3DL1/2v mediates HLA-allotype specific inhibition of NK cells. (A) Binding of the 3DL1-specific monoclonal antibody DX9 to NK cells shows that 3DL1/2v is expressed at the cell surface at intermediate level. Shown are FACS analyses of peripheral-blood NK cells from donors who express single 3DL1 allotypes. (Right) The table shows amino acid differences that correspond to the 3DL1 expression level. 3DL1*004 is not expressed because of leucine-for-serine substitution at residue 86 (Pando et al. 2003). 3DL1*015 has further differences in D0 that are shown in Figure 1. Error bars indicate the standard deviation of DX9-PE fluorescence intensity, from all of the lymphocytes that stained positive with DX9. (B,C) Results from Cr51-release cytotoxicity assays. The basis for this assay is killing of HLA Class I–deficient cells (221) by an NK cell line (NKL). Inhibition of cytotoxicity occurs if NKL is transduced with inhibitory KIR and 721.221 is transfected with cognate HLA Class I ligand. Blocking the interaction with specific antibody restores target killing to that obtained using non-transduced NKL. The specificity of inhibition was determined in replicate assays using DX9, which specifically blocks 3DL1, or DX31, which specifically blocks 3DL2. The percent specific inhibition that is shown was calculated from killing in the presence of control/specific-blocking antibody. Results are mean (±SE) of five experiments at an effector:target ratio of 20:1. (***) P < 0.001; (**) P < 0.01 from Student's t-test for comparison of means. All of the effectors killed HLA-negative targets. Each of these assays shows different combinations of KIR and ligand. (B) The degree of inhibition mediated by natural KIR allotypes; these are 3DL1*001, 3DL1*015, 3DL1/2v (3DL1*059), and 3DL2*001 or no KIR (nkl). Target cells were 721.221 cells transfected with (left) HLA-B*1513, (center) A*3201, and (right) A*1102. B*1513 and –A*3201 are ligands for 3DL1, and A*1102 is a ligand for 3DL2. (C, left) The table shows the composition of the mutant KIR allotypes. Tail-swap mutant m1 (*001-L283) has the 3DL1*059 Ig-domain with a 3DL1*001 tail. m2 (*059-W283) has the 3DL1*001 Ig-domain with a 3DL2*001 tail. Results of the cytotoxicity analysis are shown for (center) HLA-A*3201 and (right) HLA-B*1513.
