FcγRIIc expression and function. (A) Distribution of FcγRIIc mRNA expression in leucocytes. T cells CD3⁺, monocytes, and neutrophils CD14⁺, B cells CD19⁺, NK cells CD 56⁺. (B) FcγRII expression on NK cells is limited to the FCGR2C-ORF genotype. Blood cells were incubated with CD56 and CD32. Lymphocytes were gated on the basis of their forward scatter/side scatter pattern. NK-cell population was determined as CD56-positive lymphocytes. (C) FcγRIIc expression on NK cells is modulated by IL-15 but not by IL-2. PBMCs were isolated and cultured as described in “In vitro activation of NK cells.” Expression of CD32 on CD56⁺ NK cells was measured by flow cytometry with CD32, at the indicated time-points. There was a significant higher expression on days 2 and 4 on the IL-15–stimulated NK cells (P = .01, n = 5). Data are expressed as mean plus or minus SEM. (D) FcγRIIc mRNA is strongly up-regulated by GM-CSF on cells of FCGR2C-ORF donors. Neutrophils and PBMCs were isolated and cultured for 4 hours with the indicated stimuli as described in “In vitro activation of neutrophils and PBMCs.” FcγRIIc mRNA was measured by quantitative RT-PCR. GM-CSF strongly up-regulated FcγRIIc mRNA in neutrophils and, to a lesser extent, in PBMCs of FCGR2C-ORF genotyped donors, but not in FCGR2C-Stop donors (P = .0001 and P = .01, respectively; n = 5-8). Data are expressed as mean plus or minus SEM. (E) rADCC. PBLs were isolated as described in “Isolation of neutrophils and PBMCs,” and FcγRIIc functionality was assessed by rADCC. Cells from both FCGR2C-Stop and FCGR2C-ORF genotyped donors killed anti-FcγRIII–coated targets with similar kinetics (left panels). In contrast, only cells from FCGR2C-ORF genotyped donors were capable of killing anti-FcγRII–coated targets (right panels) (n = 4). Data are expressed as mean plus or minus SEM. (F) rADCC with stimulated cells. PBLs were obtained and subsequently cultured for 2 days with or without IL-2 or IL-15. Thereafter, the cells were harvested and used in a rADCC. Cells from both FCGR2C-Stop and FCGR2C-ORF genotyped donors killed anti-FcγRIII–coated targets (left panel). In both cases IL-15 and, to a lesser extent, IL-2 enhanced specific lysis of the anti-FcγRIII–coated targets (n = 3). In contrast, only cells from FCGR2C-ORF genotyped donors were capable of killing anti-FcγRII–coated targets (right panel). Here again, IL-15 strongly enhanced the specific cell lysis (n = 3). Data are expressed as mean plus or minus SEM.