Figure 4

Heightened susceptibility to A32-mediated ADCC is intimately dependent on CD4 expression and CD4-Env interactions on target cells. CEM.NKR cells or primary CD4+ T cells were infected with CCR5-tropic NL4.3.ADA.IRES.GFP viruses as mentioned in Figure 1 legend. The ∆Nef∆Vpu-D368A Env (N-U-D368A) virus harbours a mutation within the CD4-binding site of Env protein leading to defective CD4-Env interactions. (A-B) CEM.NKR cells (A) and primary CD4+ T cells (B) were examined by flow cytometry for CD4 expression. The latter was determined based upon MFI values obtained for gated GFP⁺ cells. % CD4 down-regulation was calculated as: (MFI of infected cells / MFI of GFP^- (uninfected) cells) × 100. Shown are average % of CD4 down-regulation of (A) a series of experiments (each dot represents an analysis), or (B) five evaluations with five donors. Error bars indicate SEM. (C-D) CEM.NKR T cells infected with WT, ∆Nef∆Vpu or ∆Nef∆Vpu-D368A virus were evaluated for Env expression using A32 (C) and 2G12 (D) Abs as detailed in Figure 1 legend. Env staining was determined based upon the MFI values obtained for gated GFP⁺ cells. Calculations of fold increase in Env staining were as described in Figure 1 legend. (E) CEM.NKR T cells were evaluated for their susceptibility to A32-mediated ADCC as described in Figure 2 legend. Shown are average net cell lyses from four infections. Cell killing was done using PBMC from nine donors as effectors, with each dot representing an individual. Statistical analysis of data was done using paired Student’s t-tests.