Analysis of AID expression in CLL cells treated in vitro with ibrutinib. (A) MEC-1 cell line was cultured in the presence of ibrutinib at 0.1 and 1 µM for 24 and 48 hours, or in DMSO as control. AID expression at the protein level was evaluated by immunoblotting and flow cytometry techniques. Shown are representative immunoblots (upper graphic) and mean fluorescence cytometry histograms (lower panels), where increasing AID levels are detected for all treated conditions. Proliferation measured by Ki-67 expression was performed at baseline and after 24 and 48 hours of ibrutinib treatment (right graphic). (B) PB CLL cells were incubated in the presence of ibrutinib at 0.3 and 1 µM. DMSO was used as basal control, and CD40L⁺IL-4 stimulation to induce AID expression was used as positive control (n = 5). AICDA mRNA and protein expression was evaluated at 24 and 48 hours, as indicated in the x-axis. Shown are the results from the whole cohort of patients studied by quantitative qPCR (left graphic) and the immunoblot obtained when studying the 2 patients colored in red and blue (right graphic). AID increase was detected only in the CD40L⁺IL-4 activated condition for both mRNA and protein determinations. (C) PBMC from 5 patients with CLL were incubated with CD40⁺IL-4 or CD40⁺IL-4⁺ibrutinib 1 μM, and then cultured for 5 days. Proliferation was evaluated by Ki-67 expression, and viability of cells was analyzed by propidium iodide as a life/death marker (upper graphics). As previously described by Slinger et al,⁴⁴  maximum inhibition of proliferation was achieved after 24 hours. AICDA mRNA levels and AID protein were tested by qPCR and immunoblotting, respectively, at the different times and conditions (lower graphics). Significance was calculated comparing stimulated condition (CD40⁺IL-4) with stimulated condition + ibrutinib by 2-tailed, paired t-test (*P < .05; **P < .01). (D) PBMC from 3 patients with CLL were incubated in the presence of CD40L⁺IL-4 in the same conditions as mentioned here. Samples without treatment (RPMI+FBS 10%+DMSO -ctrol-), samples treated with CD40L⁺IL-4 and samples treated with CD40L⁺IL-4⁺1 μM of ibrutinib were tested. Glyceraldehyde-3-phosphate dehydrogenase (load control); AID; STAT6 and p-STAT6^-Tyr641 proteins expression were evaluated at day 5. Immunoblots depict the effect of ibrutinib on AID expression and STAT6 phosphorylation. Whereas AID and p-STAT6^-Tyr641 decrease during ibrutinib treatment, the nonphosphorylated form of STAT-6 remains unchanged. The same proteins were analyzed in the MEC-1 cell line after incubation with ibrutinib (ibru) or DMSO as control (Ctrol). As depicted, p-STAT6^-Tyr641 was undetectable in neither condition, whereas the nonphosphorylated STAT-6 form remains unchanged. As previously described,²⁸  AID expression increased when MEC1 cells are cultured in presence of ibrutinib.