Human leukocyte antigen (HLA) class I and II are cell surface proteins that display peptide antigens to immune cells, thereby mediating detection of infected cells and production of antibodies. Pathogen exposure and demographic events, including local adaptation and admixture, have driven and maintained exceptional polymorphism of HLA genes across human populations. Papua New Guinea has a complex demography, with geographically distinct populations in the highlands and lowlands and exceptional linguistic heterogeneity throughout the island. The lowland populations retain signatures of Austronesian expansion ∼3,000 years ago. Papua New Guinea populations are also differentially exposed to endemic malarial pathogens, with a greater burden in the lowlands. We analyzed genome-wide autosomal SNP data together with HLA allele sequences, linguistic, and geographical data from 337 Papuans. We find the substructure of HLA alleles to be highly correlated with altitude in Papua New Guinea, a signal that is distinct from the rest of the genome. In addition, specific HLA-B and HLA-DP alleles in lowland groups have a greater number of homozygous genotypes than expected under neutrality. Some of these HLA alleles are of Austronesian genetic ancestry. We find that the HLA-binding repertoires at candidate loci are significantly enriched for antigenic P. falciparum-derived peptides. Together, these results indicate that pathogen-driven selective pressures correlate with the observed HLA genetic substructure in Papua New Guinea, highlighting the critical importance of characterizing highly complex HLA variation in understanding differences in disease susceptibility across diverse human groups.

Mouse lemurs (Microcebus spp.) are an emerging primate model organism, but their genetics, cellular and molecular biology remain largely unexplored. In an accompanying paper1, we performed large-scale single-cell RNA sequencing of 27 organs from mouse lemurs. We identified more than 750 molecular cell types, characterized their transcriptomic profiles and provided insight into primate evolution of cell types. Here we use the generated atlas to characterize mouse lemur genes, physiology, disease and mutations. We uncover thousands of previously unidentified lemur genes and hundreds of thousands of new splice junctions including over 85,000 primate splice junctions missing in mice. We systematically explore the lemur immune system by comparing global expression profiles of key immune genes in health and disease, and by mapping immune cell development, trafficking and activation. We characterize primate-specific and lemur-specific physiology and disease, including molecular features of the immune program, lemur adipocytes and metastatic endometrial cancer that resembles the human malignancy. We present expression patterns of more than 400 primate genes missing in mice, many with similar expression patterns to humans and some implicated in human disease. Finally, we provide an experimental framework for reverse genetic analysis by identifying naturally occurring nonsense mutations in three primate immune genes missing in mice and by analysing their transcriptional phenotypes. This work establishes a foundation for molecular and genetic analyses of mouse lemurs and prioritizes primate genes, isoforms, physiology and disease for future study.

HLA class I alleles of archaic origin may have been retained in modern humans because they provide immunity against diseases to which archaic humans had evolved resistance. According to this model, archaic introgressed alleles were somehow distinct from those that evolved in African populations. Here, we show that HLA-B∗73:01, a rare allotype with putative archaic origins, has a relatively rare peptide binding motif with an unusually long-tailed peptide length distribution. We also find that HLA-B∗73:01 combines a restricted and unique peptidome with high-cell surface expression, characteristics that make it well-suited to combat one or a number of closely related pathogens. Furthermore, a crystal structure of HLA-B∗73:01 in complex with KIR2DL2 highlights differences from previously solved structures with HLA-C molecules. These molecular characteristics distinguish HLA-B∗73:01 from other HLA class I alleles previously investigated and may have provided early modern human migrants that inherited this allele with a selective advantage as they colonized Europe and Asia.

Genetic variation in host immunity impacts the disproportionate burden of infectious diseases that can be experienced by First Nations peoples. Polymorphic human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptors (KIRs) are key regulators of natural killer (NK) cells, which mediate early infection control. How this variation impacts their responses across populations is unclear. We show that HLA-A∗24:02 became the dominant ligand for inhibitory KIR3DL1 in First Nations peoples across Oceania, through positive natural selection. We identify KIR3DL1∗114, widespread across and unique to Oceania, as an allele lineage derived from archaic humans. KIR3DL1∗114+NK cells from First Nations Australian donors are inhibited through binding HLA-A∗24:02. The KIR3DL1∗114 lineage is defined by phenylalanine at residue 166. Structural and binding studies show phenylalanine 166 forms multiple unique contacts with HLA-peptide complexes, increasing both affinity and specificity. Accordingly, assessing immunogenetic variation and the functional implications for immunity are fundamental toward understanding population-based disease associations.

Diverse sets of complete human genomes are required to construct a pangenome reference and to understand the extent of complex structural variation. Here, we sequence 65 diverse human genomes and build 130 haplotype-resolved assemblies (130 Mbp median continuity), closing 92% of all previous assembly gaps1,2 and reaching telomere-to-telomere (T2T) status for 39% of the chromosomes. We highlight complete sequence continuity of complex loci, including the major histocompatibility complex (MHC), SMN1/SMN2, NBPF8, and AMY1/AMY2, and fully resolve 1,852 complex structural variants (SVs). In addition, we completely assemble and validate 1,246 human centromeres. We find up to 30-fold variation in α-satellite high-order repeat (HOR) array length and characterize the pattern of mobile element insertions into α-satellite HOR arrays. While most centromeres predict a single site of kinetochore attachment, epigenetic analysis suggests the presence of two hypomethylated regions for 7% of centromeres. Combining our data with the draft pangenome reference1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference3 to a median quality value (QV) of 45. Using this approach, 26,115 SVs per sample are detected, substantially increasing the number of SVs now amenable to downstream disease association studies.

A fundamental endeavor of the International Histocompatibility and Immunogenetics Workshop (IHIW) was assembling a collection of DNA samples homozygous through the MHC genomic region. This collection proved invaluable for assay development in the histocompatibility and immunogenetics field, for generating the human reference genome, and furthered our understanding of MHC diversity. Defined by their HLA‐A, ‐B, ‐C and ‐DRB1 alleles, the combined frequency of the haplotypes from these individuals is ∼20% in Europe. Thus, a significant proportion of MHC haplotypes, both common and rare throughout the world, and including many associated with disease, are not yet represented. In this workshop component, we are collecting the next generation of MHC‐homozygous samples, to expand, diversify and modernize this critical community resource that has been foundational to the field. We asked laboratories worldwide to identify samples homozygous through all HLA class I and/or HLA class II genes, or through whole‐genome SNP genotyping or sequencing, to have extensive homozygosity tracts within the MHC region. The focus is non‐Europeans or those having HLA haplotypes less common in Europeans. Through this effort, we have obtained samples from 537 individuals representing 294 distinct haplotypes, as determined by their HLA class I and II alleles, and an additional 50 haplotypes distinct in HLA class I or II alleles. Although we have expanded the diversity, many populations remain underrepresented, particularly from Africa, and we encourage further participation. The data will serve as a resource for investigators seeking to characterise variation across the MHC genomic region for disease and population studies. [Correction added on [24 July 2024], after first online publication: the Abstract was inadvertently remove and has been reinstated in this version.]

The Genotype List (GL) String grammar for reporting HLA and Killer‐cell Immunoglobulin‐like Receptor (KIR) genotypes in a text string was described in 2013. Since this initial description, GL Strings have been used to describe HLA and KIR genotypes for more than 40 million subjects, allowing these data to be recorded, stored and transmitted in an easily parsed, text‐based format. After a decade of working with HLA and KIR data in GL String format, with advances in HLA and KIR genotyping technologies that have fostered the generation of full‐gene sequence data, the need for an extension of the GL String system has become clear. Here, we introduce the new GL String delimiter “?,” which addresses the need to describe ambiguity in assigning a gene sequence to gene paralogs. GL Strings that do not include a “?” delimiter continue to be interpreted as originally described. This extension represents version 1.1 of the GL String grammar.

The malaria parasite Plasmodium falciparum causes substantial human mortality, primarily in equatorial Africa. Enriched in affected African populations, the B*53 variant of HLA-B, a cell surface protein that presents peptide antigens to cytotoxic lymphocytes, confers protection against severe malaria. Gorilla, chimpanzee, and bonobo are humans' closest living relatives. These African apes have HLA-B orthologs and are infected by parasites in the same subgenus (Laverania) as P. falciparum, but the consequences of these infections are unclear. Laverania parasites infect bonobos (Pan paniscus) at only one (TL2) of many sites sampled across their range. TL2 spans the Lomami River and has genetically divergent subpopulations of bonobos on each side. Papa-B, the bonobo ortholog of HLA-B, includes variants having a B*53-like (B07) peptide-binding supertype profile. Here we show that B07 Papa-B occur at high frequency in TL2 bonobos and that malaria appears to have independently selected for different B07 alleles in the two subpopulations.

Supplementary Figure from Phosphoantigen-Stimulated γδ T Cells Suppress Natural Killer–Cell Responses to Missing-Self

Long-term human diseases can shape the immune system, and natural killer (NK) cells have been documented to differentiate into distinct subsets specifically associated with chronic virus infections. One of these subsets found in large frequencies in HIV-1 are the CD56-CD16+ NK cells, and this population's association with chronic virus infections is the subject of this review. Human NK cells are classically defined by CD56 expression, yet increasing evidence supports the NK cell status of the CD56-CD16+ subset which we discuss herein. We then discuss the evidence linking CD56-CD16+ NK cells to chronic virus infections, and the potential immunological pathways that are altered by long-term infection that could be inducing the population's differentiation. An important aspect of NK cell regulation is their interaction with human leukocyte antigen (HLA) class-I mole -cules, and we highlight work that indicates both virus and genetic-mediated variations in HLA expression that have been linked to CD56-CD16+ NK cell frequencies. Finally, we offer a perspective on CD56-CD16+ NK cell function, taking into account recent work that implies the subset is comparable to CD56+CD16+ NK cell functionality in antibody-dependent cell cytotoxicity response, and the definition of CD56-CD16+ NK cell subpo-pulations with varying degranulation capacity against target cells.

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.

ABSTRACT Mouse lemurs ( Microcebus spp.) are an emerging primate model organism. However, little is known about their genetics or cellular and molecular biology. In the accompanying paper, we used large-scale single cell RNA-sequencing of 27 organs and tissues to identify over 750 molecular cell types, characterize their full transcriptomic profiles, and study evolution of primate cell types. Here we use the atlas to characterize mouse lemur genes, mutations, physiology, and disease. We uncover thousands of previously unidentified lemur genes and hundreds of thousands of new splice junctions that globally define lemur gene structures and reveal over 85,000 primate splice junctions missing in mice. We systematically explore the lemur immune system, comparing the global expression profiles of key immune genes in health and disease, and molecular mapping of immune cell development, trafficking, and their local and global activation to infection. We characterize primate/lemur-specific physiology and disease including molecular features of the immune program, of lemur adipocytes that exhibit dramatic seasonal rhythms, and of metastatic endometrial cancer that resembles the human malignancy. We identify and describe the expression patterns of over 400 primate genes missing in mice, many with similar expression patterns in human and lemur and some implicated in human disease. Finally, we provide an experimental framework for reverse genetic analysis by identifying naturally-occurring nonsense (null) mutations in three primate genes missing in mice and analyzing their transcriptional phenotypes. This work establishes mouse lemur as a tractable primate model organism for genetic and molecular analysis, and it prioritizes primate genes, splice junctions, physiology, and disease for future study.

Human NK cells are usually defined as CD3-CD56+ lymphocytes. However, a CD56-CD16+ (CD56neg) lymphocyte population that displays NK-associated markers expands during chronic viral infections such as HIV-1 and HCV, and, to lesser extent, in herpesvirus infections. This CD56neg NK cell subset has been understudied because it requires the exclusion of other lymphocytes to accurately identify its presence. Many questions remain regarding the origin, development, phenotype, and function of the CD56neg NK cell population. Our objective was to determine the frequency of this NK subset in healthy controls and its alteration in viral infections by performing a meta-analysis. In addition to this, we analyzed deposited CyTOF and scRNAseq datasets to define the phenotype and subsets of the CD56neg NK cell population, as well as their functional variation. We found in 757 individuals, from a combined 28 studies and 6 datasets, that the CD56neg subset constitutes 5.67% of NK cells in healthy peripheral blood, while HIV-1 infection increases this population by a mean difference of 10.69%. Meta-analysis of surface marker expression between NK subsets showed no evidence of increased exhaustion or decreased proliferation within the CD56neg subset. CD56neg NK cells have a distinctive pattern of KIR expression, implying they have a unique potential for KIR-mediated education. A perforin-CD94-NKG2C-NKp30- CD56neg population exhibited different gene expression and degranulation responses against K562 cells compared to other CD56neg cells. This analysis distinguishes two functionally distinct subsets of CD56neg NK cells. They are phenotypically diverse and have differing capacity for education by HLA class-I interactions with KIRs.

Abstractγδ T cells stimulated by phosphoantigens (pAg) are potent effectors that secrete Th1 cytokines and kill tumor cells. Consequently, they are considered candidates for use in cancer immunotherapy. However, they have proven only moderately effective in several clinical trials. We studied the consequences of pAg-stimulated γδ T-cell interactions with natural killer (NK) cells and CD8+ T cells, major innate and adaptive effectors, respectively. We found that pAg-stimulated γδ T cells suppressed NK-cell responses to “missing-self” but had no effect on antigen-specific CD8+ T-cell responses. Extensive analysis of the secreted cytokines showed that pAg-stimulated γδ T cells had a proinflammatory profile. CMV-pp65–specific CD8+ T cells primed with pAg-stimulated γδ T cells showed little effect on responses to pp65-loaded target cells. By contrast, NK cells primed similarly with γδ T cells had impaired capacity to degranulate and produce IFNγ in response to HLA class I–deficient targets. This effect depended on BTN3A1 and required direct contact between NK cells and γδ T cells. γδ T-cell priming of NK cells also led to a downregulation of NKG2D and NKp44 on NK cells. Every NK-cell subset was affected by γδ T cell–mediated immunosuppression, but the strongest effect was on KIR+NKG2A– NK cells. We therefore report a previously unknown function for γδ T cells, as brakes of NK-cell responses to “missing-self.” This provides a new perspective for optimizing the use of γδ T cells in cancer immunotherapy and for assessing their role in immune responses to pAg-producing pathogens.See related Spotlight by Kabelitz, p. 543.

Leukocyte immunoglobulin-like receptor B1 (LILRB1) is widely expressed on various immune cells and the engagement of LILRB1 to HLA class I and pathogen-derived proteins can modulate the immune response. In the current study, 108 LILRB1 alleles were identified by screening the LILRB1 locus from the 1000 Genomes Phase 3 database. Forty-six alleles that occurred in three or more individuals encode 28 LILRB1 allotypes, and the inferred LILRB1 allotypes were then grouped into 9 LILRB1 D1-D2 variants for further analysis. We found that variants 1, 2, and 3 represent the three most frequent LILRB1 D1-D2 variants and the nine variants show frequency differences in populations. The binding assay demonstrated that variant 1 bound to HLA class I with the highest avidity, and all tested LILRB1 D1-D2 variants bound to HLA-C with lower avidity than to HLA-A and -B. Locus-specific polymorphisms at positions 183, 189, and 268 in HLA class I and dimorphisms in HLA-A (positions 207 and 253) and in HLA-B (position 194) affect their binding to LILRB1. Notably, the electrostatic interaction plays a critical role in the binding of LILRB1 to HLA class I as revealed by electrostatic analysis and by comparison of different binding avidities caused by polymorphisms at positions 72 and 103 of LILRB1. In this paper, we present a comprehensive study of the population genetics and binding abilities of LILRB1. The data will help us better understand the LILRB1-related diversity of the immune system and lay a foundation for functional studies.

ABSTRACT The major histocompatibility complex (MHC) class I and II glycoproteins have been associated with numerous disease phenotypes, mechanisms and outcomes. These associations can be due to allotypic polymorphism or to altered levels of allotype expression. Although well studied in a range of cell types and microenvironments, no study has encompassed all cell-types present in an individual mouse lemur. In this study the droplet-based single cell RNA sequence data from the mouse lemur cell atlas, Tabula Microcebus, were used to examine the patterns of MHC class I and II expression. The cell atlas comprises data obtained from 27 organs from four mouse lemurs, enabling comparison of expression pattern between both cell types and the individual lemurs. Patterns of gene expression showed a good concordance among the four mouse lemurs. Three primary patterns of expression were identified and associated with different cell-types. Mapping MHC expression onto existing cell trajectories of cell development and spatial gradients revealed fine scale differences in expression level and pattern in single tissues. The bioinformatic pipeline developed here is applicable to other cell atlas projects.

The hormone insulin executes its function via binding and activating of the insulin receptor, a receptor tyrosine kinase that is mainly expressed in skeletal muscle, adipocytes, liver, pancreatic β-cells, and in some areas of the central nervous system. Stimulation of the insulin receptor activates intracellular signaling cascades involving the enzymes extracellular signal-regulated protein kinase-1/2 (ERK1/2), phosphatidylinositol 3-kinase, protein kinase B/Akt, and phospholipase Cγ as signal transducers. Insulin receptor stimulation is correlated with multiple physiological and biochemical functions, including glucose transport, glucose homeostasis, food intake, proliferation, glycolysis, and lipogenesis. This review article focuses on the activation of gene transcription as a result of insulin receptor stimulation. Signal transducers such as protein kinases or the GLUT4-induced influx of glucose connect insulin receptor stimulation with transcription. We discuss insulin-responsive transcription factors that respond to insulin receptor activation and generate a transcriptional network executing the metabolic functions of insulin. Importantly, insulin receptor stimulation induces transcription of genes encoding essential enzymes of glycolysis and lipogenesis and inhibits genes encoding essential enzymes of gluconeogenesis. Overall, the activation or inhibition of insulin-responsive transcription factors is an essential aspect of orchestrating a wide range of insulin-induced changes in the biochemistry and physiology of insulin-responsive tissues.