Figure 3. CD57 Positive NK cells from Control Donors Possess a Stronger Dual-functional Response Against Heterologous ADCC Targets Coated with HIV-plasma and Autologous HIV-1 Infected CD4⁺ Primary T Cells Coated with HIV-specific BNABs.

(A-B) Composite analysis of the ADCC degranulation and cytokine response of 10 control donors over time. PBMC were incubated at a 5:1 effector to target cell ratio for three hours with gp120-coated CEM NK^res cells along with plasma from an Elite Controller PLWH reference sample (ADCC target) and the percentage of CD57^pos gated and CD57^neg gated CD56^dim/CD3⁻ NK cells staining positive for CD107a degranulation (A) and IFN-gamma production (B) was determined every hour by flow cytometry (n=10). Statistical analyses of two groups was performed at each timepoint using a paired, non-parametric Wilcoxon Signed-Rank Test with a two-tailed p-value. Individual samples for ADCC assay were processed on different days in individual experiments for functional analysis. (C) gp120 envelope staining (y-axis) and intra-cellular p24 capsid staining (x-axis) of uninfected or HIV-1 NL4-3 infected CD4⁺ primary T cells from a representative control donor incubated in the presence or absence of HIV envelope-specific BNABs 10–1074 and 3BNC117 and a fluorescently conjugated secondary antibody specific for the Fc portion of human IgG. The data is representative of 3 independent experiments with one sample per experiment. (D) Analysis of dual-functional NK cell response from a representative control uninfected donor against autologous HIV-1 infected ADCC targets as measured by CD107a Degranulation and IFN-gamma production. PBMC were incubated at a 2:1 effector to target cell ratio for three hours with HIV-1 NL4-3 infected autologous CD4⁺ primary T cells coated with or without the HIV envelope-specific BNABs 10–1074 and 3BNC117. The data is representative of 3 independent experiments with one sample per experiment and shown in a three-parameter density plot with CD107a degranulation on the X-axis, CD57 on the Y-axis, and IFN-gamma production super-imposed on top as red dots. The frequency of CD56^dim/CD3⁻ gated NK cells staining positive for CD107a (black) and IFN-gamma (red) is shown in the upper right-hand quadrant and the gates were determined based upon the background from the No Target control condition.