A low caspase-3 activity cleaves HPK1 in expanded primary T cells. (A) Expansion of T cells leads to increased HPK1 expression and conversion to HPK1-C. Presence of full-length HPK1 and its processed fragment HPK1-C was visualized by Western blotting (WB) in primary human T cells at day 1 or day 6 after stimulation. Erk1 is shown as a control. (B) Cell lysates of primary human T cells at day 1 or day 6 after stimulation with PHA and expansion with IL-2 were incubated with in vitro–translated (i.v.tr.) [³⁵S]-labeled HPK1 and resolved by SDS-PAGE. Full-length HPK1 and the caspase cleavage fragments HPK1-N and HPK1-C were detected by autoradiography. As a control, apoptotic processing of [³⁵S]-labeled HPK1 is shown by incubation with lysates from SKW6.4 cells stimulated for 3 hours with anti–Apo-1 (+) or left nonstimulated (−). (C) Primary human T cells isolated from peripheral blood were stimulated with PHA and expanded with IL-2. Samples were taken at the indicated culture day and lysates were resolved by SDS-PAGE. Full-length caspase-3 and its processed fragments p20, p19, and p17 were visualized by Western blotting. Presence of HPK1 and processing toward HPK1-C were analyzed by Western blotting. Expression of actin is shown as control. Viability of the expanded T cells at the time points when processing of caspase-3 and HPK1 was detected was higher compared with the time points when no processing of caspase-3 or HPK1 was detected. Lysates of Jurkat T cells stimulated for 3 hours with anti–Apo-1²⁴  (+) or left nonstimulated (−) are shown to control for apoptotic processing of caspase-3. (D) The experiment was performed as in panel B, with cell lysates of primary human T cells at day 6 of culture in the absence or presence of increasing concentrations of the caspase-3/-7–specific inhibitor z-DEVD-fmk. T-cell lysate was omitted in lane 1 (input). (E,F) To block the cleavage of HPK1 toward HPK1-C, primary mouse T cells were incubated at day 3 after stimulation with Con A and expanded in the presence of the 5 μM panspecific caspase inhibitor qVD-oph (E) or 50 μM caspase-3–specific inhibitor z-DEVD-fmk (F) for 72 hours. Cells were washed to remove residual inhibitors, divided into 2 fractions, and subjected either to Western blotting (inset) using antibodies against HPK1 (top) or Erk1 (bottom) or to analysis of AICD. Therefore, cells were further analyzed by incubation with 0.1 μg/mL of plate-bound anti-CD3 antibodies or 2.5 ng/mL of CD95L for 18 hours. Viability of the expanded T cells was not altered by addition of qVD-oph or z-DEVD-fmk. Standard deviation is given for triplicate measurements. The experiment was repeated 3 times with similar outcome.