nef WT-transduced CD34⁺ progenitors show a strongly impaired T-cell development in FTOC. Bivariate dot plots of flow cytometric measurement of Nef^– (control) and Nef⁺ (NL4-3, LAI, NA-7) transduced cells. (A) CD8β-PE, CD4-APC versus EGFP expression of transduced CD34⁺ thymocytes at day 22 of FTOC. (B) CD8β-PE versus CD4-APC expression from thymocytes shown in panel A, gated on EGFP⁺ cells. Quadrants were set to include 99% of cells stained with isotypic controls and EGFP^– cells in lower left quadrant. Values indicate percentage (rounded off to a whole number) of cells present in corresponding quadrants. (C) Filled histograms show the CD3-APC expression profile of EGFP⁺ thymocytes. For reference, the staining profile of the control is overlaid (open histogram). Gates are placed arbitrarily, and the numbers indicate the percentage of cells within the respective gates, although the amount of CD3^high cells is often very low (< 100 events). (D) Thymic generation ratio was calculated as indicated in “Materials and methods.” The figure shows mean values of the thymic generation ratio and their standard deviations calculated from the data generated in at least 3 independent experiments. An asterisk (*) over a bar indicates a statistically significant difference between the experimental construct and control data sets (P < .05).