Defective proliferation and enhanced susceptibility to apoptosis. (A) PBMC of the patient (P1) or his healthy heterozygous sister were left untreated or stimulated with PHA or anti-CD3/CD28 beads and carboxyfluorescein diacetate succinimidyl ester (CSFE) dilution of CD4⁺ or CD8⁺ T cells was determined after 5 days incubation (unstimulated: gray; stimulated: black). The experiment was performed 3 times with similar results. (B) Control PBMC were labeled with CFSE and stimulated with phytohemagglutinin (PHA) or anti-CD3/CD28 beads in the absence (gray line) or presence (black line) of the TPP2 inhibitor butabindide. CFSE dilution of CD4⁺ and CD8⁺ T cells was analyzed after 5 days. The experiment was repeated 3 times with similar results. (C) EBV lines of the patient (P1), his healthy heterozygous sister, a healthy donor (HD) control and a FAS-mutant ALPS patient were stimulated with increasing concentrations of anti-Fas antibody cross-linked with protein A, the topoisomerase inhibitor etoposide (eto), the protein kinase inhibitor staurosporine (sts), or were sublethally irradiated (30 Gy). Viable Annexin V/PI negative cells were determined by flow cytometry after 24 hours or on day 0, 2, and 4, respectively. Results are representative of 3 independent experiments with similar results. (D) Cells of an EBV line from a healthy donor were stimulated with increasing concentrations of staurosporine in the absence (filled squares) or presence (open squares) of the TPP2 inhibitor butabindide and viable cells were determined after 24 hours. The experiment was repeated twice with similar results.