FIGURE 8.

TNFR2 targets ROR-γt expression and tyrosine phosphorylation of STAT3 and STAT5 to modulate Th17 differentiation. (A) Steady-state ROR-γt mRNA expression is elevated in TNFR2 KO CD4⁺ T cells compared to WT T cells. The cDNA used in these assays was obtained from the WT and TNFR2 KO Il2^+/− cells stimulated under Th17-polarizing conditions in the presence or absence of exogenous IL-2 or under Th0 conditions from Figs. 7F – I. Values for steady-state ROR-γt, ROR-α, AHR, and ARNT are expressed as relative expressions (fold change) following normalization to β-actin using the mRNA levels in WT Th0 cells as the calibrator. (B) Intracellular tyrosine-phosphorylated STAT5 (upper rows) and STAT3 (lower rows) were determined for 5C.C7 ^+/−Il2-GFP^+/− WT or TNFR2 KO CD4⁺ T cells left unstimulated, activated for 4 d under Th17-polarizing conditions with or without the addition of IL-2 (100 U/ml), or activated for 4 dunder Th0 conditions. IL-6 (50 ng/ml) and IL-2 (100 U/ml) were added for the final 20 min of cell culture. (Top) Representative flow cytometry histograms are shown for pY-STAT5 and pY-STAT3 using 7AAD⁻ CD4⁺-gated T cells for analyses. The dotted line indicates pY-STAT3 or pY-STAT5 staining in unstimulated cells. The mean ± SEM of pY-STAT-positive cells (i.e., those to the right of the dotted line) from three independent experiments are shown. (Bottom) The mean ± SEM of the MFI of pY-STAT3- or pY-STAT5-positive CD4⁺ T cells from the cells acquired and analyzed above (top) are shown. *p < 0.05. (C) Overall, we conclude that tmTNF activation of TNFR2 expressed on CD4⁺ T cells stimulates Il2 expression and Il2 mRNA stability. During Th17 differentiation, TNFR2 is increased. When tmTNF/TNFR2 signaling is blocked or impaired, Th17 differentiation is promoted, and IL-17 activates STAT3 and STAT6