VIVIT peptide inhibitor blocks the BCR/NFAT-dependent pathway in responding CLL B cells. (A) NFAT2 immunofluorescence staining of CLL B cells from 1 representative R case (n = 3) stimulated or not in the presence or not of NFAT inhibitor 11-R VIVIT (5μM for 24 hours). Scale bar corresponds to 5 μm. (B) Quantitative RT-PCR analysis of CD23 transcript expression levels in freshly isolated B cells from R cases (n = 5) on stimulation with anti-IgM (10 μg/mL) in the presence or not of 11-R VIVIT (5μM). CD23 mRNA expression was normalized on ABL transcript expression in each tested sample, and results were graphed. Cell surface expression of CD23 protein (C) or CD71 protein (D) was determined using flow cytometry analysis on CLL cells from 7 (C) or 8 (D) R cases stimulated with anti-IgM (10 μg/mL) in presence of increasing concentrations (0, 2, and 5μM) of 11-R VIVIT. Levels are indicated as fold increase on stimulation. (E) Fold increase of metabolic activity (% MTS) was determined on 48-hour BCR stimulation for 13 NR and 12 R cases. B lymphocytes were incubated in the presence or not of 11-R VIVIT (5μM).