Immune characterization of peripheral B cells from patients following initiation and discontinuation of ibrutinib. Multiparameter flow-cytometric analysis was completed with B cells from PBMC samples from 10 patients with relapsed CLL post–allo-HCT treated with ibrutinib at Stanford. Data are shown for 3 clinically relevant B-cell subsets: CLL, memory B cells, and pre-GC–activated B cells. (A) Blood samples from a representative patient that were collected preibrutinib and 3 months following ibrutinib initiation were analyzed by sequential gating as shown. All of the patients’ samples were analyzed with the same sequential gating depicted. Green, blue, and red gates mark the various cell populations determined to be positive compared with an FMO (fluorescence minus 1). These colors correspond to the line graphs in panels B and C, as well as to the absolute cell numbers listed in supplemental Table 5. (B) The change following ibrutinib initiation for each clinically relevant B-cell subset is graphed as the ratio of the absolute number of cells detected postibrutinib to the absolute number of cells in the same subset preibrutinib. Ratios are plotted as mean with standard error of the mean (SEM) (n = 10 at preibrutinib, and n = 5 at 1, 3, 6, and 12 months postibrutinib initiation). (C) The change following ibrutinib discontinuation for each clinically relevant B-cell subset is graphed as the ratio of the absolute number of cells detected postibrutinib to the absolute number of cells in the same subset preibrutinib. Ratios are plotted as mean with SEM (n = 3 at all time points).