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The gene products of the HLA/MHC and KIR multigene families are important modulators of the immune system and are associated with health and disease. Characterization of the genes encoding these receptors has been integrated into different biomedical applications, including transplantation and reproduction biology, immune therapies and in fundamental research into disease susceptibility or resistance. Conventional short-read sequencing strategies have shown their value in high throughput typing, but are insufficient to uncover the entire complexity of the highly polymorphic HLA/MHC and KIR gene systems. The implementation of single-molecule and real-time sequencing platforms, offered by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), revolutionized the fields of genomics and transcriptomics. Using fundamentally distinct principles, these platforms generate long-read data that can unwire the plasticity of the HLA/MHC and KIR genes, including high-resolution characterization of genes, alleles, phased haplotypes, transcription levels and epigenetics modification patterns. These insights might have profound clinical relevance, such as improved matching of donors and patients in clinical transplantation, but could also lift disease association studies to a higher level. Even more, a comprehensive characterization may refine animal models in preclinical studies. In this review, the different HLA/MHC and KIR characterization approaches using PacBio and ONT platforms are described and discussed. This article is protected by copyright. All rights reserved.

The role of natural killer (NK) cells is tightly modulated by interactions of killer cell immunoglobulin-like receptors (KIR) with their ligands of the MHC class I family. Several characteristics of the KIR gene products are conserved in primate evolution, like the receptor structures and the variegated expression pattern. At the genomic level, however, the clusters encoding the KIR family display species-specific diversity, reflected by differential gene expansions and haplotype architecture. The human KIR cluster is extensively studied in large cohorts from various populations, which revealed two KIR haplotype groups, A and B, that represent more inhibitory and more activating functional profiles, respectively. So far, genomic KIR analyses in large outbred populations of non-human primate species are lacking. In this study, we roughly quadrupled the number of rhesus macaques studied for their KIR transcriptome (n = 298). Using segregation analysis, we defined 112 unique KIR region configurations, half of which display a more inhibitory profile, whereas the other half has a more activating potential. The frequencies and functional potential of these profiles might mirror the human KIR haplotype groups. However, whereas the human group A and B KIR haplotypes are confined to largely fixed organizations, the haplotypes in macaques feature highly variable gene content. Moreover, KIR homozygosity was hardly encountered in this panel of macaques. This study exhibits highly diverse haplotype architectures in humans and macaques, which nevertheless might have an equivalent effect on the modulation of NK cell activity.

Natural killer (NK) cells represent a major component of the immune system, and are involved in the protection against pathogens, the surveillance of tumor cells, and the regulation of other players in the immune response. The education, activation and functioning of NK cells is tightly modulated by two major sets of inhibitory and activating receptors. A conserved set of NK cell receptors include members of the CD94:NKG2 family that recognize MHC-E molecules. The other set comprise the killer cell immunoglobulin-like receptors (KIR), which represent a diverse group of structurally similar transmembrane molecules. Members of the KIR family co-evolved with their diversified MHC-A, -B and -C ligands that are in a continuous arms race with pathogens. The KIR receptors are encoded within the Leukocyte Receptor Complex (LRC) on chromosome 19q13.4 and segregate independent from their MHC ligands. The KIR genes are arranged in a polygenic cluster conform a head-to-tail tandem organization, which is characterized by extensive diversification generated by point mutations and chromosomal recombination. As such, the KIR gene content is highly unique at an individual level and displays differential gene distribution at a population level. The highly variable KIR gene content, in combination with their polymorphic MHC class I ligands, is associated with differential susceptibility and protection in health and disease. In this thesis, we aimed to improve the KIR gene characterization in humans and macaques. The latter species is commonly used as model in preclinical studies to develop medicine and therapies. Their KIR gene system and NK cell biology display similarities to humans, with species-specific variation mainly reflected in the KIR receptor structure and haplotype organization. Initial transcriptome characterization studies using conventional sequencing techniques suggested a highly plastic KIR gene system in macaques that might exceed the complexity observed in humans. However, a comprehensive overview of the macaque KIR gene system is lacking. The application of single-molecule real-time (SMRT) sequencing platforms, which are commercialized by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), enables studies on the complexity of the KIR gene cluster at higher levels of resolution, with enhanced throughput and read length. These novel sequencing techniques provided comprehensive insights on KIR allele variation, chromosomal recombination events, fusion gene entities, alternative splicing profiles and KIR haplotype organizations, which together indicate a rapidly evolving KIR gene system in primates.

Long-read sequencing approaches have considerably improved the quality and contiguity of genome assemblies. Such platforms bear the potential to resolve even extremely complex regions, such as multigenic families and repetitive stretches of DNA. Deep sequencing coverage, however, is required to overcome low nucleotide accuracy, especially in regions with high homopolymer density, copy number variation, and sequence similarity, such as the MHC and KIR gene clusters of the immune system. Therefore, we have adapted a targeted enrichment protocol in combination with long-read sequencing to efficiently annotate complex genomic regions. Using Cas9 endonuclease activity, segments of the complex KIR gene cluster were enriched and sequenced on an Oxford Nanopore Technologies platform. This provided sufficient coverage to accurately resolve and phase highly complex KIR haplotypes. Our strategy facilitates rapid characterization of large and complex multigenic regions, including its epigenetic footprint, in multiple species, even in the absence of a reference genome.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele–associated diseases, as well as the immune response in transplantation and reproduction.

The Killer-cell Immunoglobulin-like Receptors (KIR) are encoded by a diverse group of genes, which are characterized by allelic polymorphism, gene duplications, and recombinations, which may generate recombinant entities. The number of reported macaque KIR sequences is steadily increasing, and these data illustrate a gene system that may match or exceed the complexity of the human KIR cluster. This report lists the names of quality controlled and annotated KIR genes/alleles with all the relevant references for two different macaque species: rhesus and cynomolgus macaques. Numerous recombinant KIR genes in these species necessitate a revision of some of the earlier-published nomenclature guidelines. In addition, this report summarizes the latest information on the Immuno Polymorphism Database (IPD)-NHKIR Database, which contains annotated KIR sequences from four non-human primate species.

The killer-cell Ig-like receptors (KIR) form a multigene entity involved in modulating immune responses through interactions with MHC class I molecules. The complexity of the KIR cluster is reflected by, for instance, abundant levels of allelic polymorphism, gene copy number variation, and stochastic expression profiles. The current transcriptome study involving human and macaque families demonstrates that KIR family members are also subjected to differential levels of alternative splicing, and this seems to be gene dependent. Alternative splicing may result in the partial or complete skipping of exons, or the partial inclusion of introns, as documented at the transcription level. This post-transcriptional process can generate multiple isoforms from a single KIR gene, which diversifies the characteristics of the encoded proteins. For example, alternative splicing could modify ligand interactions, cellular localization, signaling properties, and the number of extracellular domains of the receptor. In humans, we observed abundant splicing for KIR2DL4, and to a lesser extent in the lineage III KIR genes. All experimentally documented splice events are substantiated by in silico splicing strength predictions. To a similar extent, alternative splicing is observed in rhesus macaques, a species that shares a close evolutionary relationship with humans. Splicing profiles of Mamu-KIR1D and Mamu-KIR2DL04 displayed a great diversity, whereas Mamu-KIR3DL20 (lineage V) is consistently spliced to generate a homolog of human KIR2DL5 (lineage I). The latter case represents an example of convergent evolution. Although just a single KIR splice event is shared between humans and macaques, the splicing mechanisms are similar, and the predicted consequences are comparable. In conclusion, alternative splicing adds an additional layer of complexity to the KIR gene system in primates, and results in a wide structural and functional variety of KIR receptors and its isoforms, which may play a role in health and disease.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules.