Impact of intrathymic injection of an HA-encoding lentiviral vector in TCR-HA transgenic mice. (A) Representative staining showing CD4/CD8 expression in total cells of the thymus (TOTAL) 6 days after intrathymic injection of GFP-expressing (LvGFP) or HA-expressing (LvHA) lentiviral vectors in TCR-HA mice. Absolute numbers of total cells in injected thymus ± SD are indicated above the panels. Gates defining CD4⁺CD8^– single-positive (CD4SP) and CD4⁺CD8⁺ double-positive thymocytes are shown. Numbers indicated on the dot plot represent frequencies of DP cells and are representative of 4 of 7 LvHA-injected mice obtained in 3 independent experiments. (B) Expression of the HA-specific (6.5) TCR and of CD25 in gated thymic CD4⁺CD8^– cells (CD4SP) in the same conditions. Indicated are the frequencies of cells falling within each quadrant. Results are representative of 3 independent experiments. (C, left) Representative profile of foxp3 expression in 6.5⁺CD25^– CD4SP cells (gray histogram) and 6.5⁺CD25⁺ CD4SP cells of an LvGFP-injected (dotted line) or an LvHA-injected (solid line) mouse. (C, right) Representative CD24 expression within 6.5^–CD25⁺ CD4SP cells (gray histogram) and 6.5⁺CD25⁺ CD4SP cells of an LvGFP-injected (dotted line) or an LvHA-injected (solid line) mouse. Similar profiles were seen in 3 other mice. Total thymus was stained with CD4, CD8, 6.5, CD25, and foxp3 or CD24 mAbs.