Figure 5
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.

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.

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