Ibrutinib suppresses CLL-expressed CTLA4. (A) CLL cells were treated with 1 μM ibrutinib or acalabrutinib for 1 hour before washing and coculture with activated T cells. CTLA4 expression on CLL cells was measured 48 hours later by surface and intracellular flow cytometry (n = 10 ibrutinib, 7-8 acalabrutinib). (B) CLL cells were treated with 1 μM ibrutinib or acalabrutinib for 1 hour before washing. CTLA4 mRNA levels were measured 48 hours later by qPCR (n = 18 ibrutinib, 14 acalabrutinib). (C) T cells were treated with 1 μM ibrutinib or acalabrutinib for 1 hour before washing and stimulation with 10 μg/mL anti-CD3 adsorbed onto the plate surface and 1 μg/mL anti-CD28. CTLA4 expression was measured by surface and intracellular flow cytometry after 48 hours of stimulation (n = 5). (D) Matched primary CLL leukemia samples were obtained from patients before treatment and after 5 months of ibrutinib treatment. CLL cells were cocultured with allogeneic activated T cells. CTLA4 expression was measured after 48 hours using surface and intracellular flow cytometry (n = 5). (E) Matched primary CLL leukemia samples were obtained from patients before treatment and after 5 months of acalabrutinib treatment. CLL cells were cocultured with allogeneic activated T cells. CTLA4 expression was measured after 48 hours using surface and intracellular flow cytometry (n = 3). Graphs show mean ± standard error of the mean. *P < .05 by mixed effect modeling (A-C) or paired t-test (D-E). ns, not significant.