Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of arginine245

Abstract

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among people. We demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, interferon ␥ production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in its transmembrane domain that mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245-negative alleles, KIR2DL1 that included R245 recruited more Src-homology-2 domaincontaining protein tyrosine phosphatase 2 and ␤-arrestin 2, showed higher inhibition of lipid raft polarization at immune synapse, and had less down-regulation of cell-surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably a fundamental understanding of KIR2DL1 allelic polymorphism in human disease susceptibility, transplant outcome, and donor selection. (Blood. 2009;114:5182-5190) IntroductionNatural killer (NK) cells are a part of our immune system that eliminates virally infected cells and tumor cells through cytolytic killing and cytokine secretion. 1 An NK cell's responses to its targets are regulated by the balance of signals generated through various activating and inhibiting receptors. 2,3 NK-cell receptors may be categorized on the basis of their ligand specificity for major histocompatibility complex class I and related molecules. Expression of various combinations of receptors on the surface of NK cells creates a diverse repertoire. 4,5 In humans, one of the most important groups of receptors that regulate NK-cell function is killer immunoglobulin-like receptors (KIRs). 6,7 The KIRs make up a family of diverse glycoproteins encoded by a compact cluster of genes located on human chromosome 19q13.4. 8,9 KIRs expressed at the surface of NK cells regulate their response by interacting with human leukocyte antigen (HLA) class I molecules. The KIR family has both activating and inhibitory receptors. KIR inhibitory receptors suppress NK cells' function using the immunoreceptor tyrosine-based inhibitory motif in their long cytoplasmic tails. Activating KIR receptors have short cytoplasmic tails and do not contain the inhibitory motif.Each of the KIR genes exhibits allelic variation as well as haplotypic variability in terms of the number and types of genes on the haplotypes. 9,10 The allelic variations of KIR genes range from 2 to more than 30. 10 The polymorphisms between the alleles of a given KIR gene can occur in its extracellular, transmembrane, or cytoplasmic domains. Polymorphism at each of these 3 domains has been associated with significant bi...

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“…Secondly, the commonest KIR2DL1 allele on the B haplotype is KIR2DL1*004. This binds more weakly to HLA-C2 than KIR2DL1*003, the most common allele on the A haplotype [45]. We predict that there would be reduced inhibition of uNK cells by KIR2DL1*004 on the KIR B haplotype.…”

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“…first reported that NK cells educated via KIR2DL1*004 had lower interferon‐ γ production than those educated via KIR2DL1*003 111. This occurs because KIR2DL1*004 has weak affinity for HLA‐C2 targets34 as well as reduced capacity for intracellular signal generation 112. A single dimorphism in the transmembrane region determines the signalling capacity; allotypes with R245 transduce a functional inhibitory signal and those with C245 have reduced inhibitory signalling 112.…”

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“…Nowadays, the easiest method to quantify NK cells expressing only one inhibitory KIR gene (i.e., negative for all other MHC inhibitory receptors) is multicolor flow cytometry (24)(25)(26) The third level of diversity is allele polymorphism, which has been observed in all inhibitory KIR genes (22,23). For example, in the KIR2DL1 family, 25 alleles have been observed, and each has different strengths in inhibiting NK cells and different durability of surface expression after interaction with ligands (27). These differences correlate with the intensity of inhibitory signaling through SHP2 and b arrestin-2, resulting in differences in immune synapse formation.…”

“…Mechanistically, the arginine residue at position 245 in the transmembrane domain is important in inhibition. Alleles with arginine in position 245 are stronger than those with cysteine in that position (27). On the basis of this molecular determinant, single-nucleotide polymorphism assays have been developed for rapid, high-throughput clinical typing (28).…”