PCI-32765 induces selective cytotoxicity in B cells compared with T cells, but alters activation induced T-cell cytokine production. (A) CD19⁺ cells from CLL patient cells (N > 60) and CD19⁺ cells from normal B cells (N = 10) were incubated with 10μM PCI-32765 for 48 hours. Viability was determined by annexin-V/PI flow cytometry, and was calculated relative to time-matched untreated controls. Dark lines represent averages. (B) CD3⁺ T cells (N = 3) and CD19⁺ B cells (N = 3) from normal volunteers were examined for BTK expression by immunoblot. (C) CD3⁺ T cells (N = 10) from normal volunteers were incubated with or without increasing concentrations of PCI-32765 (0.1μM-10μM) for 48 hours. Viability was determined by annexin-V/PI flow cytometry, and was calculated relative to time-matched untreated controls. (D) CD3⁺ T cells (N = 7) from normal volunteers were incubated with or without increasing concentrations of PCI-32765 (0.1μM-10μM) for 48 hours. Cells were stimulated using an anti-CD3 T-cell activation plate for 24 hours, and IL-6, IL-10, and TNF-α production were measured by ELISA. (E) RNA was extracted and converted to cDNA from CD19⁺ normal B cells and CD3⁺ normal T cells (N = 5; each). RT-PCR analysis was done to determine quantities of BTK mRNA.