Vecabrutinib exhibits a minor impact on activation and proliferation of isolated T cells stimulated with anti-CD3 beads. Changes in TCR signaling, as measured by relative expression of GFP (A) and changes in T-cell activation, as measured by relative expression of CD25 (B) or CD69 (C) on T cells isolated from NR4A1-GFP mice treated ex vivo with DMSO or 1 µM ibrutinib, vecabrutinib, or tirabrutinib and stimulated with anti-CD3/CD28 beads for 6 hours. Dashed lines denote normalization to DMSO. P values above individual columns represent comparison with DMSO. (D) Representative flow cytometry plots for changes in T-cell activation measured by relative expression of CD69 (E) and CD25 (F) on human T cells isolated from healthy individuals treated ex vivo with DMSO or 1 µM ibrutinib, vecabrutinib, or tirabrutinib and stimulated with anti-CD3/CD28 beads for 6 hours. (G) Representative flow cytometry plot for changes in proliferation of human T cells from healthy individuals upon treatment with DMSO or 1 µM (H) or 5 µM (I) ibrutinib, vecabrutinib, or tirabrutinib and stimulation with anti-CD3/CD28 beads for 72 hours. *P ≤ .05, **P ≤ .01, ***P ≤ .001, paired Student t test. Ibru, ibrutinib; ns, not significant (P > .05); Tira, tirabrutinib; Veca, vecabrutinib.