mTOR inhibition reduces abnormally differentiated DNT cells. (A, B) CFSE-labeled ALPS CD4⁺ (circle), CD8⁺ (square), and DNT (triangle) cells were stimulated with anti-CD3/CD28 coated beads and IL-2 in the presence of 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM rapamycin. Proliferation (A) and viability (B) were determined by flow cytometry. Data show mean of 3 independent experiments. (C) PBMC from patients with ALPS before and under rapamycin or MMF therapy were gated for CD3⁺/TCRαβ⁺ (%DNT cells) or CD3⁺/TCRαβ⁺/CD4⁻/CD8⁻ (DNT cells). (D) Frequency of DNT cells among all T cells in healthy controls (circle), patients with ALPS (square), and patients with ALPS treated with rapamycin (up-pointing triangle) or MMF (down-pointing triangle) is shown. (E) Percentages of CD4⁺/CD25⁺/FoxP3⁺ Treg cells were determined in PBMC from healthy controls and untreated, rapamycin-treated, and MMF-treated patients with ALPS. (F, G) Percentages of CCR7⁻/CD45RA⁺ (F) and Ki67⁺ (G) cells among DNT cells of all studied healthy controls (n = 12) and untreated (n = 16), rapamycin-treated (n = 9), and MMF-treated (n = 3) patients with ALPS is graphed. Each symbol represents an individual subject; open symbols indicate patients with ALPS (p2-705, p16-11, p1-809) before and under therapy. ns, not significant; *P < .05; **P < .01; ***P < .001 (Mann-Whitney U test).