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Natural killer (NK) cells have long been considered short-lived effectors of innate immunity. However, recent animal models and human studies suggest that subsets of NK cells have adaptive features. We investigate clonal relationships of various NK-cell subsets, including the adaptive population, by taking advantage of naturally occurring X-linked somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs) from patients with paroxysmal nocturnal hemoglobinuria (PNH). The affected HSPCs and their progeny lack expression of glycosylphosphatidylinositol (GPI) anchors on their cell surface, allowing quantification of PIGA -mutant (GPI-negative) HSPC-derived peripheral blood cell populations. The fraction of GPI-negative cells within the CD56 dim NK cells was markedly lower than that of neutrophils and the CD56 bright NK-cell compartments. This discrepancy was most prominent within the adaptive CD56 dim NK-cell population lacking PLZF expression. The functional properties of these adaptive NK cells were similar in PNH patients and healthy individuals. Our findings support the existence of a long-lived, adaptive NK-cell population maintained independently from GPI pos CD56 dim .

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34(+) cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34(+) cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF-mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.