SMAD2 is essential for TGF beta-mediated Th17 cell generation

Abstract

TGFβ is the quintessential cytokine of T cell homeostasis. TGFβ signaling is required for the efficient differentiation and maintenance of CD4(+)FOXP3(+) T cells that inhibit immune responses. Conversely, in conjunction with the inflammatory cytokine IL-6, TGFβ promotes Th17 cell differentiation. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ-signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. Although mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-deficient T cells are impaired in their response to TGFβ in vitro and in vivo, and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is uniquely essential for TGFβ signaling in CD4(+) T effector cell differentiation.

FIGURE 1.

SMAD2 is necessary to efficiently induce TGFβ-dependent FOXP3⁺ and IL-17A⁺ T cell subsets. A, CFSE-labeled WT and Smad2 CKO naive CD4 T cells were activated (anti-CD3/CD28 cross-linking in all panels) with varying concentrations of TGFβ for 2 days. The extent of proliferation was measured by the loss of CFSE using flow cytometry. Data shown are representative of three independent experiments (a minimum of three mice/genotype/experiment) with similar results. Error bars are S.E. B, CD4 T cells were cultured under iTreg conditions with varying concentrations of TGFβ for 3 days. FOXP3 expression was analyzed by intranuclear staining (three independent experiments with similar results). C and D, CD4 T cells were cultured in varying doses of TGFβ and IL-6 with mitomycin C-treated splenocytes in Th17 conditions for 4 days. Intracellular staining (ics) results for IL-17A and IFNγ are shown. Flow cytometric profiles shown in C are representative of three independent experiments, and D shows the average frequency of IL-17A⁺ Smad2 CKO T cells relative to controls in varying culture conditions. Statistics are based on Student's t test, with *, p < 0.05; **, p < 0.01. E, CD4 T cells were cultured with IL-1β and IL-6 in activating conditions for 4 days followed by ics. Top row, anti-CD3/CD28 mAb without cytokines. F, CD4 T cells were cultured with increasing doses of TGFβ and IL-6 in the presence of a fixed amount of IL-2 blocking mAb for 4 days followed by ics for the cytokines. G, left, activin RII expression on B6 CD4 T cells ex vivo and after activation for 2 days. Right, IL-17A⁺ cells generated with activin A and IL-6 in 3 day-activated CD4 T cells (right). H, WT and Smad2 CKO peripheral Lymph node cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 5 h and analyzed for cytokine expression in γδ TCR+ cells (two independent experiments).

FIGURE 2.

SMAD2 modulates IL-6Rα expression on CD4 T cells. A, quantitative RT-PCR analysis for Il6ra mRNA expression in activated CD4 T cells (three mice/genotype) relative to Actb expression. B, semiquantitative RT-PCR analysis for Il6ra and Rorc with Actb as control. A 4-fold dilution series is represented. One of two experiments is shown. C, naive CD4 T cells from WT and Smad2 CKO mice were activated for 30 min in the presence of IL-6. Expression of phosphorylated STAT3 (pSTAT3) was analyzed by ics. D, mean fluorescent intensity of pSTAT3 expression in T cells at various time points after IL-6 stimulation. Data are representative of three independent experiments.

FIGURE 3.

SMAD2 is required in vivo for the generation of IL-17A⁺ CD4 T cells. A and B, Smad2 CKO. CD4 T cells cause more severe colitis in Rag1^−/− hosts. Naive CD4 T cells were transferred to Rag1^−/− recipients (n = 5/genotype), and T cells were analyzed 3 weeks after transfer for IL-17A and IFNγ expression by ics. Flow cytometric profiles of individual mice in A and averages in B are representative of two independent experiments with similar results. MLN, mesenteric lymph nodes; LPL, lamina propria lymphocytes. C, the onset and severity of colitis were monitored by weight measurements and gut histology. Data shown are an average of two experiments. D, diminished Th17 response to C. rodentium infection in Smad2 CKO mice. Mice infected with C. rodentium were sacrificed at 2 weeks after infection. Representative flow cytometric profiles of CD4⁺ T cells expressing the activation markers in the spleens are shown (two experiments, minimum three mice/genotype/experiment). E, CD4⁺ T cells from lymphoid tissues (splenocytes are shown here, but a similar pattern was observed in all tissues) of mock-infected and infected animals were analyzed for the expression of IL-17A and/or IFNγ by ics. Left, representative flow cytometric profiles; right, averages (n = 5/genotype) with statistics (as in Fig. 1) of the frequencies of CD4⁺IL-17A⁺. UI, uninfected; CB, Citrobacter-infected.