Complement-dependent, antigen-guided depletion of hybridoma cells by HLA-Fc in vitro. (A) Selective killing of PA2.1 cells and BB7.1 cells by cognate HLA-Fc. PA2.1 cells were untreated (gray) or treated with A2Fc (red), A2Fc-LALAPG (brown), or B7Fc (blue) at 0.01 mg/mL in diluted rabbit serum (left and middle panels); BB7.1 cells were untreated or treated with A2Fc or B7Fc at 0.01 mg/mL in diluted rabbit serum (right panels). After 3 hours of incubation at room temperature, washed cells were stained with 7-AAD and analyzed by flow cytometry to quantify viable cells. Overlaid histograms of 7-AAD staining are shown for untreated and treated cells as indicated; note that the upper left and upper middle panels represent the same condition from 2 independent experiments. (B-D) Dose relationship of HLA-Fc−mediated cytotoxicity on PA2.1 (B-C) and BB7.1 cells (D). Cells were treated with titrated HLA-Fc as indicated in diluted rabbit serum for 3 hours at room temperature, followed by 7-AAD staining and flow cytometry. The percentage of cell survival was normalized to that of untreated cells in each experiment. Data from 5 (B) and 3 (C-D) independent experiments. Circles and error bars represent mean ± SD. Curve fitting was performed by nonlinear regression using concentration vs normalized response and a variable slope model.