HPK1 facilitates spontaneous and αCD3-mediated apoptosis in primary murine CD4⁺ T cells.

(A) Restimulation of primary murine CD4⁺ T cells with αCD3 mAb for 3 hours results in an increase of HPK1 levels. Naive primary CD4⁺ T cells from LNs of DO 11.10 TCR tg mice were stimulated with OVA peptide for 24 hours. After 4 days of expansion in the presence of IL-2, the cells were either left untreated (–) or restimulated with plate-bound αCD3ε mAb for 3 hours (+). Using Abs specific for HPK1 or β-actin, 80 μg whole cellular proteins was immunblotted. Representative blots from 3 independent experiments are shown. (B) Schematic structure of HPK1 and HPK1-encoding retroviral vectors. Four proline-rich motifs (PR 1-4) within potential SH3 binding sites, the caspase cleavage motif DDVD, and the position of kinase-death mutation K to M at position 46 are indicated. (C) HPK1 expressed from the retroviral construct pHA-HPK1 wt is catalytically active. The indicated retroviral constructs were transfected into 293T cells. After 48 hours, the cells were lysed, proteins were precipitated with an αHA mAb, and in vitro kinase assays were performed using GST-cJun_5-89 as an exogenous substrate. HPK1 expression in precipitates is shown below. Phosphorylation was visualized by autoradiography (top and middle panels). Representative blots from 4 independent experiments are shown. (D) CD4⁺ T cells from LNs of DO 11.10 TCR tg mice were stimulated with OVA peptide and infected. After 4 days of expansion in the presence of IL-2, the cells were either left untreated (upper panel) or restimulated with plate-bound αCD3ε mAb for 8 hours (lower panel), stained with Annexin V–PE, and analyzed by FACS. Representative data from 3 independent experiments are shown.