Vecabrutinib treatment alters T-cell subpopulations in the murine Eµ-TCL model. (A) Changes in relative frequencies of CD8⁺ and CD4⁺ T-cell subpopulations in spleen relative to vehicle control following 10 days of treatment with vecabrutinib or ibrutinib in the murine Eµ-TCL1 model. (B) Representative flow cytometry plots showing effector (CD8⁺CD127^lowCD44^int-hi), memory (CD8⁺CD44⁺CD127⁺), and naive (CD8⁺CD44⁻CD127⁺) T-cell populations. (C) Changes in effector, memory, and naive CD8⁺ T cells, (D) percentage of Tregs (CD4⁺CD25⁺FOXP3⁺) and (E) percentage of Tregs expressing the Treg activation markers CD103 and GITR in the different treatment groups. (F-H) Activation of the immediate early TCR gene Nur77 in effector cells (CD8⁺CD127^lowCD44^int-hi) and Tregs (CD4⁺CD25⁺FOXP3⁺) in splenocytes isolated from 8 recipient mice transplanted with Eµ-TCL1 tumor. The cells were treated ex vivo with increasing concentrations of vecabrutinib (F), ibrutinib (G), or tirabrutinib (H), in triplicates, followed by stimulation with anti-CD3 antibody. Changes in T-cell activation measured by relative expression of CD25 in CD4⁺ T cells (I) and CD8⁺ T cells (J) and by relative expression of CD69 in CD4⁺ T cells (K) and CD8⁺ T cells (L) upon treatment of peripheral blood mononuclear cells derived from patients with CLL with DMSO or 1 µM ibrutinib or vecabrutinib and stimulation for 6 hours using soluble anti-CD3 or anti-CD3/CD28 antibodies. Dashed lines in (I-L) denote normalization to DMSO. P values above individual columns represent comparison with DMSO. *P ≤ .05, **P ≤ .01, ***P ≤ .001, Mann-Whitney U test (A,C-H), paired Student t test (I-L). a′, anti; Ibru, ibrutinib; MFI, mean fluorescence intensity; ns, not significant (P > .05); Unstim., unstimulated; Veca, vecabrutinib.