Natural killer (NK) cell functions are modulated by polymorphic killer cell immunoglobulin-like receptors (KIR). Among 13 human KIR genes, which vary by presence and copy number, KIR3DL3 is ubiquitously present in every individual across diverse populations. No ligand or function is known for KIR3DL3, but limited knowledge of expression suggests involvement in reproduction, likely during placentation. With 157 human alleles, KIR3DL3 is also highly polymorphic and we show heterozygosity exceeds that of HLA-B in many populations. The external domains of catarrhine primate KIR3DL3 evolved as a conserved lineage distinct from other KIR. Accordingly, and in contrast to other KIR, we show the focus of natural selection does not correspond exclusively to known ligand binding sites. Instead, a strong signal for diversifying selection occurs in the D1 Ig domain at a site involved in receptor aggregation, which we show is polymorphic in humans worldwide, suggesting differential ability for receptor aggregation. Meanwhile in the cytoplasmic tail, the first of two inhibitory tyrosine motifs (ITIM) is conserved, whereas independent genomic events have mutated the second ITIM of KIR3DL3 alleles in all great apes. Together, these findings suggest that KIR3DL3 binds a conserved ligand, and a function requiring both receptor aggregation and inhibitory signal attenuation. In this model KIR3DL3 resembles other NK cell inhibitory receptors having only one ITIM, which interact with bivalent downstream signaling proteins through dimerization. Due to the extensive conservation across species, selection, and other unusual properties, we consider elucidating the ligand and function of KIR3DL3 to be a pressing question.

We characterized HLA and KIR combinatorial diversity in Malay and Malay Chinese, identified substantial allelic and structural diversity of the KIR locus in both populations and discovered novel variations at each analysis level. The Malay are more diverse than Malay Chinese, likely representing a unique history that includes admixture with immigrating populations spanning several thousand years. Characterizing the Malay are KIR haplotypes with large structural variants present in 10% individuals, and the Malaysian Chinese, a low frequency of interactions of KIR2DL1 with C2+HLA‐C.

Killer cell immunoglobulin-like receptors (KIR) interact with human leukocyte antigen (HLA) class I molecules, modulating critical NK cell functions in the maintenance of human health. Characterizing the distribution and characteristics of KIR and HLA allotype diversity across defined human populations is thus essential for understanding the multiple associations with disease, and for directing therapies. In this study of 176 Zhejiang Han individuals from Southeastern China, we describe diversity of the highly polymorphic KIR and HLA class I genes at high resolution. KIR-A haplotypes, which carry four inhibitory receptors specific for HLA-A, B or C, are known to associate with protection from infection and some cancers. We show the Chinese Southern Han from Zhejiang are characterized by a high frequency of KIR-A haplotypes and a high frequency of C1 KIR ligands. Accordingly, interactions of inhibitory KIR2DL3 with C1+HLA are more frequent in Zhejiang Han than populations outside East Asia. Zhejiang Han exhibit greater diversity of inhibitory than activating KIR, with three-domain inhibitory KIR exhibiting the greatest degree of polymorphism. As distinguished by gene copy number and allele content, 54 centromeric and 37 telomeric haplotypes were observed. We observed 6% of the population to have KIR haplotypes containing large-scale duplications or deletions that include complete genes. A unique truncated haplotype containing only KIR2DL4 in the telomeric region was also identified. An additional feature is the high frequency of HLA-B*46:01, which may have arisen due to selection pressure from infectious disease. This study will provide further insight into the role of KIR and HLA polymorphism in disease susceptibility of Zhejiang Chinese.

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina and ultra-long Nanopore sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (B38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.32% – 0.69% in a 1000 Genomes Project read re-mapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina, ultra-long Nanopore, and PacBio HiFi sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (GRCh38/hg38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally Torsten Houwaart and Stephan Scholz contributed equally. Paul J. Norman and Alexander T. Dilthey jointly supervised this work.

Highly polymorphic interaction of KIR3DL1 and KIR3DS1 with HLA class I ligands modulates the effector functions of natural killer (NK) cells and some T cells. This genetically determined diversity affects severity of infections, immune-mediated diseases, and some cancers, and impacts the course of immunotherapies, including transplantation. KIR3DL1 is an inhibitory receptor, and KIR3DS1 is an activating receptor encoded by the KIR3DL1/S1 gene that has more than 200 diverse and divergent alleles. Determination of KIR3DL1/S1 genotypes for medical application is hampered by complex sequence and structural variation, requiring targeted approaches to generate and analyze high-resolution allele data. To overcome these obstacles, we developed and optimized a model for imputing KIR3DL1/S1 alleles at high-resolution from whole-genome SNP data. We designed the model to represent a substantial component of human genetic diversity. Our Global imputation model is effective at genotyping KIR3DL1/S1 alleles with an accuracy ranging from 88% in Africans to 97% in East Asians, with mean specificity of 99% and sensitivity of 95% for alleles >1% frequency. We used the established algorithm of the HIBAG program, in a modification named Pulling Out Natural killer cell Genomics (PONG). Because HIBAG was designed to impute HLA alleles also from whole-genome SNP data, PONG allows combinatorial diversity of KIR3DL1/S1 with HLA-A and -B to be analyzed using complementary techniques on a single data source. The use of PONG thus negates the need for targeted sequencing data in very large-scale association studies where such methods might not be tractable.

The functionality of natural killer (NK) cells is tuned during education and associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation of KIR and HLA, which is known to influence the functional strength of NK cells, fine tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA-class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but was stable over time in each individual and genetically determined by allelic variation in HLA-class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as a robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of MHC-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells resulting in predictable hierarchies in global NK cell function.

Objectives: Individuals with Down syndrome (DS) display high risk of celiac disease (CD), but the mechanisms underlying this increased susceptibility await elucidation. Here, we examined the prevalence of HLA genotypes associated with CD risk in the general population and tested a previously developed genetic risk score (GRS) for CD in people with DS. Methods: HLA genotypes were obtained for 204 individuals with DS in the Human Trisome Project cohort study, of whom 9% had CD. We compared HLA genotype frequencies in those with and without CD against frequencies observed in the general population. CD permissive HLA haplotypes explored were DQ2.5, DQ2.2, DQ8.1, and DQ7.5. We also analyzed 38 non-HLA-DQ alleles used to generate the CD GRS. Results: Frequencies of risk genotypes were different for CD in DS versus CD in the general population. For example, we observed lower frequency of DQ2.5/DQ2.5 and higher prevalence of DQ7.5/X and X/X in CD in DS. Although GRS values were significantly increased in those with CD and DS, their predictive power was decreased relative to the general population. Transcriptome analysis revealed dysregulated expression of many genes composing the GRS in DS. Proteomics analysis showed that GRS values correlate with elevation of specific immune factors in DS. Conclusions: The genetic risk profile of CD in DS is different relative to the general population, which is likely due to dysregulation of immune pathways in DS. Larger studies are needed to elucidate pathogenic mechanisms and to develop a validated GRS for CD in DS.