are the mean (+s.d.) of 8-14 mice of each genotype from 7 experiments. (c) Foxp3 expression in bone marrow chimeras following retroviral transduction of S1P₁. Bone marrow stem cells from WT mice were transduced with retrovirus expressing S1P₁ (S1P₁-GFP) or empty vector (GFP), and transferred into sublethally irradiated Rag1^-/- mice. At 6-8 weeks after reconstitution, Foxp3 expression was analyzed in gated CD4SP thymocytes. Data are representative of 2 independent experiments. (d) Expression of Foxp3 in mixed bone marrow chimeras. Bone marrow stem cells from WT (CD45.1⁺) and S1P₁-KO or S1P₁-Tg mice (CD45.2⁺) were mixed at 1:1, and transferred into Rag1^-/- mice to generate mixed bone marrow chimeras. At 6-8 weeks after reconstitution, Foxp3 expression was analyzed in CD4SP thymocytes, and cells from different donors were distinguished by their CD45.1 and CD45.2 expression. Data are representative of 3 independent experiments. *, P < 0.001 (Student’s t-test).

Figure 2. S1P₁ blocks thymic differentiation of T_reg cells

(a) Flow cytometry of total and gated CD4SP thymocytes isolated from WT control, S1P₁-KO and S1P₁-Tg FTOC. Panels on the right show the proportions and absolute numbers of Foxp3⁺ CD4SP T_reg cells with the mean (+s.d.) calculated from ≥8 mice of each genotype. (b) Flow cytometry of gated CD4SP thymocytes from WT control, S1P₁-KO and S1P₁-Tg mice. Panels on the right show the proportions and absolute numbers of the CD4⁺CD25⁺Foxp3⁻ precursor population, with the mean (+s.d.) calculated from ≥8 mice of each genotype. (c) Induction of Foxp3 expression in the CD4⁺CD25⁺Foxp3⁻ population in vitro. CD4⁺CD25⁺Foxp3⁻ cells were purified and stimulated with medium alone, IL-2 or IL-15 for 20 h, and induction of Foxp3 expression was measured by flow cytometry. The lower panel shows an IL-2 dependent dose response curve. Data are representative of 5 independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t-test).

Figure 3. Enhanced peripheral population and suppressive activity of S1P₁-KO T_reg cells

(a) Flow cytometry of gated CD4 T cells from the spleen and peripheral lymph nodes (PLN) of WT and S1P₁-KO mice. The panel on the right shows the proportions of Foxp3⁺ T_reg cells among total CD4⁺ T cell population, with the mean (+s.d.) calculated from 4 mice of each genotype. (b) Flow cytometry analysis of T_reg markers (Foxp3, CD25, GITR and CTLA4) in PLN of WT and S1P₁-KO mice. Data are representative of 2 independent experiments. Similar findings were observed in other peripheral lymphoid organs (not shown). (c) In vitro T-cell suppression assays using Foxp3⁺ CD4SP cells from WT and S1P₁-KO mice. The left panel shows a representative proliferative assay of 4 independent experiments, the middle panel is the percentage of suppression with the mean (±s.d.) calculated from 4 experiments, and the right panel shows a representative of 2 independent experiments measuring IL-2 production. (d) In vitro T-cell suppression assays using S1P₁-deleted peripheral T_reg cells. Foxp3⁺ T_reg cells from the periphery of S1pr1^+/+ and S1pr1^fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP), and sorted GFP⁺ T_reg cells were used in the T-cell suppression assays with different T_conv and T_reg ratios; freshly isolated T_reg cells were used as a comparison. The left panel is a representative of 3 independent experiments, and the right panel shows the percentage of suppression with the mean (+s.d.) calculated from 3 experiments. *, P < 0.01; **, P < 0.001 (Student’s t-test).

Figure 4. Reduced suppressive activity of S1P₁-Tg T_reg cells in vitro and in vivo

(a) In vitro T-cell suppression assays using Foxp3⁺ T_reg cells from WT and S1P₁-Tg mice. The left panel shows a representative proliferative assay of 8 independent experiments, and the right panel is the percentage of suppression with the mean (±s.d.) calculated from 8 experiments. (b) In vitro T-cell suppression assays using Foxp3⁺ T_reg cells transduced with S1P₁-expressing retrovirus. WT T_reg cells were transduced with S1P₁-expressing (S1P₁-GFP) and empty vector (GFP) retroviruses, and sorted GFP⁺ T_reg cells were used in the T-cell suppression assays with different T_conv and T_reg ratios. The left panel shows a representative proliferative assay of 5 independent experiments, and the right panel is the percentage of suppression with the mean (+s.d.) calculated from 5 experiments. (c-e) Failure of S1P₁-Tg T_reg cells to control colitis in vivo. T_conv cells were transferred alone or in combination with WT or S1P₁-Tg T_reg cells into Rag1^-/- mice. (c) Changes in body weight after transfer. (d) Histology scores of experimental mice. (e) Representative colon histology. Data are the mean (+s.d.) of 5 mice of each genotype and are representative of 2 independent experiments. *, P < 0.001 (Student’s t-test).

Figure 5. S1P₁-Tg mice show disrupted immune homeostasis and develop age-related autoimmunity due to defects in the T_reg compartment

(a-e) Analysis of WT and S1P₁-Tg mice. (a) Flow cytometry of T cell activation markers from peripheral lymphoid organs of aged mice (10 months). MLN, mesenteric lymph nodes. Data are representative of 6 independent experiments. (b) Proliferative response to TCR stimulation of T_conv cells from WT and S1P₁-Tg mice (2 months). Data are representative of 6 independent experiments. (c) Titers of anti-nuclear antigen and anti-ds DNA antibodies of aged mice (10 months). Data are the mean (±s.d.) of >10 mice of each genotype and are representative of 4 independent experiments. (d) Effector cytokine production of activated T cells from WT and S1P₁-Tg mice (5-6 months). Data are representative of 2 independent experiments. (e) Serum titers of IgG1 and IgG2a (5-6 months). Data are the mean of 5 mice of each genotype and are representative of 3 independent experiments. (f-h) Analysis of WT and S1P₁-Tg T cells in the mixed BM chimeras (6-9 months after reconstitution), including expression of activation markers (f), proliferation (g), and effector cytokine production (h). Data are representative of 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t-test).

Figure 6. S1P₁ induces activation of Akt-mTOR to inhibit T_reg development and function

(a) IL-2 activated signaling pathways in thymic T_reg precursors from WT and S1P₁-Tg mice. CD4⁺CD25⁺Foxp3⁻ cells were purified and stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 4 independent experiments. (b) Effects of drug treatments on IL-2 induced Foxp3 expression in T_reg precursors. CD4⁺CD25⁺Foxp3⁻ cells were treated with U0126, LY294002 and Rapamycin for 30 minutes, followed by IL-2 stimulation. Data are representative of 3 independent experiments. (c) IL-2 activated signaling pathways in peripheral T_reg cells from WT and S1P₁-Tg mice. T_reg cells were stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 5 independent experiments. (d) Suppressive activity of T_reg cells transduced with dn-Akt retrovirus. WT and S1P₁-Tg T_reg cells were transduced with control (MiT) and dn-Akt expressing (dn-Akt) retroviruses (non-transduced cells are shown on the right as a comparison), and transduced cells were sorted and used in the T-cell suppression assays with different T_conv and T_reg ratios. Data are representative of 3 independent experiments. *, P < 0.001 (Student’s t-test).

Figure 7. S1P₁ is necessary for Akt activation in T_reg cells

(a,b) IL-2 activated signaling pathways in thymic T_reg precursors (a) and Foxp3⁺ reg T_reg cells (b) from WT and S1P₁-KO mice. Purified CD4⁺CD25⁺Foxp3⁻ cells (a) or CD4⁺Foxp3⁺ cells (b) were stimulated with medium alone or IL-2, and activation of Akt, STAT5 and Erk were examined by flow cytometry using phospho-specific antibodies. Data are representative of 3 independent experiments. (c) Suppressive activity of T_reg cells transduced with Cre-GFP retrovirus alone or in combination with constitutively active Akt. Foxp3⁺ T_reg cells from the periphery of S1pr1^+/+ and S1pr1^fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP) alone or in combination with empty control (MiT) or constitutively active Akt (ca-Akt) retroviruses, and transduced cells were sorted and used in the T-cell suppression assays with different T_conv and T_reg ratios. Data are representative of 2 independent experiments. *, P < 0.001 (Student’s t-test).

Figure 8. Differential regulation of S1P₁ expression in T_reg and T_conv cells

(a) T_reg and T_conv cells from WT mice were stimulated with anti-CD3, anti-CD28 and IL-2, and S1pr1 mRNA expression was analyzed by quantitative PCR. Data are representative of 3 independent experiments. (b) OT-II TCR-transgenic mice were crossed with Foxp3^gfp knockin mice, and sorted T_reg and T_conv cells from these mice were transferred into C57BL/6 mice, followed by s.c. immunization with ovalbumin emulsified in CFA. Two days after immunization, T_reg and T_conv cells from the draining lymph nodes were purified based on GFP expression, and S1pr1 mRNA expression was analyzed in each T-cell subset. Data are representative of 2 independent experiments.