Normal binding and endocytosis of IgG-immune complexes (IgG-ICs) by GPI-anchor-deficient DCs. (A) Staining of AMs (left panels) or DCs (right panels) with anti-FcγRII/III mAb 2.4G2 (top panels) or monomeric mouse IgG2a as a marker for FcγRI (bottom panels). (B) Staining of DCs with IgG-ICs; mean fluorescence intensity for WT cells was 111 and for KO cells was 165. (A-B) Shown are fluorescence intensity plots of cells from littermate control (thin lines) or LysMCre/Pig-a^flox mice (bold lines), and antibody controls (dotted lines). (C) Blocking of IgG-IC binding by 2.4G2. Control DCs were preincubated with medium (left), 2.4G2 (25 μg/mL; middle), or isotype-matched control mAb anti-CD11b (M1/70; 25 μg/mL; right) for 30 minutes on ice, and next stained with IgG-ICs (solid line) or medium (dotted line). (D) Endocytosis of IgG-ICs. Control (WT, top panels) or GPI-anchor-deficient (KO, bottom panels) DCs were incubated on ice with FITC-containing IgGICs, washed, and kept on ice (4°C; left panels) or incubated at 37°C for 20 minutes (middle panels) or 30 minutes (right panels). Percentages of cells having endocytosed IgG-ICs (ie, gated cells exhibiting decreased extracellular IgG-IC staining [FL2] and unchanged FL1 signal) are shown in the upper right quadrants.