Ibrutinib irreversibly binds to ITK-C₄₄₂ and RLK expression provides compensatory kinase activity, which protects Th1 and CD8 T cells. (A) Immunoblot analysis of 45-minute nuclear and 2-hour whole-cell extracts from ibrutinib or alternate BTK inhibitor–pretreated, freshly purified healthy donor primary CD4⁺ cells stimulated with anti-CD3/anti-CD28. Nuclear extracts were probed for NFAT1 and Brg1; whole-cell extracts were probed for pSTAT6-Y₆₄₁, total STAT6, pIkBα-S_32/36, total IkBα, JunB, and actin. (B) Immunoblot analysis of Jurkat parental, Jurkat-ITKwt, and Jurkat-ITKC₄₄₂A nuclear lysates after ibrutinib pretreatment and anti-CD3/anti-CD28 stimulation. Blots were probed for NFAT1 and Brg1. (C) AUC for Fluo4-AM calcium release analysis of Jurkat-ITK and Jurkat-ITKC442A cell lines after pretreatment with ibrutinib or DMSO and stimulation with anti-CD3. Each symbol represents a single replicate experiment. Error bars represent SEM. (D) Cytokine analysis of IL-4 (black bars and right y-axis) and IFN-γ (open bars and left y-axis) media levels in anti-CD3/anti-CD28–stimulated Th1- and Th2-polarized cell cultures. These are the same cell cultures used in panels E and F. (E) Intracellular cytokine analysis of Th1(IFN-γ)– and Th2(IL-4)–polarized T-cell cultures pretreated with the indicated concentration of ibrutinib or DMSO and stimulated for 6 hours via anti-CD3/anti-CD28. Cytokine measurements were taken on separately cultured subsets of cells after 3 weeks of polarizing cell culture with weekly anti-CD3/anti-CD28 stimulation. Error bars represent SEM. (F) Th1-, Th2-, and CD8-purified primary cells were stimulated with anti-CD3/anti-CD28 after pretreatment with ibrutinib. Immunoblot analysis was conducted probing for NFAT and Brg1 as well as pIkBα-S_32/36, total IkBα, and actin. (G) Immunoblot analysis of Jurkat parental, Jurkat-RLK, and Jurkat-EV (empty vector) nuclear lysates after ibrutinib pretreatment and anti-CD3/anti-CD28 stimulation. Blots were probed for NFAT1 and Brg1. (H) AUC for Fluo4-AM calcium release analysis of Jurkat-EV (empty vector) and Jurkat-RLK cell lines after pretreatment with ibrutinib or DMSO and stimulation with anti-CD3. Each symbol represents a single replicate experiment. Error bars represent SEM. Unstim, unstimulated.