Figure 4

Residues important for PAK2-association are important for enhancing T cell activation when Nef is expressed alone. HXB2 pseudotyped lentiviral particles containing pHAGE-EF1α-Nef-IRES-zsGreen genomes were used to transduce freshly isolated PBMC. Three days post-transduction, the cells were stimulated with PHA-P (1 μg/ml) in the presence of 10 U/ml IL-2 for an additional 3 days. Replicate cultures contained 10 U/ml IL-2 alone. All cultures were then stained with α-CD3-PE-Cy5.5, CD8-PE, and CD25-APC-Cy7. Cell surface markers and zsGreen expression were monitored by flow cytometry. A. CD3 and CD8 expression define CD8 T cell (upper right quadrant) and CD4 T cell (lower right quadrant) populations. CD8 T cells were analyzed directly for CD25 expression. CD4 T cells were analyzed for zsGreen expression in B. B. zsGreen expression analysis of CD4 T cells. Transduced (Nef+ zsGreen+) and untransduced (Nef- zsGreen-) populations were defined for further analysis in C. Open histogram is the mock transduction. Shaded histogram is the sample transduced with the empty vector. C. CD25 expression in indicated populations. Total CD8 T cell population is shown in the upper panel. Nef+ (zsGreen+) populations are shown in the middle panel. Nef- (zsGreen-) populations are shown in the bottom panel. Percent CD25+ and CD25 MFI of the entire population is shown above the histogram. Representative plots for three samples are shown. D. The average % CD25 positive or CD25 MFI ± standard deviation (SD) of triplicate cultures is shown for cultures with 10 U/ml IL-2 alone (left panel) or 1 μg/ml PHA-P with 10 U/ml IL-2. *p < 0.05 ** and p < 0.005 (Student's t-test).