Figure 6.

mTORC1 and mTORC2 influence cytokine signaling through differential inhibition of SOCS proteins. (a) IB of mTOR activation in lysates from wild-type, naive T cells stimulated with anti-CD3 and anti-CD28 in varying doses of rapamycin for 8 h in serum-containing media. (b) Cytokine production of wild-type, 5C.C7 T cells stimulated with PCC peptide for 48 h in T_H1 or T_H2 skewing conditions in the presence or absence of very low dose (VLD, 100pM) rapamycin, washed and expanded in fresh drug and IL-2 for 5 days, then restimulated with anti-CD3 and anti-CD28. (c) IB of STAT phosphorylation in lysates from freshly isolated wild-type, T-Rheb^−/− and T-Rictor^−/− CD4⁺ T cells resuspended in serum-free media for 1 h and stimulated with IL-12, IL-4, or IL-6 for 30 min. (d) SOCS3 and SOCS5 mRNA levels from wild-type, T-Rheb^−/− and T-Rictor^−/− CD4⁺ T cells stimulated with mock or anti-CD3 and anti-CD28 simulation overnight. Values are normalized to 18s rRNA and scaled to wild-type unstimulated (for SOCS3) or stimulated (for SOCS5). (e) IB of SOCS3 and SOCS5 of cells treated as in d. (f) Kinetic analysis of SOCS3 and SOCS5 protein expression in lysates from wild-type, T-Rheb^−/− and T-Rictor^−/− CD4⁺ T stimulated with anti-CD3 and anti-CD28 for the indicated times. Actin is included as a loading control. (g) IFN-γ production in wild-type, T-Rheb^−/− and T-Rictor^−/− CD4⁺ T cells transfected with control, SOCS3, and SOCS5 siRNA by nucleofection, rested 16 h, stimulated in T_H1 and T_H2 skewing conditions 48 h, then expanded in IL-2 for 48 h and restimulated with anti-CD3 and anti-CD28. (h) IL-4 production from cells in g. All data are representative of at least three independent experiments, except for g and h, which were performed twice.