A cell-intrinsic defect impairs maturation of ItpkB^−/− NK cells. (A) Purified ItpkB^−/− donor HSCs (CD45.1⁻CD45.2⁺) were infected with bicistronic retroviruses expressing GFP alone (control) or together with WT ItpkB and injected intravenously into lethally irradiated CD45.1⁺CD45.2⁺ recipients. Five to 10 weeks later, ItpkB^−/− donor HSC-derived cells were identified by CD45.1⁻CD45.2⁺ expression (supplemental Figure 3A). Retrovirally transduced cells were identified by GFP expression. NK cells reconstituted with WT ItpkB (GFP⁺; lower right), but not empty vector (top panel) or nontransduced cells (GFP⁻) efficiently developed into CD11b⁺CD27⁻ NK cells. (B-C) Lethally irradiated CD45.1⁺CD45.2⁺ recipients were injected with the indicated ratios of lymphocyte-depleted WT CD45.1⁺:ItpkB^−/− CD45.2⁺ BM cells and analyzed 6.5 weeks later. (B) Total WT (□) or ItpkB^−/− (■) CD3⁻NK1.1⁺ NK cell numbers in individual recipients. Horizontal lines denote the means of the respective populations. (C) WT:ItpkB^−/− total number ratios of splenic CD3⁻NK1.1⁺ NK cells (Total), CD3⁻NK1.1⁺NKG2D⁺CD11b⁻ immature, and CD3⁻NK1.1⁺NKG2D⁺CD11b⁺ mature NK cells. Statistical significance of the indicated comparisons was assessed via paired Student t test (n = 4). (D) Pie charts showing the percentage of immature (CD11b⁻; top) or mature (CD11b⁺; bottom) splenic NK cells expressing the indicated Ly49D and H combinations in the ItpkB^+/+ (left) or ItpkB^−/− (right) compartment. Qualitatively similar results were found for ItpkB^+/+:ItpkB^−/− HSC injection ratios of 5:1 and 1:3.5 or when CD3⁻NK1.1⁺NKG2D⁺ cells were analyzed (not shown). Results shown are representative of 2 independent experiments.