Figure 4.

Th1/Tc1 polarization during induction of rapamycin resistance requires PI3 kinase. (A) Human Th1/Tc1 cells were generated by co-stimulation and six-day culture in IL-2 + IFNα either alone (“−”) or in the presence of rapamycin (“+”). The protein lysate was obtained and subjected to western blot analysis for the following molecules: β-Actin, mTOR/phospho-mTOR, p70S6 kinase/phospho-p70S6 kinase, and 4EBP1. A similar result was obtained in two additional experiments. (B) Protein lysates from human Th1/Tc1 cells (“−”) or T1.R cells (“+”) were also tested for expression of total PI3 kinase (“PI3K”) or two forms of phosphorylated PI3 kinase (“p85 Tyr458” and “p85 Tyr 199”). A similar result was obtained in two additional experiments. (C) Representative examples of STAT1 phosphorylation in T1.R cells by flow cytometry analysis after restimulation alone (part i) or with the PI3 kinase inhibitor, LY294002 (part ii); summation of results using PI3K inhibition by LY in T1 cells (“LY”; part iii, n = 3), T1.R cells (part iv, n = 3) or by wortmannin (“W”; part v, n = 3). (D) Evaluation of PI3K dependency of type I polarization of T1 cells using LY (part ii, n = 3), T1.R cells using LY (part i; representative example of three experiments); results are expressed as frequency of CD4⁺ or CD8⁺ T cells that express IFNγ by IC flow cytometry after re-stimulation. Similar PI3K-dependency was observed using wortmannin as inhibitor (part iii; summation data of n = 3 experiments). (E) PI3K-dependency of T1.R polarization was also evaluated by testing supernatants for IL-2 and IFNγ content (representative example with LY as inhibitor, (parts i and ii); summation of n = 3 experiments using wortmannin as inhibitor, parts iii and iv).