Figure 2

Antibody-dependent cellular cytotoxicity assay: development and validation. CEM.NKR cells were infected for 48 h with CCR5-tropic NL4.3.ADA.IRES.GFP ∆Nef∆Vpu and analyzed for their susceptibility to ADCC by PBMC under different testing parameters. Target cells were exposed to either control IgG, A32 or, alternatively, pre-exposed to the A32 Fab prior to the A32 exposure. Env staining was done as described in Figure 1 legend and ADCC was performed as described in Methods. (A) Evaluation of A32 staining specificity using the A32 Fab. (B) Gating strategy to select eFlour670-negative, GFP + target cells by flow cytometry. (C) Determination of net Ab-specific cell lysis using gating strategy depicted in (B). The number shown inside dot plot depicts % of GFP⁺ cells remaining at the end of assay. Percent of cell lysis was calculated as [(% GFP⁺ cells in the absence of effectors -% GFP⁺ cells in the presence of effectors plus control IgG, A32 or A32 Fab + A32)/% GFP⁺ cells in the absence of effectors] × 100. (D) Cell lysis in the absence (A32) or presence of the A32 Fab fragment (A32 Fab + A32). Net Ab-specific cell lysis was obtained following subtraction of % cell lysis in the presence of IgG from that by either A32 or A32 Fab + A32. Each line represents PBMC-mediated lysis from a donor. (E) Effect of varying ET ratios (10 or 30) on net Ab-specific cell lysis using A32 as test Ab and IgG as control. Shown are results from two representative donors. (F) Contributions of different cell subsets to induction of ADCC. Total PBMC or PBMC depleted of monocytes/macrophages (PBMC-Mo) or NK cells (PBMC-NK) were used as effector cells. A32 was used as test Ab and IgG as control. Histograms represent average net A32-specific lysis +/- SD of four experiments with five donors.