8F4 induces cytotoxicity in PR1-presenting cells. (A-D) For CDC,²²  5 × 10⁴ target cells (A-B: T2 cells loaded with PR1 or control peptide; C-D: primary AML or ND cells) in 10-RPMI/HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) were incubated with 8F4 or control antibody in the presence of complement at 37°C for 90 minutes. Cytotoxicity was assessed with the Cyto Tox-Glo Cytotoxicity Assay (Promega). (A) 8F4-mediated lysis of T2 cells is PR1 specific, requires the presence of complement, and depends on 8F4 antibody concentration. (B) At a constant 8F4 concentration (10 μg/mL), CDC depends on the PR1 concentration. T2 cells were loaded with increasing amounts of PR1 or pp65 control peptide. (C) 8F4 induces CDC of HLA-A2⁺ cells from AML1 and AML5, but not HLA-A2- cells from AML6 or PBMCs from an HLA-A2⁺ normal donor (ND4). *P = .0019 AML5 compared with ND4; **P < .0001 AML1 compared with ND4. (D) CDC of leukemia cells from AML1 depends on 8F4 concentration. Mouse IgG2a (isotype control) and pooled human intravenous immunoglobulin were compared at the same concentration as 8F4. (E) 8F4 induces lysis of PR1-loaded T2 cells by ADCC. Target T2 cells were loaded with PR1 or control peptide. Fresh PBMCs from a healthy donor were activated with IL-2 (200 IU/mL) for 2 days and used as effector cells at an E:T ratio of 40:1. Cells were mixed and incubated for 15 hours at 37°C with or without 8F4 or control antibody BB7.2 (50 μg/mL). (A-E) Specific lysis from representative experiments is shown as mean ± SEM from 3 replicates. The negative values for specific lysis are due to background luminescence of cells in the presence of complement likely caused by the enzymatic cleavage of substrate by complement in the absence of an antibody-coated target.¹¹