CD95L-dependent and HPK1-C–mediated, CD95L-independent mechanisms synergize in AICD of primary human T cells. (A) Primary human T cells were analyzed for AICD at day 6 after stimulation with PHA and expansion with IL-2. Cells were incubated with 1 μg/mL of plate-bound anti-CD3 antibodies for 18 hours with or without caspase-9–specific inhibitor z-LEHD-fmk (20 μM) in the presence (▩) or the absence (□) of 10 μg/mL of the CD95L-blocking antibody NOK-1. (B) The experiment was carried out similar to panel A, but primary human T cells were transfected 48 hours before with siRNA oligonucleotides comprising a HPK1-specific (siHPK1) or nonspecific sequence (control). The experiments presented are representative of 3 repeats. The siRNA-mediated down regulation of HPK1 was compared with actin by Western blotting (inset). (C) T cells from non-tg or HPK1-C tg littermates were activated with Con A and expanded in the presence of IL-2. At day 3 after activation, cells were transfected with siRNA oligonucleotides comprising a Bim (siBim) or a nonspecific sequence (control). Forty-eight hours following transfection, T cells were subjected to TCR-induced AICD by incubation with 0.1μg/mL anti-CD3 antibodies. The experiments presented are representative of 3 repeats. The siRNA-mediated down-regulation of HPK1 was compared with actin by Western blotting (inset). Standard deviation is given for triplicate measurements. (D) Model for the role of HPK1-C in the regulation of activation-induced cell death (AICD). HPK1-C suppresses the induction of antiapoptotic NF-κB target genes Bcl-2, Bcl-x_L, and cIAP1. Stimulation of the T-cell receptor (TCR) in preactivated T cells leads to activation of CD95 by newly synthesized CD95L involving caspase-8. In parallel, TCR stimulation leads to CD95-independent activation of the mitochondrial death pathway and caspase-9, which is augmented by HPK1-C. Stimulation of the B-cell receptor (BCR) leads to AICD primarily via the mitochondrial pathway, which is also augmented by the presence of HPK1-C.