TGF-beta 1 inhibition of IFN-gamma-induced signaling and Th1 gene expression in CD4+ T cells is Smad3 independent but MAP kinase dependent

Abstract

In addition to classic Smad signaling pathways, the pleiotropic immunoregulatory cytokine TGF-beta1 can activate MAP kinases, but a role for TGF-beta1-MAP kinase pathways in T cells has not been defined heretofore. We have shown previously that TGF-beta1 inhibits Th1 development by inhibiting IFN-gamma's induction of T-bet and other Th1 differentiation genes, and that TGF-beta1 inhibits receptor-proximal IFN-gamma-Jak-Stat signaling responses. We now show that these effects of TGF-beta1 are independent of the canonical TGF-beta1 signaling module Smad3, but involve a specific MAP kinase pathway. In primary T cells, TGF-beta1 activated the MEK/ERK and p38 MAP kinase pathways, but not the JNK pathway. Inhibition of the MEK/ERK pathway completely eliminated the inhibitory effects of TGF-beta1 on IFN-gamma responses in T cells, whereas inhibition of the p38 pathway had no effect. Thus, TGF-beta1's inhibition of IFN-gamma signaling in T cells is mediated through a highly specific Smad3 independent, MEK/ERK-dependent signaling pathway.

Fig. 1. TGF-β1 suppresses IFN-γ induced Th1 gene expression through a Smad3-independent pathway

Splenic CD4⁺ T cells from BALB/c littermate wild-type (Smad3^+/+) and Smad3^−/− mice were isolated and stimulated for 24 hours with anti-CD3/anti-CD28. Cells were washed, rested for 2 h, and treated with IFN-γ and/or TGF-β1 for 3 h (Smad3^+/+) or 6 h (Smad3^−/−), respectively. Relative IRF-1, T-bet, and IL-12Rβ2 levels were measured by real-time RT-PCR, normalized to β-actin. Data show the mean + S.D derived from individually isolated, treated, and analyzed T cells from (A) Smad3^+/+ mice (n = 6) and (B) Smad3^−/− mice (n = 4). * p < 0.05, ** p < 0.01. This experiment was repeated with similar results.

Fig. 2. TGF-β1 suppresses receptor-proximal IFN-γ signaling in T helper cells through a Smad3-independent pathway

Splenic CD4⁺ T cells from littermate Smad3^+/+ or Smad3^−/− mice were prepared as in the legend to Fig. 1 and treated with IFN-γ and/or TGF-β1 for the indicated times. Cell lysates were prepared and analyzed by western blot to detect phosphotyrosine-JAK1, total JAK1, phosphotyrosine-STAT1 (Y701), and total STAT1, respectively. This experiment was repeated with similar results.

Fig. 3. TGF-β1 activates ERK and p38 MAP kinase pathways in T helper cells

Splenic and LN CD4⁺ T cells were prepared as in Fig. 1, and treated with IFN-γ and/or TGF-β1 for 0 – 60 min before lysis. In some wells, cells were incubated for 30 min with PD98059 prior to cytokine addition, as indicated. Cell lysates were subject to western blot to detect phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-MKK3/6, and MKK3/6, respectively. The asterisk (*) indicates a cell lysate from HeLa cells treated with CoCl₂, as a positive control for phospho-ERK.

Fig. 4. TGF-β1 suppresses IFN-γ induced Th1 gene expression through a MEK/ERK-dependent pathway

Splenic and LN CD4⁺ T cells from BALB/c mice were prepared and stimulated as in Fig. 1. In some wells, cells were incubated with PD98059 (20 μM) or SB202190 (20 μM) for 30 min prior to cytokine addition. After 3 hr of cytokine stimulation, RNA was isolated and analyzed by real-time RT-PCR for (A) T-bet or (B) IRF-1, and IL-12Rβ2. Bars indicated the mean + SD of triplicate cell culture wells. * p < 0.05. ** p < 0.01. This experiment was repeated twice more with similar results.

Fig. 5. TGF-β1 suppresses receptor-proximal IFN-γ signaling in T helper cells through a MEK/ERK-dependent pathway

BALB/c CD4⁺ T cells were prepared and stimulated as in Fig. 1. In some wells, cells were incubated with PD98059 (20 μM) or SB202190 (20 μM) for 30 min prior to cytokine addition. After 0 – 60 minutes of cytokine stimulation, cell lysates were subject to Western Blot analysis for phosphotyrosine-STAT1 (Y701) or total STAT1. This experiment was repeated with similar results.

Fig. 6. Additional experimental approaches confirm that TGF-β1’s effects are MEK/ERK-dependent

BALB/c CD4⁺ T cells were prepared and stimulated as in Fig. 1. (Top) In some wells, cells were incubated with the MEK inhibitor UO126 (20 μM) for 30 min prior to cytokine addition. After 0 – 60 minutes of cytokine stimulation, cell lysates were prepared. (Bottom) BALB/c CD4⁺ T cells were stimulated overnight with anti-CD3/anti-CD28 then infected with a retrovirus expressing a dominant negative form of MEK1, or with a control retrovirus. After two additional days of incubation, cells were washed, replated, and treated with cytokines for the times indicated and cell lysates prepared. Cell lysates were subject to Western Blot analysis for phosphotyrosine-STAT1 (Y701) or total STAT1.