Figure 1. BM cells from RAG^−/− mice stimulated with LPS contain NK cells that make IFN-γ.

(A) Fresh BM cells from RAG^−/− mice were cultured in a 96-well ELISPOT plate at 1 × 10⁵ cells/well to detect IFN-γ–secreting cells in the presence or absence of 200 ng/ml LPS for 18 h at 37°C. Each dot represents an independent experiment (24 experiments). Statistical significance was assessed by Student’s t test. (B) Same as (A) except that BM cells and splenocytes from RAG^−/− mice were used for comparison. Data represent the mean ± SD of four independent experiments. (C) Same as (A) except that BM cells were either NK-depleted using anti-NK1.1 plus -NKp46 antibodies or left untouched and cultured at 1 × 10⁵ cells/well. Each dot represents an independent experiment. Statistical significance was assessed by one-way ANOVA. (D) NK cells from BM or splenocytes from RAG^−/− mice were either preenriched by negative sorting for NK1.1⁺ cells or a Miltentyi NK-cell isolation kit. Various numbers of NK1.1⁺ cells were directly sorted into the ELISPOT plate using anti-NK1.1⁺ Ab and stimulated with 200 ng/ml LPS or a combination of ~125 pg/ml IL-12 plus ~600 ng/ml of IL-18. IFN-γ–secreting cells were counted after stimulation for 18 h at 37°C. Data represent the mean ± SD of three independent experiments. Statistical significance was assessed by **p<0.01, ***p<0.001.