Sca-1^lo subset of LN NKPs is greatly reduced in athymic mouse LN. (A) WT (B6) and Foxn1^−/− (nude) mouse LN Lin⁻CD49b⁺ cells were gated as shown by rectangles in dot plots, and CD127, Sca-1, and CD117 expressions were analyzed (histograms). (B) B6 LN Sca-1^hi and Sca-1^lo Lin⁻CD49b⁺CD127⁺ cells and nude LN Lin⁻CD49b⁺CD127⁺ cells were purified and cultured in vitro for NK-cell differentiation. NK cells thus generated were analyzed for the expression of indicated NK-cell markers. The numbers show average (± SD; n = 3) percentages of cells expressing the indicated markers. (C) The cytotoxicity of NK cells generated in vitro from Sca-1^lo and Sca-1⁺ LN NKPs from B6 and LN NKPs from nude mice (B) were tested with the use of YAC-1 as target. (D) FACS-purified Sca-1^lo or Sca-1^hi LN NKPs from B6 and LN NKPs from nude mice were cultured for NK-cell differentiation as in panel B (left and middle). After 12 days, NKp46⁺ cells from the cultures were FACS-purified, and genomic DNA was extracted. Equivalent amounts of DNA were subjected to PCR for rearranged TCRγ genes and separated by agarose gel electrophoresis. DNA was also extracted from freshly isolated Sca-1^lo and Sca-1^hi LN NKPs from B6 mouse LN cells and analyzed for rearranged TCRγ genes as described before (right). Thymocytes served as a positive control, and NK cells generated from BM NKPs as a negative control.