The effect of Nef on HIV-1–specific CTL antiviral activity is unrelated to HLA-I restriction of the epitope. The impact of Nef was compared across presenting HLA-I types. (A) Nef-effect ratios plotted for the indicated HLA-I types. Each dot represents one epitope; the horizontal bar represents the mean. There was no significant difference between groups (Kruskal-Wallis test). (B) A flow cytometric approach was used to measure Nef-mediated down-regulation of HLA-I A*02 and HLA-I B*57 on acutely HIV-1–infected cells. A representative experiment shows the analysis of HLA-A*02 down-regulation after gating on infected cells (positive for the HSA reporter) and determination of MFI of A*02 staining. The relative expression of A*02 on cells infected with wild-type Nef virus (gray dotted histogram) was then calculated as a fraction of MFI compared with Nef-M20A virus (black solid histogram) after subtraction of background MFI (from an isotype control). (C) Expression of cell-surface CD4 and the HSA reporter over time is demonstrated after acute infection of primary CD4⁺ T lymphocytes from an HIV-1–infected donor with both A*02 and B*57 (subject number 00036, a slow progressor not on treatment). (D) The infected (HSA^high+ and CD4^dim/−, percentages shown) primary CD4⁺ T lymphocytes were gated and analyzed for A*02 (top panel) and B*57 (bottom panel) expression. HLA-I expression is plotted for HIV-1 with wild-type Nef (gray shaded histograms) versus Nef-M20A (black histograms). (E) Gompertz plots of Nef-mediated down-regulation of A*02 versus B*57 are shown for the laboratory T-cell line 1CC4.14 (top panel) or primary CD4⁺ T lymphocytes* (bottom panel). In the top panel, the estimates for time to 10% A*02 versus B*57 down-regulation by Nef are 22.1 and 19.6 hours after infection, respectively. The slopes of the fitted curves at 10% down-regulation of A*02 versus B*57 are −0.016 and −0.015, respectively. The maximum levels of down-regulation of A*02 versus B*57 are estimated to be 50% and 40%, respectively. These parameters are not significantly different for A*02 versus B*57. In the bottom panel, the estimates for time to 10% A*02 versus B*57 down-regulation by Nef are 18.1 and 18.0 hours after infection, respectively. The slopes of the fitted curves at 10% down-regulation of A*02 versus B*57 are −0.023 and −0.021, respectively. The maximum levels of down-regulation of A*02 versus B*57 are estimated to be 60% and 50%, respectively. These parameters are not significantly different for A*02 versus B*57. The efficiency of infection was higher (approximately 40%-50% vs the 10% shown in Figure 3C) and Nef-mediated HLA-I down-regulation occurred earlier (approximately 12 hours after infection) in virus-infected primary CD4⁺ T lymphocytes of HIV-1–uninfected donors (data not shown).