Comparison of the NK cell expression of specific inhibitory receptors and functional inhibition by HLA-C. Based upon KIR genotype, donors were selected for having inhibitory KIR2DL with specificity for HLA-C and no cross-reactive stimulatory KIR2DS. NK cells from these donors were assayed by ELISPOT for secretion of IFN-γ. Assays were performed in the presence and absence of IL-2 or IL-12. Target cells were either 221, 221-Cw*0304 (A), or 221-Cw*0401 (C). Of the cells responding to 221, the percentage inhibited by presence of HLA-C ligand was calculated. For each donor these observed values were compared with predictions based upon the percentage of NK cells expressing either the cognate inhibitory KIRs or other inhibitory HLA-C–reactive receptors (CD94:NKG2A and LILRB) as determined by flow cytometry using specific monoclonal antibodies (B,D). (A,B) The analysis of inhibition of NK cells by 221-Cw*0304. The data shown for donor 11 is representative of that obtained with 4 donors (donors 5, 10, 11, and 25) (Figure 3). For donor 11 the frequency of NK cells expressing inhibitory HLA-Cw*0304–reactive receptors was 66% after culture without cytokine; 69% after culture with IL-2; and 82% in the IL-12–treated cultures (where CD94:NKG2A expression increases; Figure 2). These values were obtained by summing the frequencies of the following NK cell subsets: KIR2DL3⁺ CD94:NKG2A⁺, KIR2DL3⁺ CD94:NKG2A^–, KIR2DL3^– CD94: NKG2A⁺, and KIR2DL3^– CD94:NKG2A^– LILRB1⁺. (C-D) The inhibition of NK cells from donor 20 (Figure 3) by HLA-Cw*0401; 2 independent experiments gave similar results. For donor 20 the frequency of NK cells expressing inhibitory HLA-Cw*0401–reactive receptors was 64% after culture without cytokine, 71% after culture with IL-2, and 75% after culture with IL-12. These values were obtained by summing the frequencies of the following NK cell subsets: KIR2DL1⁺ CD94:NKG2A⁺, KIR2DL1⁺ CD94:NKG2A^–, and KIR2DL1^– CD94:NKG2A⁺. Because LILRB1 recognizes HLA-Cw*0304 but not HLA-Cw*0401,⁴⁴  the KIR2DL1^– CD94:NKG2A^– LILRB1⁺ subset (22% of NK cells of donor 20) was not included in the NK cells predicted to be inhibitable by HLA-Cw*0401. (E) Representative data obtained in the ELISPOT assay. NK cells producing IFN-γ were identified in each well of a 96-well microtiter plate. Analysis was performed on duplicate wells of NK cells challenged with 221 or 221-Cw*0304 cells. To obtain sufficient spots from NK cells cultured without cytokines, 10-fold more NK cells were plated per well than for NK cells cultured with cytokine.