WHIM mutation of CXCR4 enhances engraftment of PBSCs in NOD/SCID mice. BM of NOD/SCID mice that received transplants of naive nontransduced PBSCs, PBSCs transduced with GFP only, or GFP plus either wt CXCR4 or mutated CXCR4 were analyzed at 6 weeks by fluorescence-activated cell-sorting (FACS) for the markers indicated on the x-axes. (A) After harvest from mice BM, cells were stained with anti–human CD45–APC or isotype antibody. Shown is a representative analysis from 1 mouse in each group, where the numerical averages for the groups are provided in the text. Enhanced human cell engraftment was observed in both mutated CXCR4 and wt CXCR4 groups compared with naive and GFP-only groups (n = 10, P < .01; see “Effect of excess expression of wt or mutated CXCR4 transgene on engraftment of human PBSCs in the NOD/SCID xenograft model for group values). (B) Shown is a representative set of graphs showing GFP expression in the gated population of human CD45⁺ leukocytes as the black solid line in each panel as labeled, with background fluorescence seen in the naive nontransduced group shown as the dotted line in each panel. There is no significant difference between the groups. It is important to note that these curves suggest that there probably are a significant number of weakly GFP⁺ transduced cells that are not counted when a gate is simply set to the 99.7 percentile of the naive control. The group numbers of percentage of GFP⁺ cells exceeding the 99.7 percentile for the naive group are given in the text under “Effect of excess expression of wt or mutated CXCR4 transgene on engraftment of human PBSCs in the NOD/SCID xenograft model.” (C) Shown is a representative set of graphs indicating the CXCR4 expression in the gated population of human CD45⁺ leukocytes as the black solid line in each panel as labeled, with the background level of fluorescence seen with labeling with isotype control antibody indicated by the dotted line. Note that there is as expected a significant amount of expression of native CXCR4 in the naive and GFP-only transduced populations of human CD45⁺ leukocytes from the BM xenograft. There is a highly significant amount of extra expression of transgene-derived wt CXCR4 and mutated CXCR4 in those transduced populations (n = 10, P < .01 for either the wt CXCR4 or mutated CXCR4 groups compared with the naive or GFP-only groups). Note that it is difficult to accurately assess the actual number of cells expressing the CXCR4 transgene over background native CXCR4. Here again, it is important to note that these curves suggest that there probably are a significant number of weakly CXCR4⁺ cells that are not counted when a gate is simply set to the 99.7 percentile of the antibody isotype control. The group numbers of percentages of CXCR4⁺ cells exceeding the 99.7 percentile for the naive group are given in the text under “Effect of excess expression of wt or mutated CXCR4 transgene on engraftment of human PBSCs in the NOD/SCID xenograft model.”