Effect of forced AF1q expression on T-cell development in humanized mice. (A-B) CD34⁺ HPCs (1.5 × 10⁵ cells) exposed to the AF1q, A2M, or empty vectors were injected intravenously into irradiated NSG mice, and the BM, spleen, and thymus were harvested 8 weeks later. Single-cell suspensions were analyzed by multicolor flow cytometry to determine the extent of CD45⁺GFP⁺ chimerism and for phenotyping the cells. Percentages of GFP⁺ cells among CD45⁺ human cells present in the BM (A) or the thymus (B) of individual recipient mice are shown (transduction efficiency: 14% ± 2%; 8 mice per condition; 2 experiments). Median percentages are indicated by a horizontal bar. (C) Effect of AF1q or A2M on thymus colonization. The efficiency of thymus engraftment was calculated as the ratio of GFP⁺CD45⁺ cell percentages detected in the thymus and BM of individual mice. Statistically significant differences (P < .05; Student t test) between AF1q/A2M and control mice are marked by asterisks. (D-F) Percentages of CD7⁺ and CD10⁺ lymphoid precursors (D) or of multipotent CD7⁻CD10⁻ HPCs (E) among CD34⁺GFP⁺CD45⁺ BM cells. (F) percentages of CD4⁺CD8⁺ and CD4^+/−CD8^+/− single-positive T cells among thymic GFP⁺CD45⁺ cells. Means ± SD percentages are shown. Statistically significant differences are marked as above. (G-H) Flow cytometric analysis of CD45⁺GFP⁺ cells in the BM (H) and thymi (I) of individual mice at week 8 after grafting. Numbers in quadrants indicate the percentages of each population.