Background Rituximab has broad and increasing application in rheumatic diseases

Background Rituximab has broad and increasing application in rheumatic diseases. Fc–receptor CD16 (FcRIIIA). The co-activating receptor CD137 (41BB) was upregulated on a fraction of NK cells. NK cell function was altered in some donors in whom we observed rituximab-dependent reduction in NK cell cytotoxicity towards K562 tumor cells. Conclusions NK cells mediate rituximab-induced B cell depletion. Rituximab induces altered NK cell phenotype and function. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1101-3) contains supplementary material, which is available to authorized users. values have to be interpreted descriptively. Normal distribution was not assumed and therefore non-parametric statistical assessments were used. The MannCWhitney test was used to compare two groups. The Wilcoxon signed rank test was used to compare paired samples. All tests were performed with a significance level of 5?% (confidence interval 95?%). Results Addition of rituximab to PMBCs leads to B cell depletion in the absence of serum Freshly isolated PBMCs from 14 healthy donors were cultured with or without rituximab overnight. In all donors we observed a strong rituximab-mediated reduction in B cell numbers, and no B cells were detectable after rituximab treatment ( 0.55?% of lymphocytes) in 10/14 donors (Fig.?1a). In the first experiments, we used anti-TNF alpha antibody infliximab or IvIgs as unfavorable controls. We discontinued these controls in further experiments, as no effects on either the presence of B cells (Fig.?1a; infliximab, culture in medium without RTX (culture in medium with RTX (apoptotic B cells with positive staining for Annexin-V PE. a, b B cell numbers were reduced after incubation with RTX in 14/14 impartial 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 B cell percentages before and after culture overnight with rituximab (statistically significant, Wilcoxon signed rank test, 10C90th percentile. PBMCs with incomplete B cell depletion after Fmoc-Lys(Me,Boc)-OH incubation with RTX overnight (test, to belong to the same donor. Additive effect on degranulation is usually defined by (CD107a pos. NK cells after culture with therapeutic antibody) -(CD107a pos. NK cells after culture without therapeutic Rabbit polyclonal to HS1BP3 antibody). c CD16 expression on CD107a-positive NK cells. Shown is usually one representative donor. a-c CD107a expression together with CD16 expression has been investigated in healthy individuals (forward scatter The Fc-gamma-receptor CD16 was downregulated on degranulated (CD107a-positive) NK cells, as shown in Fig.?2c. The proportion of CD16bright cells among CD56dim NK cells was decided after culture with or without rituximab in 16 healthy controls. Rituximab led to a significant decrease in CD16bright NK cells (Fig.?2d). The extent of CD16 downregulation varied between donors. We conclude that rituximab induces NK cell degranulation in healthy PBMCs. Similar to published data in tumor models, rituximab induced downregulation of CD16. NK cells and serum cooperate in mediating rituximab-induced B cell depletion To investigate a causal relationship between NK cell degranulation and the depletion of B cells upon rituximab treatment we depleted NK cells from freshly isolated PBMCs using anti-CD56 and anti-CD16 antibodies and magnetic beads. The remaining PBMCs were cultured overnight with or without rituximab and with or without autologous human Fmoc-Lys(Me,Boc)-OH serum. Rituximab-induced B cell depletion was abrogated if NK cells were depleted from the PBMCs (Fig.?3a, heat inactivated. The next day PBMCs were stained and Fmoc-Lys(Me,Boc)-OH analyzed as described in Fig.?1. a All samples were cultured without human serum. b NK cells were depleted in all samples. c All samples were treated with RTX over night. a-c Data from the same experiment and donor with incomplete NK cell depletion; the complete experiment with all negative controls.