Intrathymic development of transduced ISP4⁺progenitor cells in vitro. Bivariate dot plots of flow cytometric measurement of Nef⁻ transduced cells (top) and Nef⁺ transduced cells (bottom). (A) CD4-PE versus EGFP expression of transduced ISP4⁺ thymocytes, 1 day after transduction. (B) CD4-APCy, CD8β-PE, and CD3-APCy versus EGFP expression of thymocytes from day 7 FTOC started with cells shown in (A). (C) CD8β-PE and CD3-PE versus CD4-APCy expression from thymocytes shown in (B), gated on EGFP⁺ cells (> 20 arbitrary fluorescence units). (D) CD8β-PE versus CD4-APCy expression of EGFP⁺ thymocytes from day 21 FTOC started with cells shown in (A). Quadrants were set to include 99% of cells stained with isotypic controls and EGFP⁻ cells in lower left quadrant, except arbitrarily positioned to delimit DP cells in CD8β versus CD4 stainings. Figures indicate percentage of cells present in corresponding quadrants.