Figure 1

Reduction of thymic output is a conserved property of primate lentiviral Nef proteins. Flow cytometric analysis of FTOC initiated with transduced CD34⁺ thymic progenitors. (A) Gating strategy: R1 and R2 to gate on living cells of human origin (not staining with anti-mouse CD45 Cy-Chrome). For indicated plots in panel (B) eGFP^- and eGFP⁺ cells were gated using the histogram gates shown (R3 and R4). (B) Dot plots show control eGFP (left) or Nef NA7-IRES-eGFP transduced cultures (right) stained as indicated gated on eGFP^- cells (upper plots) and eGFP⁺ cells (middle plots); and eGFP vs. CD4 or CD3 gated on live human cells (lower plots). Quadrants were set to include 99% of non-transduced cells stained with isotypic controls in lower left quadrant. Numbers in plots indicate mean fluorescent intensity (MFI): for upper and middle plots of the marker on the adjacent axis for the total population (all quadrants), for the lower plots MFI’s for the marker on the Y-axis for all eGFP^- cells (upper and lower left quadrants) or all eGFP⁺ cells (upper and lower right quadrants) are indicated. (C) Representative examples of eGFP vs. CD4 and eGFP vs. CD3 stainings, gated on live human cells of Nef transduced cultures, as indicated. Quadrants and numbers in plots as in lower plots panel B, if sufficient events available. (D) T-cell generation ratio (TGR) calculated from Nef transduced cultures, as indicated. Bars represent averages of at least 3 independent experiments, error bars indicate standard deviation. Nef proteins that do not downregulate CD3 are shown in dark grey (in red functional defective mutant HIV-1 O8), those that do in light grey (in blue functional mutant HIV-2 171). Difference compared to control eGFP (white bar) was tested with Kruskal-Wallis with Dunn’s correction for multiple testing, *p < 0.05, **p < 0.005.