Engagement of FcγRIV is critical to the blocking of FcγRIII by 9E9. (A) Representative histograms showing detectable FcγRIV (i) or FcγRIII (ii) on neutrophils isolated from mice in the adoptive transfer studies using either 9E9 or de-gly9E9. Splenocytes were incubated with anti-mLy-6G (PEcy7), Ly6C (PerCP), CD11b (PE), F4/80 (APC), and anti-mFcγR (FITC). Antibodies to detect FcγRIII and FcγRIV were AT152-4 F(ab′)2 and 9E9, respectively. Samples were opsonized for 30 minutes on ice before washing, RBC lysis, and analysis on a Becton Dickinson FACSDiva II flow cytometer. (B) WT or FcγRIV−/− C57BL/6 mice were injected IP with either 9E9 or de-gly9E9 (50 µg). Three to 4 hours later, splenocytes were harvested and the level of detectable FcγRs measured on myeloid subsets, (i) macrophages, (ii) monocytes, and (iii) neutrophils, as in panel A. (C) Schematic diagram of the proposed mechanism by which 9E9 blocks FcγRIII (N = 3). APC, allophycocyanin; FITC, fluorescein isothiocyanate; IP, intraperitoneally; irr, irrelevant control; KO, knockout; MFI, mean fluorescence intensity; NT, nontreated; PE, phycoerythrin; PerCP, peridinin chlorophyll; RBC, red blood cell.
