HPK1-C initiates AICD independent of CD95L. (A) To control for the capacity of the CD95L-blocking antibody MFL3 to efficiently inhibit CD95L-dependent cell death, primary mouse T cells at day 5 after stimulation were activated with 0.1 μg/mL of plate-bound anti-CD3 antibodies and cocultured with CD3-deficient, but CD95L-sensitive, CFSE-labeled target cells at the given ratios in the absence or presence of 10 μg/mL of the CD95L-blocking antibody MFL3. (B) Primary mouse T cells were analyzed at day 5 after stimulation for activation-induced cell death (AICD) after incubation with 0.1 or 1 μg/mL of plate-bound anti-CD3 antibodies for 18 hours in the absence (□) or the presence (▩) of 10 μg/mL of the CD95L-blocking antibody MFL3. Standard deviation is given for triplicate measurements. The experiment presented is representative of 5 repeats. (C) Primary mouse T cells of HPK1-C transgenic (tg) mice or non-transgenic (non-tg) littermates were analyzed at day 5 after stimulation with Con A and expansion with IL-2 for AICD by incubation with 0.1 μg/mL of plate-bound anti-CD3 antibodies for 18 hours in the presence (▩) or the absence (□) of 10 μg/mL of the CD95L-blocking antibody MFL3. Standard deviation is given for triplicate measurements. The experiment was repeated 5 times with similar outcome. (D) Primary mouse T cells of HPK1-C tg mice or non-tg littermates were stimulated by anti-CD3 antibodies for the indicated time points or left nonstimulated. RNA was extracted and subjected to reverse transcriptase–polymerase chain reaction (RT-PCR) analysis using primers specific for CD95L. Expression of actin is shown as a control.