Ibrutinib can limit Th2 activation, thereby selectively promoting Th1 expansion and skewing Th1/Th2 cytokines in human CLL patients and IgG subisotypes in TCL1 leukemic mice. (A) Intracellular staining analysis of IFN-γ in 5-day anti-CD3/anti-CD28–stimulated CD4⁺ T-cell cultures pretreated with ibrutinib or vehicle. Experiment was repeated 5 times; isotype staining control is provided. (B) Immunoblot analysis of JunB (top) and T-bet (bottom) levels in bulk CD4⁺ cultures pretreated with ibrutinib and anti-CD3/anti-CD28 stimulated (or unstimulated) for 3 days in vitro. Actin is used as loading control. Densitometry analyses are normalized to the DMSO-treated (0 µM) sample. (C) Normalized intracellular staining analysis of IL-4 (open bars n = 6) and IFN-γ (closed bars n = 9) CD4⁺ cells derived from CLL patients pretreated with ibrutinib and stimulated with anti-CD3/anti-CD28. Error bars represent SEM. (D) Percent relative alteration in plasma cytokine levels from pretreatment to day 28 of therapy in relapsed refractory CLL patients enrolled in a phase 1 trial of oral ibrutinib. (E) Plasma IgG1 (Th2) and IgG2c (Th1) subisotype analysis of C57BL/6 EµTCL1 mice at 8 months of age after 7 consecutive months of ibrutinib (25 mg/kg/day) (n = 12) or vehicle (n = 13) administration via drinking water. IFNG, interferon γ; Unstim, unstimulated.