In T cells, ibrutinib specifically targets ITK, inhibiting TCR-induced cellular signaling and activation. (A) Immunoblot analysis of freshly isolated ibrutinib pretreated primary CD4+ cells from a healthy donor, anti-CD3/anti-CD28 stimulated (or unstimulated), whole-cell lysates. Blot probed for pITK-Y₁₈₀, total ITK, pSTAT6-Y₆₄₁, total STAT6, pIkBα-S_32/36, total IkBα, JunB, and actin. Densitometry analysis normalized to dimethylsulfoxide (DMSO)-treated (0 µM) sample. (B) Immunoblot analysis of freshly isolated ibrutinib pretreated primary CD4⁺ cells from a healthy donor, anti-CD3/anti-CD28–stimulated (or unstimulated), cytoplasmic, and nuclear lysates. Blots probed for NFAT (and activated hyperdephosphorylated NFAT), Brg1, and actin. Densitometry analyses are normalized to the DMSO-treated (0 µM) sample. (C) Immunoblot analysis of freshly isolated ibrutinib-pretreated primary CD4⁺ cells from a healthy donor, anti-CD3/anti-CD28–stimulated (or unstimulated), whole-cell lysates. Blots were probed for pZAP70-Y₃₁₉, total ZAP70, pLAT-Y₁₉₁, total LAT, pLCK-Y₅₀₅, total LCK, pIkBα-S_32/36, total IkBα, and actin. Densitometry analyses are normalized to the DMSO-treated (0 µM) sample. (D) Nuclear or whole-cell lysate immunoblot analysis of Jurkat cells pretreated with ibrutinib and stimulated with either anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate/ionomycin for 45 minutes. Blots were probed with Brg1, NFAT1, and actin (nuclear lysates) or pIkBα-S_32/36, total IkBα, and actin (cellular lysates). (E) Immunofluorescent microscopy of ibrutinib-pretreated, freshly isolated, primary CD4⁺ cells from healthy donors (panels A and B) were stimulated for 45 minutes with anti-CD3/anti-CD28 (or unstimulated), fixed, and stained for NFAT (green) and nuclei (4,6 diamidino-2-phenylindole [DAPI], blue). Activated cells are characterized by influx of NFAT into nuclear region (green overlay with blue = cyan) and are denoted by white arrows. (F) Percent relative NFAT1/DAPI colocalization derived from Pearson correlation analysis of 10 independent immunofluorescent microscopy fields (different donors than pictured in panel E and normalized to the average unstimulated value. Cyclosporin A (CSA) treatment was used as an additional negative control. Error bars represent SEM. (G) Phosphoflow analysis of pPLCγ1-Tyr₇₈₃ in 1hr anti-CD3/anti-CD28–stimulated cryopreserved PBMCs obtained immediately predose or after 8 days of receiving ibrutinib therapy for CLL (n = 11). A minimum of 400 000 events were collected. Graph displays the overall percent of live CD3⁺CD4⁺pPLCγ1⁻Tyr783⁺ events in each sample. Error bars represent SEM. (H) Calcium flux analysis of ibrutinib (n = 8), vehicle (n = 24), or BAPTA-AM (n = 8) pretreated Jurkat cells after TCR stimulation by anti-CD3. Area under the curve (AUC) is presented for each dataset in the center. All data were normalized to baseline and BAPTA-treated fluorescent averages. Time points depicted on horizontal axis are relative to stimulation with anti-CD3. (I) AUC for calcium flux of various concentrations of ibrutinib. Each symbol indicates a single replicate experiment. Statistical analysis represented on graph is relative to DMSO treatment. PMA/Iono., phorbol 12-myristate 13-acetate and ionomycin; Unstim, unstimulated.