BM single Lin⁻CD122⁺NK1.1⁻DX5⁻ NKPs have combined NK- and T-cell potential. Single BM Lin⁻CD122⁺NK1.1⁻DX5⁻ NKPs were directly sorted into OP9DL1 stroma layers and cultured with KL, IL-7 (first week), FL, IL-2, and IL-15. After 14 and 21 days, clones were harvested and analyzed by FACS. ToPro was used to eliminate dead cells. (A-B) FACS profiles of representative clones generated from a single Lin⁻CD122⁺NK1.1⁻DX5⁻ NKP. Text above profiles indicates prior gating. (C) Mean (SD) proportion of total proliferating clones and clones containing NK (TCR-β⁻NK1.1⁺DX5⁺) and T (NK1.1⁻CD25⁺Thy1.2⁺ or NK1.1⁻CD25⁺ Thy1.2⁺ TCR-β⁺) cells generated from single NKPs. Data expressed in relation to plated single cells, from 3 independent experiments, with total 167 plated cells and 105 clones analyzed. (D) Mean (SD) proportion of total clones and clones containing NK (TCR-β⁻NK1.1⁺ DX5⁺), T (TCR-β⁺NK1.1⁻CD4⁺/CD8⁺, NK1.1⁻ CD25⁺Thy1.2⁺, or NK1.1⁻CD25⁺Thy1.2⁺ TCR-β⁺), and TCR⁺NK1.1⁺ T cells, or combinations of these, generated from single NKPs. Data are expressed in relation to number of plated single cells, from 6 independent experiments, with a total 382 single cells plated and 201 clones analyzed. (E) Clones generated from single NKPs (shown in panels A and C), shown to have combined NK- and T-cell potential by FACS, were analyzed by RT-PCR for Cd3e, Ptcra, and Hprt gene expression. Thymocytes were used as positive control and OP9DL1 stroma cells cultured without hematopoietic cells as a negative control. Ethidium bromide–stained agarose gels with resulting PCR products from RT-PCR analysis of Cd3e, Ptcra, and Hprt. The photo has been taken using Gel logic 100 (Kodak). Data from 1 of 2 experiments with similar results. Note that clone 2 is the same clone derived from a single Lin⁻CD122⁺NK1.1⁻DX5⁻ NKP and analyzed by FACS in panel A.