Sera from nude mice contain IgG antibodies that bind to melanoma cells and inhibit proliferation of FcγRIIB1-positive melanoma cells in an intracytoplasmic-dependent manner. (a) Day-0 and day-14 sera from nude mice bearing HT144IIB(Cyto^–) or HT144IIB1 tumors, or medium alone (FITC-GAM), were incubated with HT144 cells, and binding of IgG was assessed by flow cytometry. (b) HT144IIB1 (left panel)or HT144IIB(Cyto^–) (right panel) cells (5 × 10³/well) were incubated with the indicated concentrations of IgG isolated from sera of nude mice at day 0 (cross) or bearing day-14 HT144IIB1 (open circles) or HT144IIB(Cyto^–) (open triangles) tumors or with the same volume of control eluates from sera of SCID mice bearing day-14 HT144IIB1 (filled circles) or HT144IIB(Cyto^–) (filled triangles) tumors for 24 hours to FCS-free medium. This was followed by a 24-hour pulse with ³H-thymidine. The percentages of inhibition were estimated with the formula [(1 – cpm of stimulated cells / cpm of unstimulated cells) × 100]. Each point represents results from three independent experiments performed in triplicate (mean ± SD). (c) HT144 (left panel) or HT144IIB1 (right panel) cells were preincubated with AT10, then with biotin-labeled mouse IgG3, and then with FITC-conjugated streptavidin, or were not preincubated. Binding of mouse IgG3 was compared with background binding of FITC-conjugated streptavidin.