FIGURE 2. TGF- inhibits RORt-directed IL-17 production by upregulating Foxp3.

a, The effect of TGF- on IL-17 expression after transduction of RORt. Naive CD4⁺ T cells incubated with TGF- from day -1 were transduced with control vector MSCV-IRES-GFP (MIG) or RORt-IRES-GFP (RORt) on day 0 (24 h after TCR activation), and IL-17 intracellular staining was performed on day 5. b, The inhibitory effect of TGF- when included at different times relative to transduction of RORt. The percentage of IL-17⁺ cells among GFP (RORt)⁺ cells is shown. c, Knockdown of TGF--induced Foxp3 expression with an shRNA against Foxp3 (LMP1066). Naive CD4⁺ T cells were stimulated as in a and co-transduced on days 0 and 1 with control retroviral construct MSCV-IRES-hCD2 (MCD2) or RORt-IRES-hCD2 (RORt) and the specific shRNA vector (LMP1066) or control vector (LMP). After transduction, the cells were cultured with or without TGF-, and Foxp3 expression was measured by intracellular staining on day 5. d, Restoration of RORt-induced IL-17 expression on knockdown of Foxp3. IL-17 expression was assessed in cells co-transduced as in c and gated for the level of GFP expression. The percentage of IL-17⁺ T cells in GFP^hi cell populations is shown. Results with additional shRNA vectors that failed to downregulate Foxp3 expression were similar to those with the control LMP vector. Representative data from three experiments are shown.