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S. Elias et al.
preparations, however, caused significant activation of NK cells, demonstrated by a substantial increase in CD107a levels (Figure 1B). Primary NK cells obtained from eight dif- ferent healthy donors displayed similar effects (Figure 1C). In order to corroborate these findings, we examined the degranulation level of primary activated NK cells incubated with increasing doses of the anti-RhD preparation KamRho, and observed a dose-dependent response (Figure
1D). Similar results were obtained using the anti-RhD preparation Rhophylac (Online Supplementary Figure S1B).
Natural killer cell degranulation is mediated specifically by anti-RhD antibodies
Since both anti-RhD antibody mixtures contain mainly non-specific antibodies, we sought to investigate whether the degranulation effect is mediated specifically by anti-
A
B
CD
Figure 1. Anti-RhD antibodies induce degranulation of natural killer cells. (A) Staining of erythrocytes (red blood cells [RBC]) with a pre-defined RhD expression profile using two commercial anti-RhD preparations: Rhophylac (left two panels) and KamRho (right two panels). Gray filled histograms represent the background staining with the secondary antibody. One representative experiment is shown out of three performed. (B) Degranulation of natural killer (NK) cells incubated with various commercial human polyclonal antibodies. NK-cell degranulation was assessed by calculating the percentage of CD107a+ NK cells out of the total NK cells (analyzed by flow cytometry). The percent of degranulated NK cells is indicated in each scatter plot. (C) The average NK-cell degranulation of eight different NK-cell donors after incubation with different polyclonal antibodies. The NK-cell degranulation level was calculated as in (B) and then normalized to the basal percent of CD107a+ NK cells without antibody (no Ab). ***P<0.001; ANOVA with Tukey’s HSD post-hoc test. Error bars represent standard error. (D) Degranulation of NK cells following incu- bation with increasing doses of KamRho. Error bars represent standard deviation of triplicates. One representative experiment is shown out of three performed.
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haematologica | 2021; 106(7)