BaEV-LV GPs confer high-level transduction of human immature thymocyte populations. (A) Schematic representation of T-cell development in the thymus. Phenotypic surface markers are indicated for each differentiation stage. HSCs (CD34⁺CD1a^–) are migrating from the BM to the thymus. The first stages are characterized by the lack of expression of CD8 and CD4 markers (CD8^–CD4^– DN stage). The DN cells subsequently gain expression of preT and develop into CD4 immature single-positive (ISP) cells. The appearance of the CD8 surface marker together with CD4 and maturation of the TCR is called the DP stage of thymocyte development. Upon selection in the thymus, DP thymocytes develop into CD8⁺CD4^– (SP8) and CD8^–CD4⁺ (SP4) thymocytes. (B) Freshly isolated human thymocytes were transduced in the presence of IL-7 on RetroNectin-coated culture plates with the indicated LV pseudotypes at an MOI of 10, except for VSV-G LVs for which an MOI of 50 was applied. Four days after transduction, the thymocytes were stained for human surface markers (CD3, CD4, and CD8), and the percentage of GFP⁺ cells in the different thymocyte subpopulations was analyzed by FACS (mean ± SD; n = 5; DN, CD4^–CD8^–; SP-CD4, CD4⁺CD3⁺CD8^–; SP-CD8, CD4^–CD3⁺CD8⁺; DP, CD4⁺CD8⁺; ISP, CD4⁺CD3^–CD8^–). For BaEV- and RDTR-LVs, the percentage of GFP⁺, DN, and ISP is significantly higher than the percentage of DP, SP4, and SP8. (C) Freshly isolated human thymocytes were transduced as in panel B. Four days after transduction, the thymocytes were stained for human surface markers (CD34, CD7, CD1a, and CD5), and the percentage of GFP⁺ cells in the different thymocyte subpopulations was determined (mean ± SD; n = 4). For BaEV-LVs, the percentage of GFP⁺ ETPs and proT cells was significantly higher than that of preT cells. Gating strategies for FACS analysis are shown in supplemental Figure 5A. P < .01.