Single fetal liver Lin⁻CD122⁺NK1.1⁻DX5⁻ NK-cell progenitors have combined NK and T-cell potential in vitro. Single Lin⁻CD122⁺NK1.1⁻DX5⁻ NKPs from the liver from 18.5-day-old fetuses were directly sorted into OP9-DL1 stroma layers and cultured with IL-7 (first week), KL, FL, IL-2, and IL-15. After 21 days clones were harvested and analyzed by FACS. TO-P-RO1 was used to eliminate dead cells and GFP to exclude stroma cells from the analysis. (A) FACS profiles of representative clone generated from single fetal liver Lin⁻CD122⁺NK1.1⁻DX5⁻ NKP. Text above profiles indicates gating. (B) Values are mean ± SD proportion of total proliferating clones and clones containing NK (TCRβ⁻NK1.1⁺DX5⁺), T (NK1.1⁻CD25⁺Thy1.2⁺ or NK1.1⁻CD25⁺Thy1⁺TCRβ⁺), and NK1.1⁺ T (NK1.1⁺TCRβ⁺) cells generated from single fetal liver NKPs. Data expressed in relation to total plated cells from 4 independent experiments, with 375 plated cells and 102 clones analyzed. (C) Values are mean ± SD proportion of clones generated from single fetal liver NKPs containing NK (TCRβ⁻NK1.1⁺DX5⁺), T (NK1.1⁻CD25⁺Thy1.2⁺ or NK1.1⁻CD25⁺Thy1⁺TCRβ⁺), and NK1.1⁺ T (NK1.1⁺TCRβ⁺) cells, or a combination of these. Data are expressed in relation to the total clones, from 4 independent experiments, with 375 plated cells and 102 clones analyzed. (D) Clones generated from single fetal liver NKPs (shown in panels A-C), shown to have combined NK and T-cell (CD25⁺Thy1.2⁺) potential detected by FACS were analyzed by RT-PCR for Ptcra and Hprt gene expression. Thymocytes from adult mouse were used as a positive control, and OP9-DL1 cells cultured without hematopoietic cells were used as negative control. Gel Red-stained agarose gels with resulting PCR products from RT-PCR analysis of Ptcra and Hprt. The photo has been taken using Gel logic 100 (Kodak). Representative data from 1 of 5 experiments with similar results (29 clones derived from single fetal NKPs were tested).