Natural killer (NK) cells serve essential functions in immunity and reproduction. Diversifying these functions within individuals and populations are rapidly-evolving interactions between highly polymorphic major histocompatibility complex (MHC) class I ligands and variable NK cell receptors. Specific to simian primates is the family of Killer cell Immunoglobulin-like Receptors (KIR), which recognize MHC class I and associate with a range of human diseases. Because KIR have considerable species-specificity and are lacking from common animal models, we performed extensive comparison of the systems of KIR and MHC class I interaction in humans and chimpanzees. Although of similar complexity, they differ in genomic organization, gene content, and diversification mechanisms, mainly because of human-specific specialization in the KIR that recognizes the C1 and C2 epitopes of MHC-B and -C. Humans uniquely focused KIR recognition on MHC-C, while losing C1-bearing MHC-B. Reversing this trend, C1-bearing HLA-B46 was recently driven to unprecedented high frequency in Southeast Asia. Chimpanzees have a variety of ancient, avid, and predominantly inhibitory receptors, whereas human receptors are fewer, recently evolved, and combine avid inhibitory receptors with attenuated activating receptors. These differences accompany human-specific evolution of the A and B haplotypes that are under balancing selection and differentially function in defense and reproduction. Our study shows how the qualitative differences that distinguish the human and chimpanzee systems of KIR and MHC class I predominantly derive from adaptations on the human line in response to selective pressures placed on human NK cells by the competing needs of defense and reproduction.

The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrum of diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are killer lymphocytes that provide defense against viral infections and tumor transformation. Analogous to that of CTL, interactions of killer-cell immunoglobulin-like receptors (KIR) with specific human leukocyte antigen (HLA) class I ligands calibrate NK cell education and response. Gene families encoding KIRs and HLA ligands are located on different chromosomes, and feature variation in the number and type of genes. The independent segregation of KIR and HLA genes results in variable KIR-HLA interactions in individuals, which may impact disease susceptibility. We tested whether KIR-HLA combinations are associated with Vogt-Koyanagi-Harada (VKH) disease, a bilateral granulomatous panuveitis that has strong association with HLA-DR4. We present a case control study of 196 VKH patients and 209 controls from a highly homogeneous native population of Japan. KIR and HLA class I genes were typed using oligonucleotide hybridization method and analyzed using two-tailed Fisher's exact probabilities. The incidence of Bx-KIR genotypes was decreased in VKH patients (odds ratio [OR] 0.58, P = 0.007), due primarily to a decrease in centromeric B-KIR motif and its associated KIRs 2DS2, 2DL2, 2DS3, and 2DL5B. HLA-B22, implicated in poor immune response, was increased in VKH (OR = 4.25, P = 0.0001). HLA-Bw4, the ligand for KIR3DL1, was decreased in VKH (OR = 0.59, P = 0.01). The KIR-HLA combinations 2DL2+C1/C2 and 3DL1+Bw4, which function in NK education, were also decreased in VKH (OR = 0.49, P = 0.012; OR = 0.59, P = 0.013). Genotypes missing these two inhibitory KIR-HLA combinations in addition to missing activating KIRs 2DS2 and 2DS3 were more common in VKH (OR = 1.90, P = 0.002). These results suggest that synergistic hyporesponsiveness of NK cells (due to poor NK education along with missing of activating KIRs) and CTL (due to HLA-B22 restriction) fail to mount an effective immune response against viral-infection that may trigger VKH pathogenesis in genetically susceptible individuals, such as HLA-DR4 carriers.

The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8+ T cell clone established by stimulation of HLA-A2+ CD8+ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) α- and β-chains were cloned into a retroviral vector. TCR-transduced HLA-A2+ T cells efficiently killed HLA-A2+H3.3K27M+ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell-based therapy targeting this shared neoepitope.

Allogeneic cell therapeutics for cancer therapy or regenerative medicine are susceptible to antibody-mediated killing, which diminishes their efficacy. Here we report a strategy to protect cells from antibody-mediated killing that relies on engineered overexpression of the IgG receptor CD64. We show that human and mouse iPSC-derived endothelial cells (iECs) overexpressing CD64 escape antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity from IgG antibodies in vitro and in ADCC-enabled mice. When CD64 expression was combined with hypoimmune genetic modifications known to protect against cellular immunity, B2M-/-CIITA-/- CD47/CD64-transgenic iECs were resistant to both IgG antibody-mediated and cellular immune killing in vitro and in humanized mice. Mechanistic studies demonstrated that CD64 or its intracellularly truncated analog CD64t effectively capture monomeric IgG and occupy their Fc, and the IgG bind and occupy their target antigens. In three applications of the approach, human CD64t-engineered thyroid epithelial cells, pancreatic beta cells and CAR T cells withstood clinically relevant levels of graft-directed antibodies and fully evaded antibody-mediated killing.

BACKGROUND Solid-phase assays to investigate the complement-activating capacity of HLA antibodies have been utilized to optimize organ allocation and improve transplant outcomes. The clinical utility of C1q/C3d-binding characteristics of de novo donor-specific anti-HLA antibodies (dnDSA) associated with C4d-positive antibody-mediated rejection (C4d⁺ AMR) in kidney transplants (KTx) has not been defined. MATERIAL AND METHODS Sera from 120 KTx recipients that had dnDSA concurrent with protocol/cause biopsy (median 3.8 years after transplantation) were screened for C1q and C3d-binding dnDSA. The difference in the incidence of C4d⁺ AMR between recipients with and without C1q/C3d-binding dnDSA was assessed. RESULTS Over 86% of dnDSAs were class II antibodies. The immunodominant dnDSAs characterized by the highest median fluorescence intensity (MFI) in most recipients were HLA-DQ antibodies (67%). Most recipients (62%, n=74) had either C1q⁺ (56%), C3d⁺ (48%), or both C1q⁺C3d⁺ (41.2%) dnDSA, while the remaining 38% were negative for both C1q and C3d. Of those with C1q⁺/C3d⁺ dnDSA, 87% had high-MFI IgG (MFI=14144±5363 and 13932±5278, respectively), while 65% of C1q⁻C3d⁻ dnDSA had low-MFI IgG (MFI=5970±3347). The incidence of C4d+ AMR was significantly higher in recipients with C1q⁺ (66%), C3d+ (74%), and C1q⁺C3d⁺ (72%) dnDSA than in those with C1q⁻C3d⁻ dnDSA (30%) recipients. Recipients with C3d⁺/C1q⁺ dnDSA had higher C4d⁺ scores on biopsy. CONCLUSIONS C1q⁺/C3d⁺ dnDSA were associated with C4d⁺ AMR and high-IgG MFI. Our data call into question the predictive utility of C1q/C3d-binding assays in identifying KTx recipients at risk of allograft failure. In conclusion, IgG MFI is sufficient for clinical management, and the C1q/C3d-assays with added cost do not provide any additional information.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT.

Natural Killer (NK) cells are the third population of lymphocyte in the mononuclear cell compartment that triggers first-line of defense against viral infection and tumor transformation. Historically, NK cells were thought of as components of innate immunity based on their intrinsic ability to spontaneously kill target cells independent of HLA antigen restriction. However, it is now clear that NK cells are quite sophisticated and use a highly specific and complex target cell recognition receptor system arbitrated via a multitude of inhibitory and activating receptors. Killer cell immunoglobulin-like receptors (KIR) are the key receptors of human NK cells development and function. To date, fourteen distinct KIRs have been identified: eight are inhibitory types, and six are activating types. The number and type of KIR genes present varies substantially between individuals. Inhibitory KIRs recognize distinct motifs of polymorphic HLA class I molecules. Upon engagement of their specific HLA class I ligands, inhibitory KIR dampen NK cell reactivity. In contrast, activating KIRs are believed to stimulate NK cell reactivity when they sense their ligands (unknown). KIR and HLA gene families map to different human chromosomes (19 and 6, respectively), and their independent segregation produces a wide diversity in the number and type of inherited KIR-HLA combinations, likely contributing to overall immune competency. Consistent with this hypothesis, certain combinations of KIR-HLA variants have been correlated with susceptibility to diseases as diverse as autoimmunity, viral infections, and cancer. This review summarizes our emerging understanding of KIR-HLA diversity in human health and disease.

Killer cell immunoglobulin-like receptors (KIR) regulate natural killer cell response against infection and malignancy. KIR genes are variable in the number and type, thereby discriminating individuals and populations. Herein, we analyzed the KIR gene content diversity in four native populations of Iran. The KIR genomic diversity was comparable between Bakhtiari and Persian and displayed a balance of A and B KIR haplotypes, a trend reported in Caucasian and African populations. The KIR gene content profiles of Arab and Azeri were comparable and displayed a preponderance of B haplotypes, a scenario reported in the natives of America, India, and Australia. A majority of the B haplotype carriers of Azeri and Arab had a centromeric gene-cluster (KIR2DS2-2DL2-2DS3-2DL5). Remarkably, this cluster was totally absent from the American natives but occurred at highest frequencies in the natives of India and Australia in combination with another gene cluster at the telomeric region (KIR3DS1-2DL5-2DS5-2DS1). Therefore, despite having similar frequencies of B haplotypes, the occurrence of B haplotype-specific KIR genes, such as 2DL2, 2DL5, 3DS1, 2DS1, 2DS2, 2DS3, and 2DS5 in Azeri and Arab were substantially different from the natives of America, India, and Australia. In conclusion, each Iranian population exhibits distinct KIR gene content diversity, and the Indo-European KIR genetic signatures of the Iranians concur with geographic proximity, linguistic affinity, and human migrations.

The genes coding for the main molecules involved in the human immune system--immunoglobulins, human leucocyte antigen (HLA) molecules and killer-cell immunoglobulin-like receptors (KIR)--exhibit a very high level of polymorphism that reveals remarkable frequency variation in human populations. 'Genetic marker' (GM) allotypes located in the constant domains of IgG antibodies have been studied for over 40 years through serological typing, leading to the identification of a variety of GM haplotypes whose frequencies vary sharply from one geographic region to another. An impressive diversity of HLA alleles, which results in amino acid substitutions located in the antigen-binding region of HLA molecules, also varies greatly among populations. The KIR differ between individuals according to both gene content and allelic variation, and also display considerable population diversity. Whereas the molecular evolution of these polymorphisms has most likely been subject to natural selection, principally driven by host-pathogen interactions, their patterns of genetic variation worldwide show significant signals of human geographic expansion, demographic history and cultural diversification. As current developments in population genetic analysis and computer simulation improve our ability to discriminate among different--either stochastic or deterministic--forces acting on the genetic evolution of human populations, the study of these systems shows great promise for investigating both the peopling history of modern humans in the time since their common origin and human adaptation to past environmental (e.g. pathogenic) changes. Therefore, in addition to mitochondrial DNA, Y-chromosome, microsatellites, single nucleotide polymorphisms and other markers, immunogenetic polymorphisms represent essential and complementary tools for anthropological studies.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.

Four boys with Pelizaeus-Merzbacher disease, an X-linked leukodystrophy, underwent transplantation with human allogeneic central nervous system stem cells (HuCNS-SC). Subsequently, all subjects were followed for an additional 4 years in this separate follow-up study to evaluate safety, neurologic function, magnetic resonance imaging (MRI) data, and immunologic response. The neurosurgical procedure, immunosuppression, and HuCNS-SC transplantation were well tolerated and all four subjects were alive at the conclusion of the study period. At year 2, all subjects exhibited diffusion MRI changes at the implantation sites as well as in more distant brain regions. There were persistent, increased signal changes in the three patients who were studied up to year 5. Two of four subjects developed donor-specific HLA alloantibodies, demonstrating that neural stem cells can elicit an immune response when injected into the CNS, and suggesting the importance of monitoring immunologic parameters and identifying markers of engraftment in future studies.

Phase IIb clinical trial with isatuximab (Isa)-lenalidomide (Len)-dexamethasone (Dex) showed an improved progression-free survival (PFS) in patients with relapsed or refractory multiple myeloma (RRMM), but the efficacy varied by patient. Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) cells plays a crucial role in arbitrating antitumor activities of therapeutic-antibodies. We tested if patient-specific genetic makeup known to set NK cell functional threshold influence response to Isa-Len-Dex therapy.

Human leukocyte antigen (HLA) class I-specific killer-cell immunoglobulin-like receptors (KIR) regulate natural killer (NK) cell function in eliminating malignancy. Breast cancer (BC) patients exhibit reduced NK-cytotoxicity in peripheral blood. To test the hypothesis that certain KIR-HLA combinations impairing NK-cytotoxicity predispose to BC risk, we analyzed KIR and HLA polymorphisms in 162 women with BC and 278 controls. KIR-Bx genotypes increased significantly in BC than controls (83.3% vs. 71.9%, OR 1.95), and the increase was more pronounced in advanced-cancer (OR 5.3). No difference was observed with inhibitory KIR (iKIR) and HLA-ligand combinations. The activating KIR (aKIR) and HLA-ligand combinations, 2DS1 + C2 (OR 2.98) and 3DS1 + Bw4 (OR 2.6), were significantly increased in advanced-BC. All patients with advanced-cancer carrying 2DS1 + C2 or 3DS1 + Bw4 also have their iKIR counterparts 2DL1 and 3DL1, respectively. Contrarily, the 2DL1 + C2 and 3DL1 + Bw4 pairs without their aKIR counterparts are significantly higher in controls. These data suggest that NK cells expressing iKIR to the cognate HLA-ligands in the absence of putative aKIR counterpart are instrumental in antitumor response. These data provide a new framework for improving the utility of genetic risk scores for individualized surveillance.

The high magnitude zoonotic event has caused by Severe Acute Respitarory Syndrome CoronaVirus-2 (SARS-CoV-2) is Coronavirus Disease-2019 (COVID-19) epidemics. This disease has high rate of spreading than mortality in humans. The human receptor, Angiotensin-Converting Enzyme 2 (ACE2), is the leading target site for viral Spike-protein (S-protein) that function as binding ligands and are responsible for their entry in humans. The patients infected with COVID-19 with comorbidities, particularly cancer patients, have a severe effect or high mortality rate because of the suppressed immune system. Nevertheless, there might be a chance wherein cancer patients cannot be infected with SARS-CoV-2 because of mutations in the ACE2, which may be resistant to the spillover between species. This study aimed to determine the mutations in the sequence of the human ACE2 protein and its dissociation with SARS-CoV-2 that might be rejecting viral transmission. The in silico approaches were performed to identify the impact of SARS-CoV-2 S-protein with ACE2 mutations, validated experimentally, occurred in the patient, and reported in cell lines. The identified changes significantly affect SARS-CoV-2 S-protein interaction with ACE2, demonstrating the reduction in the binding affinity compared to SARS-CoV. The data presented in this study suggest ACE2 mutants have a higher and lower affinity with SARS-Cov-2 S-protein to the wild-type human ACE2 receptor. This study would likely be used to report SARS-CoV-2 resistant ACE2 mutations and can be used to design active peptide development to inactivate the viral spread of SARS-CoV-2 in humans.

Here, we present results for DPA1 and DPB1 four-digit allele-level typing in a large (n = 5,944) sample of unrelated European American stem cell donors previously characterized for other class I and class II loci. Examination of genetic data for both chains of the DP heterodimer in the largest cohort to date, at the amino acid epitope, allele, genotype, and haplotype level, allows new insights into the functional units of selection and association for the DP heterodimer. The data in this study suggest that for the DPA1-DPB1 heterodimer, the unit of selection is the combined amino acid epitope contributed by both the DPA1 and DPB1 genes, rather than the allele, and that patterns of LD are driven primarily by dimer stability and conformation of the P1 pocket. This may help explain the differential pattern of allele frequency distribution observed for this locus relative to the other class II loci. These findings further support the notion that allele-level associations in disease and transplantation may not be the most important unit of analysis, and that they should be considered instead in the molecular context.