NKR-P1B-deficient NK cells are not inhibited by Clr-b on target cells. (A) Flow cytometric analysis of Clr-b expression on Clr-b-transfected CHO cells. Two clones with high (HI) and low expression levels are shown. (B-C) Ability of LAK cells from WT and NKR-P1B-deficient mice to kill CHO target cells was tested by ⁵¹Cr-release assay. Data are represented as mean ± SD of percent killing measured in triplicate wells at different effector cell (E) to target cell (T) ratios. (D) NKR-P1B expression on sorted WT NKR-P1B⁺ and NKR-P1B-deficient LAK cells (NK1.1⁺TCRβ⁻), which were further expanded in culture with interleukin-2. (E-F) Ability of sorted WT NKR-P1B⁺ and NKR-P1B-deficient LAK cells to mediate cytotoxicity toward CHO target cells was tested by ⁵¹Cr-release assay. Data are represented as the mean ± SD of percent killing measured in triplicate. (G) NKR-P1B expression on LAK cells from WT, Clr-b-deficient, and NKR-P1B-deficient mice. (H-I) Ability of LAK cells from WT, Clr-b-deficient, and NKR-P1B-deficient mice to kill CHO and Clr-b-expressing CHO (CHO-Clrb) target cells was tested by ⁵¹Cr-release assay. Data are represented as mean ± SD of percent killing measured in triplicate wells at different E:T ratios. (J) Splenocytes from NKR-P1B-deficient and WT mice, pretreated with poly(I:C), were incubated with plate-bound isotype control antibody (Isotype Ab), anti-NKR-P1C antibody (NK1.1), or PMA/ionomycin for 5 hours in the presence or absence of plate-bound anti-NKR-P1B antibody (2D12). Intracellular IFN-γ in NK cells was analyzed by flow cytometry, and the mean percentage ± SD of IFN-γ⁺ NK cells for each stimulation is shown. (K) Splenocytes from WT mice were incubated with YAC-1 cells, plate-bound anti-NKR-P1C antibody (NK1.1), isotype control antibody, or PMA/ionomycin for 5 hours. Intracellular IFN-γ in NK cell subsets, based on the expression of NKR-P1B and Ly49C/I receptors, was analyzed by flow cytometry, and the mean percentage ± SD of IFN-γ⁺ NK cells for each stimulation is shown. Data in panels A-K are representative data from 1 of multiple independent experiments. Statistical analysis was performed by Student t test, and P values are indicated where applicable. Ab, antibody; PMA+Iono, PMA/ionomycin.