Figure 7. S6K1 knockdown in TSC1-deficient T cells leads to attenuated IL-17 production.

(A) Analysis of the phosphorylation status of the indicated downstream targets of TSC1. CD4⁺ T cells from WT or CD4^creTsc1^f/f mice were stimulated with anti-CD3/CD28 for the indicated time periods, and the cell lysates were subjected to immunoblotting with the indicated antibodies. (B) Analysis of GFP expression in peripheral blood from bone marrow chimeric mice 2Πmonths after reconstitution of control (shCON) or S6K1 or GRB10 shRNAs expressing WT or Cd4^CreTsc1^f/f bone marrow cells (upper panel). Immunoblot analysis of S6K1 and GRB10 was performed in sorted CD4⁺GFP⁺ T cells (lower panel). (C and D) Flow cytometric analysis (C, left panel), frequencies (C, right panel), or cytokine production (D) of Th17-polarized naive CD4⁺GFP⁺ T cells from bone marrow chimeric mice as in B. (E) Analysis of the phosphorylation status of S6K1 in CD4⁺YFP⁺ Treg cells. (F) Analysis of mAmetrine expression in peripheral blood from bone marrow chimeric mice 2 months after reconstitution of control or S6K1 shRNAs expressing Foxp3^YFPCreTsc1^+/+ or Foxp3^YFPCreTsc1^f/f bone marrow cells. (G) Cytokine production in CD4⁺YFP⁺mAmetrine⁺ T cells from bone marrow chimeric mice as in F was measured by Bio-Plex multicytokine assay 36 hours after stimulation with anti-CD3/CD28. Data are representative of (B, C, and F) or compiled from (D and G) three to five independent experiments. Error bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed, unpaired Student’s t test.