ALPS DNT cells exhibit hyperactive mTOR signaling. (A) Phosphorylation of Akt at Ser473, mTOR at Ser2448, S6 at Ser235/236, and S6 at Ser240 was determined in T cells from patients with ALPS and healthy controls after stimulation by cross-linked anti-CD3/CD28 mAbs. Histograms depicting levels of phosphorylated proteins show 1 representative experiment, y-axis is normalized to mode. (B) Cells from patients with ALPS and healthy controls were left unstimulated (w/o) or stimulated with cross-linked anti-CD3/CD28 mAbs and analyzed for phosphorylation of indicated proteins. Graphs show mean fluorescence intensities of p-Akt(Ser473) and p-S6(Ser240) (n = 10). Box plots depict the 75th percentile, median and 25th percentile values; whiskers represent maximum and minimum values. *P < .05, **P < .01, ***P < .001 (Mann-Whitney U test). (C) Expression of HIF1α was determined in ALPS CD4⁺ (gray, filled), CD8⁺ (black, dotted), and DNT (black, solid) cells. Overlay shows representative data from 1 of 8 independent experiments with similar results. (D) Gene expression of KLF2 in the indicated purified cell populations relative to CD4⁺ T cells was determined by quantitative RT-PCR. Data represent relative KLF2 expression of 5 patients with ALPS. *P < .05 (Mann-Whitney U test).