Fig. 1

Addition of rituximab to peripheral blood mononuclear cells (PMBCs) leads to B cell depletion in the absence of serum. a PBMCs were isolated by density gradient centrifugation, left untreated overnight, and cultured over the second night in medium supplemented with heat-inactivated fetal calf serum without any therapeutic antibody (w/o, left panel), with 10 μg/ml anti-CD20 antibody rituximab (RTX) (middle) or with 10 μg/ml anti-TNF alpha antibody infliximab (right panel). After that, PBMCs were stained with a mixture of lineage antibodies (anti-CD3 PE-Dazzle, anti-CD56 Bv421, anti-CD19 FITC) and analyzed by flow cytometry. B cells were identified as CD3-CD19+ cells in the lymphocyte gate. b PBMCs were treated as described (a). After washing PBMCs were re-diluted in Annexin-V (AxV) buffer supplemented with PE-labeled Annexin-V and analyzed by flow cytometry. B cells were identified as CD3-CD19+ cells in the lymphocyte gate. Upper row culture in medium without RTX (w/o RTX). Lower row culture in medium with RTX (+ RTX). AxV+ B cells apoptotic B cells with positive staining for Annexin-V PE. a, b B cell numbers were reduced after incubation with RTX in 14/14 independent experiments, each performed with PBMCs from different healthy donors. In 10/14 experiments RTX-induced B cell depletion was complete as shown (a); in 4/14 B cell depletion was incomplete as shown (b). Infliximab was used as a negative control in 2/14 experiments. Increased binding of Annexin-V was identified in three donors with incomplete B cell depletion. c Left graph B cell percentages before and after culture overnight with rituximab (statistically significant, Wilcoxon signed rank test, p = 0.001). Right graph, whiskers 10–90th percentile. PBMCs with incomplete B cell depletion after incubation with RTX overnight (incomplete) had significantly lower ratios of natural killer cells to B cells (NK/B) than PBMCSs with complete B cell depletion (complete, <0.55 % of PBMCs) (Mann–Whitney U test, p = 0.03)