Synergistic Polymorphism at Two Positions Distal to the Ligand-Binding Site Makes KIR2DL2 a Stronger Receptor for HLA-C Than KIR2DL3

Abstract

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the “cross-reactions” were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor’s interactions with HLA-C.

Combined Genotypic and Phenotypic Killer Cell Ig-Like Receptor Analyses Reveal KIR2DL3 Alleles Displaying Unexpected Monoclonal Antibody Reactivity: Identification of the Amino Acid Residues Critical for Staining

“…Both types of clones did not lyse 721.221-Cw3, whereas they partially lysed 721.221-Cw4 cell transfectants. This is in line with recent data indicating that KIR2DL2 and KIR2DL3 recognize not only C1 but also C2, although with lower affinity (13,14). In both clones, lysis was restored by anti-HLA class I mAb or by anti-KIR2DL2/L3/S2 mAb (CH-L).…”

Deciphering the killer‐cell immunoglobulin‐like receptor system at super‐resolution for natural killer and T‐cell biology

“…Three key advancements have progressed understanding of the recognition of HLA class I by KIR; (i) the crystal structures of KIR in complex with their HLA class I ligands,10, 35, 70, 99 (ii) the development of soluble KIR36, 100, 101 and (iii) their use in a multiplex immunoassay against a broad panel of HLA class I allotypes102, 103 (Fig. 3).…”

“…Other residues are involved in the interaction however, and their polymorphism means that there is a range of binding characteristics determined by KIR allotype. The interactions of KIR2DL are further diversified by polymorphism within the subsets of C1‐bearing and C2‐bearing HLA allotypes 34, 103. The basis for these hierarchies may occur due to polymorphism at sites other than position 80, or the distinct repertoires of peptide presented by the different HLA‐C allotypes 106.…”

“…Studies using soluble KIR showed that allotype KIR2DL3*001 is C1‐specific and binds relatively weakly, whereas KIR2DL2*001 binds more strongly and has cross‐reactivity with C2 103. In addition to those directly at the binding site, the substitutions that cause these functional differences may affect the angle or orientation of the binding domain,55, 103 or the efficiency of receptor clustering at the cell surface 99, 108. Allelic polymorphism also influences the functional properties of KIR2DL1, which has evolved to be a highly specific receptor for HLA‐C2 110.…”

Receptor systems controlling natural killer cell function are genetically stratified in Europe

“…10,11 A relative hierarchy of inhibitory responses to HLA-C has been defined for KIR receptors with 2DL1, 2DL2 and 2DL3 providing progressively decreasing levels of inhibition to NK cells in response to HLA-C ligation. 10,12,13 3DL1-mediated NK cell inhibition through Bw4 is considered a strong inhibitory interaction. 14 A number of activatory KIR (2DS1, 2DS2) also bind to HLA class I, albeit with weaker affinity and variation in the presence or absence of each can influence NK cell function.…”