Fig. 3

CD4mc-dependent ADCC killing by immunized monkey sera. Primary CD4⁺ T lymphocytes from three healthy HIV-1-negative individuals were activated³⁷, and then infected with SHIV-C5 virus for 48 h before performing the antibody binding and ADCC assays. a Surface staining of infected cells was done with 1:250 dilutions of sera collected from the monkeys on the day of SHIV-C5 Challenge 1. Serum was added in the presence of 50 μM BNM-III-170 or an equivalent volume of DMSO (negative control) at 37 °C for 1 h. Goat anti-human antibody (AF-647) was used as the secondary antibody. The Mean Fluorescence Intensity (MFI) of AF-647 staining on Aqua Vivid (Invitrogen)-negative cells (living cells) is shown, relative to that of mock-infected cells. See Supplementary Fig. 9 for an example of the FACS gating strategy. b To evaluate ADCC, SHIV-C5-infected primary CD4⁺ T lymphocytes were incubated with autologous peripheral blood mononuclear cells (Effector:Target ratio of 10:1) from the same donor for 4–6 h at 37 °C in the presence of a 1:250 dilution of monkey serum and 50 μM BNM-III-170 or equivalent volume of DMSO. The serum was collected from the monkeys on the day of SHIV-C5 Challenge 1. Infected cells were identified by staining with an Alexa Fluor (AF)-488-conjugated antibody against SIV p27 CA. The percentage of ADCC killing was determined with the following formula: [(% of p27 ⁺ cells in Targets plus Effectors)−(% of p27 ⁺ cells in Targets plus Effectors plus serum)]/(% of p27 ⁺ cells in Targets) by gating infected (p27⁺) live (Aqua Vivid-negative) target cells, as described³⁷. c To evaluate the effect of the A32 Fab on the susceptibility of SHIV-C5-infected cells to ADCC mediated by the serum samples, 5 μg/ml of A32 Fab was added to the ADCC assay described in b. The results shown represent the average of three experiments. Statistical significance was tested by One-way ANOVA. (****P < 0.0001; ns, not significant)