c4 complement activation  (Assaypro)

Journal: Clinical Cancer Research

Article Title: KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes

doi: 10.1158/1078-0432.CCR-23-3504

Figure Lengend Snippet: Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and complement-mediated cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.

Article Snippet: Human male AB plasma as a source of human complement was obtained from Sigma-Aldrich.

Techniques: Flow Cytometry Complement-Mediated Cytotoxicity Assay, Incubation, Positive Control, Flow Cytometry, Labeling, Derivative Assay, Reporter Assay, Expressing, CDC Assay