DN1 and DN2 thymocytes develop to functional NK cells. (A) Flow cytometric analysis of control sample of NK cell colonies derived from DN1 and DN2 thymocytes cultured on OP9 stromal cells in the presence of IL-7 and IL-2 not stained with antibody. (B) DN1 and DN2 thymocyte-derived NK colonies stained with combination of anti-NK1.1 and anti-CD3ϵ fluorochrome-labeled antibodies. (C) YAC-1 cells (10⁴) labeled with ³H-thymidine were cultured together with increasing numbers of NK1.1⁺ cells derived from DN1 (□) and DN2 (⋄) thymocytes. NK-mediated specific lysis was determined as the percentage of lysed cells, with background counts representing 100% lysis. (D) Limiting dilution assay of DN1 (□) and DN2 (⋄) thymocytes plated at 2.5, 5, 10, and 20 cells in 96-well plates on OP9 stromal cells in the presence of IL-7 and IL-2. Number of NK cell negative wells was determined after 2 weeks of culture under an inverted light microscope. E/T indicates effector-target cell ratio.