We verified that Jurkat cell pSLP76 was activated by anti-CD3/28 sharply, and treated with a titration of doses of 7m and 9h both exhibited dose-dependent inhibition of pSLP76 in Jurkat cells (Fig. ). It is worth noting that 33 nM of 7m could significantly inhibit over 50% of pSLP76 relative to stimulation (Fig. ). Whereas 9h boasted stronger inhibition of pSLP76 at only 3.7 nM than 7m, and yielded an almost complete abolishment at 100 nM (Fig. ), defining 9h as a high cellular activity HPK1 inhibitor. As IL-2 production is a key marker indicating T cell activation, we next utilized human peripheral blood mononuclear cells (hPBMCs) to test the IL-2 production after administration of 7m, 9h and the positive control 6. Under stimulation of anti-CD3/28, all 3 compounds elevated IL-2 production of hPBMCs at certain concentrations (Fig. ). Of which, 9h exhibited the most potent activity in triggering IL-2 secretion of hPBMCs ranged from 0.03 to 0.3 μM. Notably, a relative low dose of 9h (0.03 μM)