HPK1-C sensitizes for activated T-cell death via the mitochondrial pathway. (A) Primary mouse T cells of HPK1-C transgenic (tg) mice or non-transgenic (non-tg) littermates were analyzed at day 5 after activation with Con A and expansion with IL-2 for loss of mitochondrial membrane potential (Δψ_M) by JC-1 staining after incubation with 1 μg/mL plate-bound anti-CD3 antibodies for 18 hours in the presence of 10 μg/mL CD95L-blocking antibody MFL3. All values represent mean and standard deviation of triplicate measurements and are expressed relative to the loss of Δψ_M without CD95L-blocking antibody set to 100%. (B) Primary mouse T cells of HPK1-C tg mice or non-tg littermates were stimulated by anti-CD3 antibodies for the indicated time or left nonstimulated. RNA was extracted and subjected to RT-PCR analysis using primers specific for the indicated NF-κB target genes Bcl-2, Bcl-x_L, and cIAP1. Expression of actin is shown as control. (C) The experiment was carried out essentially as in panel B and cell lysates were resolved by SDS-PAGE. Expression of Bcl-2, Bcl2-A1, and Bim_EL/L was visualized by Western blotting (WB). Tubulin is shown as a control. (D) Jurkat T cells with stable integration of either empty vectors or HPK1-C expression vectors, respectively,¹⁴  were subjected to cell death induction by incubation with 1 μg/mL plate-bound anti-CD3 antibodies for 24 hours in the absence (□) or the presence (▩) of 10 μg/mL of the CD95L-blocking antibody NOK1. The experiment presented is representative of 4 repeats. Standard deviation is given for triplicate measurements. (E) T cells with stable integration of either empty vectors or HPK1-C expression vectors were stimulated with increasing amounts of plate-bound anti-CD3 antibodies for 8 hours. Subsequently, cells were lysed and caspase 9 (top panel) and caspase 3 (bottom panel) activities were analyzed by a fluorometric substrate cleavage assay. Standard deviation is given for triplicate measurements. (F) Jurkat T cells with stable integration of either empty vectors or HPK1-C expression vectors were incubated with increasing amounts of the caspase-9–specific inhibitor z-LEHD-fmk (20 μM, 40 μM, 80 μM) and subjected to TCR-induced cell death by incubation with 1 μg/mL plate-bound anti-CD3 antibodies for 24 hours. Cell death was quantified by flow cytometry using FSC/SSC.