Generation of migratory and proliferating plasmablasts during B-cell depletion. (A) Cytometric detection of HLA-DR^high plasmablasts in peripheral blood samples of 4 patients with RA during B-cell depletion (#1 to #4) at 4 and 6 months, respectively, after a first cycle of RTX therapy and 2 and 4 months, respectively, after a second cycle of RTX treatment, compared with 4 healthy controls (#5 to #8). (B) Kinetics of HLA-DR^high plasmablast and HLA-DR^low plasma cell counts¹³  during peripheral blood CD20⁺ B-cell depletion. Data from 16 healthy controls are shown for comparison. (C) Plasmablasts from 4 healthy controls and 5 patients treated with RTX were tested for in vitro migration toward 300nM CCL28 and 10nM CXCL12, respectively, in 4 independent experiments. Migration toward bovine serum albumin (BSA) was used as a control and yielded 2%-7% migration of antibody-secreting cells. Mean values and SEM are depicted in panels B and C. (D) CD27^high/intracellular Ig^high plasmablasts were analyzed for cell proliferation as detected by Ki-67 expression. Ki-67⁺ expressing plasmablasts were significantly reduced in RTX-treated patients with RA (RA/RTX; 7 samples; open boxes; 3 independent experiments) compared with healthy controls (HCs; 6 samples; gray boxes; P = .0156 by Mann-Whitney test). Mouse (m)IgG1 of irrelevant specificity was used to control Ki-67 staining. Box plots depict minimum, maximum, and median values and upper and lower quartiles.