Genetic diversity affects the nanoscale membrane organization and signaling of natural killer cell receptors.

Genetic diversity in human natural killer (NK) cell receptors is linked to resistance and susceptibility to many diseases. Here, we tested the effect of this diversity on the nanoscale organization of killer cell immunoglobulin-like receptors (KIRs). Using superresolution microscopy, we found that inhibitory KIRs encoded by different genes and alleles were organized differently at the surface of primary human NK cells. KIRs that were found at low abundance assembled into smaller clusters than those formed by KIRs that were more highly abundant, and at low abundance, there was a greater proportion of KIRs in clusters. Upon receptor triggering, a structured interface called the immune synapse assembles, which facilitates signal integration and controls NK cell responses. Here, triggering of low-abundance receptors resulted in less phosphorylation of the downstream phosphatase SHP-1 but more phosphorylation of the adaptor protein Crk than did triggering of high-abundance receptors. In cells with greater KIR abundance, SHP-1 dephosphorylated Crk, which potentiated NK cell spreading during activation. Thus, genetic variation modulates both the abundance and nanoscale organization of inhibitory KIRs. That is, as well as the number of receptors at the cell surface varying with genotype, the way in which these receptors are organized in the membrane also varies. Essentially, a change in the average surface abundance of a protein at the cell surface is a coarse descriptor entwined with changes in local nanoscale clustering. Together, our data indicate that genetic diversity in inhibitory KIRs affects membrane-proximal signaling and, unexpectedly, the formation of activating immune synapses.

Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity.

The immune system comprises a complex network of specialized cells that protects against infection, eliminates cancerous cells, and regulates tissue repair, thus serving a critical role in homeostasis, health span, and life span. The subterranean-dwelling naked mole-rat (NM-R; Heterocephalus glaber) exhibits prolonged life span relative to its body size, is unusually cancer resistant, and manifests few physiological or molecular changes with advancing age. We therefore hypothesized that the immune system of NM-Rs evolved unique features that confer enhanced cancer immunosurveillance and prevent the age-associated decline in homeostasis. Using single-cell RNA-sequencing (scRNA-seq) we mapped the immune system of the NM-R and compared it to that of the short-lived, cancer-prone mouse. In contrast to the mouse, we find that the NM-R immune system is characterized by a high myeloid-to-lymphoid cell ratio that includes a novel, lipopolysaccharide (LPS)-responsive, granulocyte cell subset. Surprisingly, we also find that NM-Rs lack canonical natural killer (NK) cells. Our comparative genomics analyses support this finding, showing that the NM-R genome lacks an expanded gene family that controls NK cell function in several other species. Furthermore, we reconstructed the evolutionary history that likely led to this genomic state. The NM-R thus challenges our current understanding of mammalian immunity, favoring an atypical, myeloid-biased mode of innate immunosurveillance, which may contribute to its remarkable health span.

HLA-B57 micropolymorphism defines the sequence and conformational breadth of the immunopeptidome.

Immunophenotypic differences between closely related human leukocyte antigen (HLA) alleles have been associated with divergent clinical outcomes in infection, autoimmunity, transplantation and drug hypersensitivity. Here we explore the impact of micropolymorphism on peptide antigen presentation by three closely related HLA molecules, HLA-B*57:01, HLA-B*57:03 and HLA-B*58:01, that are differentially associated with the HIV elite controller phenotype and adverse drug reactions. For each allotype, we mine HLA ligand data sets derived from the same parental cell proteome to define qualitative differences in peptide presentation using classical peptide binding motifs and an unbiased statistical approach. The peptide repertoires show marked qualitative overlap, with 982 peptides presented by all allomorphs. However, differences in peptide abundance, HLA-peptide stability, and HLA-bound conformation demonstrate that HLA micropolymorphism impacts more than simply the range of peptide ligands. These differences provide grounds for distinct immune reactivity and insights into the capacity of micropolymorphism to diversify immune outcomes.

High-Resolution Genetic and Phenotypic Analysis of KIR2DL1 Alleles and Their Association with Pre-Eclampsia.

Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A, not KIR2DL1B, associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk.

Different Selected Mechanisms Attenuated the Inhibitory Interaction of KIR2DL1 with C2+ HLA-C in Two Indigenous Human Populations in Southern Africa.

The functions of human NK cells in defense against pathogens and placental development during reproduction are modulated by interactions of killer cell Ig-like receptors (KIRs) with HLA-A, -B and -C class I ligands. Both receptors and ligands are highly polymorphic and exhibit extensive differences between human populations. Indigenous to southern Africa are the KhoeSan, the most ancient group of modern human populations, who have highest genomic diversity worldwide. We studied two KhoeSan populations, the Nama pastoralists and the ≠Khomani San hunter-gatherers. Comprehensive next-generation sequence analysis of HLA-A, -B, and -C and all KIR genes identified 248 different KIR and 137 HLA class I, which assort into ∼200 haplotypes for each gene family. All 74 Nama and 78 ≠Khomani San studied have different genotypes. Numerous novel KIR alleles were identified, including three arising by intergenic recombination. On average, KhoeSan individuals have seven to eight pairs of interacting KIR and HLA class I ligands, the highest diversity and divergence of polymorphic NK cell receptors and ligands observed to date. In this context of high genetic diversity, both the Nama and the ≠Khomani San have an unusually conserved, centromeric KIR haplotype that has arisen to high frequency and is different in the two KhoeSan populations. Distinguishing these haplotypes are independent mutations in KIR2DL1, which both prevent KIR2DL1 from functioning as an inhibitory receptor for C2+ HLA-C. The relatively high frequency of C2+ HLA-C in the Nama and the ≠Khomani San appears to have led to natural selection against strong inhibitory C2-specific KIR.

Missing or altered self: human NK cell receptors that recognize HLA-C.

Natural killer (NK) cells are fast-acting and versatile lymphocytes that are critical effectors of innate immunity, adaptive immunity, and placental development. Controlling NK cell function are the interactions between killer-cell immunoglobulin-like receptors (KIRs) and their HLA-A, HLA-B and HLA-C ligands. Due to the extensive polymorphism of both KIR and HLA class I, these interactions are highly diversified and specific combinations correlate with protection or susceptibility to a range of infectious, autoimmune, and reproductive disorders. Evolutionary, genetic, and functional studies are consistent with the interactions between KIR and HLA-C being the dominant control mechanism of human NK cells. In addition to their recognition of the C1 and C2 epitopes, increasing evidence points to KIR having a previously unrecognized selectivity for the peptide presented by HLA-C. This selectivity appears to be a conserved feature of activating KIR and may partly explain the slow progress made in identifying their HLA class I ligands. The peptide selectivity of KIR allows NK cells to respond, not only to changes in the surface expression of HLA-C, but also to the more subtle changes in the HLA-C peptidome, such as occur during viral infection and malignant transformation. Here, we review recent advances in understanding of human-specific KIR evolution and how the inhibitory and activating HLA-C receptors allow NK cells to respond to healthy cells, diseased cells, and the semi-allogeneic cells of the fetus.

KIR2DS5 allotypes that recognize the C2 epitope of HLA-C are common among Africans and absent from Europeans.

INTRODUCTION: KIR2DS5 is an activating human NK cell receptor of lineage III KIR. These include both inhibitory KIR2DL1, 2 and 3 and activating KIR2DS1 that recognize either the C1 or C2 epitope of HLA-C. In Europeans KIR2DS5 is essentially monomorphic, with KIR2DS5*002 being predominant. Pioneering investigations showed that KIR2DS5*002 has activating potential, but cannot recognize HLA-A, -B, or -C. Subsequent studies have shown that KIR2DS5 is highly polymorphic in Africans, and that KIR2DS5*006 protects pregnant Ugandan women from preeclampsia. Because inhibitory C2-specific KIR2DL1 correlates with preeclampsia, whereas activating C2-specific KIR2DS1 protects, this association pointed to KIR2DS5*006 being an activating C2-specific receptor. To test this hypothesis we made KIR-Fc fusion proteins from all ten KIR2DS5 allotypes and tested their binding to a representative set of HLA-A, -B and -C allotypes. RESULTS: Six African-specific KIR2DS5 bound to C2+ HLA-C but not to other HLA class I. Their avidity for C2 is ∼20% that of C2-specific KIR2DL1 and ∼40% that of C2-specific KIR2DS1. Among the African C2 receptors is KIR2DS5*006, which protected a cohort of pregnant Ugandans from pre-eclampsia. Three African KIR2DS5 allotypes and KIR2DS5*002, bound no HLA-A, -B or -C. As a group the C2-binding KIR2DS5 allotypes protect against pre-eclampsia compared to the non-binding KIR2DS5 allotypes. Natural substitutions that contribute to loss or reduction of C2 receptor function are at positions 127, 158, and 176 in the D2 domain. CONCLUSIONS: KIR2DS5*005 has the KIR2DS5 consensus sequence, is the only allele found at both centromeric and telomeric locations of KIR2DS5, and is likely the common ancestor of all KIR2DS5 alleles. That KIR2DS5*005 has C2 receptor activity, points to KIR2DS5*002, and other allotypes lacking C2 receptor function, being products of attenuation, a characteristic feature of most KIR B haplotype genes. Alleles encoding attenuated and active KIR2DS5 are present in both centromeric and telomeric locations.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

Two Orangutan Species Have Evolved Different KIR Alleles and Haplotypes.

The immune and reproductive functions of human NK cells are regulated by interactions of the C1 and C2 epitopes of HLA-C with C1-specific and C2-specific lineage III killer cell Ig-like receptors (KIR). This rapidly evolving and diverse system of ligands and receptors is restricted to humans and great apes. In this context, the orangutan has particular relevance because it represents an evolutionary intermediate, one having the C1 epitope and corresponding KIR but lacking the C2 epitope. Through a combination of direct sequencing, KIR genotyping, and data mining from the Great Ape Genome Project, we characterized the KIR alleles and haplotypes for panels of 10 Bornean orangutans and 19 Sumatran orangutans. The orangutan KIR haplotypes have between 5 and 10 KIR genes. The seven orangutan lineage III KIR genes all locate to the centromeric region of the KIR locus, whereas their human counterparts also populate the telomeric region. One lineage III KIR gene is Bornean specific, one is Sumatran specific, and five are shared. Of 12 KIR gene-content haplotypes, 5 are Bornean specific, 5 are Sumatran specific, and 2 are shared. The haplotypes have different combinations of genes encoding activating and inhibitory C1 receptors that can be of higher or lower affinity. All haplotypes encode an inhibitory C1 receptor, but only some haplotypes encode an activating C1 receptor. Of 130 KIR alleles, 55 are Bornean specific, 65 are Sumatran specific, and 10 are shared.