FIGURE 3.

Differentiation of NK cells from thymocytes is E4BP4 independent. Differentiation of NK cells from wt and E4BP4-deficient CD4⁻CD8⁻ DN thymocytes or BM precursors. (A) Lin⁻CD3⁻CD4⁻CD8⁻ DN cells were sorted from wt, and E4BP4-deficient thymi or (B) lin⁻ precursors (CD11b⁻CD11c⁻CD19⁻Ter119⁻Ly6G⁻CD8α⁻CD3e⁻CD45R⁻NK1.1⁻CD122⁻) were sorted from BM. After 14 d of culture on OP9 feeder cells, NK cells were identified by FACS as CD3⁻CD19⁻NK1.1⁺NKp46⁺ cells (A, B). (C) Kinetic of NK cell development from DN or BM precursors. A single time course experiment is shown in the left panels; data for days 6 and 10 pooled from three experiments are shown in the right panels. (D) Kinetics of phenotypic maturation of DN-derived NK cells in vitro. (E) The frequency of NK precursors in total lin⁻CD3⁻CD4⁻CD8⁻ DN thymocytes or DN subsets was determined by limiting dilution analysis. Sorted cells were plated out at frequencies varying from 30 to 3000 cells/well, and frequencies of NK cell–positive and –negative wells were used to calculate precursor frequencies in the cell subsets used. Data from four experiments were pooled for this analysis. (F) Cytokine induced IFN-γ production by CD3⁻NKp46⁺NK1.1⁺ NK cells derived in vitro from wt or E4BP4-deficient DN thymocytes. After 14 d of culture, NK cells were incubated for 16 h with the indicated cytokines, and IFN-γ production was measured by intracellular cytokine staining. Error bars indicate the SD of replicates (unpaired t test: *p < 0.05, **p < 0.01). ns, not significant.