Rapamycin reduces mTOR hyperactivity of ALPS DNT cells in vivo. (A) Phosphorylation status of Akt(Ser473) and S6(Ser240) was determined in T cells of untreated (n = 10) and rapamycin-treated (n = 4) patients with ALPS after stimulation with cross-linked anti-CD3/CD28 mAbs. Graphs show mean fluorescence intensities, box plots depict the 75th percentile, median, and 25th percentile values; whiskers represent maximum and minimum values. (B) PBMC from healthy donors and untreated and rapamycin-treated patients with ALPS were analyzed for HIF1a expression. Graph shows HIF1a expression relative to CD4⁺ T cells. (C) Gene expression of KLF2 in the indicated purified cell populations relative to β-2 microglobulin was determined by quantitative RT-PCR. Data represent relative KLF2 expression of 4 rapamycin-treated ALPS. (D) Expression of IL-10 mRNA was assessed in CD4⁺, CD8⁺, and DNT cells from untreated (n = 7) and rapamycin-treated (n = 4) patients with ALPS. (E) Serum levels of IL-10 were determined in patients with ALPS before and during rapamycin therapy. Lines depict individual subjects. ns, not significant; *P < .05; **P < .01 (Mann-Whitney U test).