Figure 3: Accumulation of WHIM CD8 SP thymocytes in the thymus is cell-intrinsic and associated with prolonged thymic residence.

Approximately 6-week-old male and female CD45.1/.2 +/+ and CD45.2 +/w mice transgenic for RAG-GFP were sacrificed, and their BM cells were harvested. After T cell depletion, the +/+ and +/w BM cells were mixed at a 1:1 ratio and injected i.v. into lethally irradiated CD45.1/.2 +/+ and CD45.1/.2 +/w recipient mice (not expressing RAG-GFP). 8–10 weeks after transplantation, thymi were harvested and the proportions of major thymocyte subsets were quantified by FACS analysis for the subsets indicated above each graph in A-D. E) CD4/CD8 SP ratios. Ratios were calculated from data in panels C and D. In A-E, the Cxcr4 genotypes for the donor-derived thymocytes and host groups are indicated in the insets and on the x-axes, respectively. F) +/+/ to +/w SP ratios for the SP cells and host groups indicated on the x-axis. G and H) The WHIM mutation promotes accumulation of mature CD24^loCD8 SP donor cells in the thymus. G) Representative contour-plots for the CD24 and TCRβ staining of CD8 SP cells. The recipient and donor Cxcr4 genotypes (+, wild type Cxcr4; w, WHIM allele) are designated for each graph column and row, respectively; ISP, immature single positive cells (CD24^hiTCRβ^lo). The subset names and frequencies are adjacent to each gate. H) Summary data for ISPs, CD24^hi and CD24^lo CD8 SP subsets, respectively. The Cxcr4 genotypes of the donor cells are given in the legend on the right. I and J) Prolonged thymic residence time of mature +/w CD24^loCD8 SPs. The proportions of RAG-GFP-positive CD4 and CD8 SP TCRβ⁺ thymocytes were quantified in I. The Cxcr4 genotypes of the donor cells are given in the inset. J) Donor +/+ to +/w ratios of the geometric mean fluorescence intensity (MFI) of RAG-GFP expression for the SP subsets and hosts indicated on the x-axis. Data are summarized as the mean ± SEM of 16 mice per genotype from 7 independent experiments. *, p< 0.05, ****, p< 0.01, ***, p< 0.001 and ****, p< 0.0001, as determined by multiple t-tests for panels D, E and H, and the one-way ANOVA for panels F and J.