Molecular genetics and population studies of the KIR and HLA gene complexes

Division Of Basic Sciences - Nci

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Abstract

Natural killer (NK) cells play a critical role in immune surveillance by recognizing and eliminating virus-infected or transformed cells. Their activity is regulated by a balance between activating and inhibitory signaling from corresponding receptors. In humans, inhibitory NK cell receptors (iNKRs) include members of the highly polymorphic killer cell immunoglobulin-like receptor (KIR) family and the conserved CD94/NKG2A heterodimeric receptor (hereafter referred to as NKG2A), which bind the ubiquitously expressed HLA class I molecules. Inhibitory KIRs (iKIRs) recognize allotypes of HLA-A, HLA-B, and HLA-C molecules in a receptor-ligand specific manner, whereas the NKG2A receptor binds HLA-E, a non-classical HLA molecule with very limited polymorphism. The recognition of HLA class I molecules on normal cells by iNKRs promotes NK cell self-tolerance, whereas reduced HLA class I expression on aberrant cells diminishes inhibitory signaling and permits NK cell activation. The interactions between iNKRs and their specific HLA ligands are also thought to control NK cell "education", a process that calibrates NK cell reactivity. Consequently, cells expressing self HLA-specific iNKR are more functionally competent than those lacking such receptors, and stronger iNKR engagement with self HLA appears to be associated with enhanced NK cell responsiveness to stimuli. The strength of KIR/HLA binding impacts levels of NK cell functional capacity, a process that is genetically regulated as a function of the highly polymorphic HLA and KIR genes. Binding of the conserved NKG2A to HLA-E is modulated by variation in the VL9 epitopes presented by HLA-E, which are derived from polymorphic signal peptides (SPs) of HLA-A, HLA-B, and HLA-C. We have recently shown that among 16 common HLA SP variants, only six can be processed to generate VL9 epitopes that enable significant NKG2A/HLA-E engagement. The various combinations of these "functional" SPs containing VL9 epitopes that differentially determine the level of NKG2A/HLA-E binding may influence the responsiveness of NKG2A+ NK cells by calibrating their education. We used Jurkat reporter system to evaluate NKG2A binding to 360 B-lymphoblastoid cell lines (BLCLs) representing common SP genotypes. These data were used to generate coefficients that estimate the level of effector cell response attributable to each functional SP. Summing the respective coefficients in each individual (termed "SP score") based on their SP genotype predicted the level of peripheral blood CD56bright NK cell response to HLA class I-negative (i.e. missing-self) targets, where SP scores correlated positively with strength of response to the targets. Thus, SP score is a reliable measure of NK cell functional capacity mediated through differential NKG2A/HLA-E interactions, and can be used to test for effects of these interactions on disease risk. Calculations of SP scores based on SP genotypes in large cohorts of two very distinct types of human diseases, nasopharyngeal carcinoma (NPC) and ulcerative colitis (UC), revealed that higher predicted levels of NKG2A/HLA-E interaction associate with decreased risk of developing these diseases. Thus, the SP score may serve as a genetic tool to guide clinical NK cell intervention strategies, including therapeutic NKG2A blockade. The manuscript describing findings on genetic regulation of NKG2A/HLA-E interaction and impact on diseases has been submitted to Nature Immunology and is currently being reviewed. Genetic epidemiological data strongly indicate that certain KIR-HLA combinations influence various disease outcomes. Nevertheless, the functional basis underlying these associations have not been fully defined. Notably, quantification of the differential engagement between all specific receptor-ligand pairs of KIR-HLA is absent in the literature. Previous studies attempting to measure interactions between specific combinations of KIR-HLA have involved cell-free systems that measure binding of KIR-Ig fusion proteins or KIR tetramers to HLA-coated monoallelic beads, superficial methods yielding inconsistent results across publications. In order to accurately quantify the differential levels of KIR-HLA interactions, we propose to systematically characterize KIR binding to all common HLA class I allotypes using Jurkat reporter cells, similar to the measurements of NKG2A binding. Coefficients for each KIR-HLA pair will be calculated to develop an algorithm capable of predicting the overall NK cell activity in any given individual based on the composite KIR and HLA genotypes. The predicted level of NK cell activity in each individual will then be tested for potential associations with various disease outcomes. We have generated Jurkat reporter cells expressing KIR2DL2 and KIR2DL3 and measured their response to lentivirally transduced 721.221 cells expressing 24 individual HLA-C allotypes. In a preliminary test, we used the values obtained in the reporter assay as a measure of NK cell inhibition in treatment-naïve people living with HIV-1 homozygous for HLA-C allotypes that represent ligands for KIR2DL2 and KIR2DL3. We observed a significant association of the inhibition levels based on these KIR/HLA genotypes with HIV control. We are currently expanding our experiments to test KIR2DL1, KIR2DS1, and KIR3DL1 binding to the large pane of common HLA class I allotypes in cellular assays. This approach is expected to provide much deeper insight into the innate immune regulation of NK cell function.