Stem cells acquire class i recognizing nk cell receptors in vitro

Abstract

To learn more about human NK cell receptor acquisition during development, cord blood (CB) CD34+Lin−CD38− cells where cultured in direct contact with AFT024 (murine fetal liver), separated from AFT024 by a membrane and in the absence of AFT024 (n = 8). All cultures contained c-kit ligand, flt3 ligand and IL-3 ± IL-7, IL-15 or IL-2. In the absence of one of the lymphoid cytokines, NK cells were KIR and lectin receptor negative. Addition of IL-7, IL-15 or IL-2 resulted in inefficient differentiation of NK cells in the absence of a feeder or when contact with AFT024 was prohibited. In contrast, 300 CB progenitors cultured in direct contact with AFT024 resulted in 22692 ± 10591 NK cells with IL-7, 523110 ± 120220 NK cells with IL-15 and 930310 ± 234250 NK cells with IL-2. The number of NK cells expressing at least one KIR was low under all conditions except for when progenitors were cultured in direct contact with AFT024 and IL-15 (15380 ± 5096) or IL-2 (47108 ± 13852) (n = 9). Ten-fold more cells expressed a lectin receptor as detected by the anti-CD94 mab. However, these studies do not show whether receptor acquisition was determined at the stem cell level or later in development. To address this question, single CD34+Lin−CD38− cells were cultured in direct contact with AFT024. Over 400 single cell progeny were analyzed from cultures containing IL-15 or IL-2. No single cell progeny contained a clonal population of NK cells expressing a single KIR. When single cell NK cell progeny were analyzed for single KIR's, KIR2DL2 (5846 ± 1017, n = 198) was always present at higher numbers than KIR2DL1 (2488 ± 539, n = 197) or KIR3DL1 (1011 ± 152, n = 252) expressing NK cells suggesting a nonrandom sequence of KIR acquisition. Higher numbers of NK cell progeny expressing KIR developed from single fetal liver and CB cells compared to adult BM. Lasting, LIR-1 receptors were acquired during NK cell development (CD94 > LIR-1 > KIR). These results show that NK cell receptor fate occurs at a stage beyond the hematopoietic stem cell.