WHIM mutation of CXCR4 increases apoptosis of transduced human CD45⁺ cells in the BM of NOD/SCID mice at 6 weeks after transplantation. BM of mice that received transplants of GFP-only cells, or GFP plus either wt CXCR4– or mutated CXCR4–transduced cells, and naive nontransduced human CD34⁺ cells were evaluated at 6 weeks. The BM containing both GFP⁺ and GFP⁻ cells was stained with anti-CD45–APC (to allow gating on only human leukocytes), annexin V–PE (as a measure of the membrane lipid changes characteristic of apoptosis), and 7AAD (to exclude dead cells), and analyzed using dual-laser 4-color flow cytometry. Shown in the bar graph for each group are either the GFP⁻ (□) or GFP⁺ (⊡) annexin V⁺ live human leukocytes. There was no statistical difference in rates of in vivo apoptosis between any of the groups except for the GFP⁺ mutated CXCR4–transduced group, which was statistically higher than all other groups (n = 5 for naive, GFP-only, and wt CXCR4 groups, and n = 7 for mutated CXCR4; *P < .01 for all comparisons). The slight trend to higher apoptosis rates in the GFP⁻ mutated CXCR4 group compared with the other GFP⁻ cell groups may be due to the fact that the GFP expression from the mutated CXCR4-IRES-GFP construct may be so low in some transduced cells that they contribute to overall slightly higher apoptosis in the supposedly GFP⁻ population.