Molecular Signatures of Natural Killer Cells in CMV-Associated Anterior Uveitis, A New Type of CMV-Induced Disease in Immunocompetent Individuals.

Cytomegalovirus (CMV) causes clinical issues primarily in immune-suppressed conditions. CMV-associated anterior uveitis (CMV-AU) is a notable new disease entity manifesting recurrent ocular inflammation in immunocompetent individuals. As patient demographics indicated contributions from genetic background and immunosenescence as possible underlying pathological mechanisms, we analyzed the immunogenetics of the cohort in conjunction with cell phenotypes to identify molecular signatures of CMV-AU. Among the immune cell types, natural killer (NK) cells are main responders against CMV. Therefore, we first characterized variants of polymorphic genes that encode differences in CMV-related human NK cell responses (Killer cell Immunoglobulin-like Receptors (KIR) and HLA class I) in 122 CMV-AU patients. The cases were then stratified according to their genetic features and NK cells were analyzed for human CMV-related markers (CD57, KLRG1, NKG2C) by flow cytometry. KIR3DL1 and HLA class I combinations encoding strong receptor-ligand interactions were present at substantially higher frequencies in CMV-AU. In these cases, NK cell profiling revealed expansion of the subset co-expressing CD57 and KLRG1, and together with KIR3DL1 and the CMV-recognizing NKG2C receptor. The findings imply that a mechanism of CMV-AU pathogenesis likely involves CMV-responding NK cells co-expressing CD57/KLRG1/NKG2C that develop on a genetic background of KIR3DL1/HLA-B allotypes encoding strong receptor-ligand interactions.

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.

The combination of type I IFN, TNF-α, and cell surface receptor engagement with dendritic cells enables NK cells to overcome immune evasion by dengue virus.

Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC). NK cells responded by cytokine production and the lysis of target cells. Notably, in the absence of significant monokine production by DenV-infected DC, it was the combination of type I IFNs and TNF-α produced by DenV-infected DC that was important for stimulating the IFN-γ and cytotoxic responses of NK cells. Cell-bound factors enhanced NK cell IFN-γ production. In particular, reduced HLA class I expression was observed on DenV-infected DC, and IFN-γ production was enhanced in licensed/educated NK cell subsets. NK-DC cell contact was also identified as a requirement for a cytotoxic response, and there was evidence for both perforin/granzyme as well as Fas/Fas ligand-dependent pathways of killing by NK cells. In summary, our results have uncovered a previously unappreciated role for the combined effect of type I IFNs, TNF-α, and cell surface receptor-ligand interactions in triggering the antidengue response of primary human NK cells.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function.

Interactions between killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands regulate the development and response of human natural killer (NK) cells. Natural selection drove an allele-level group A KIR haplotype and the HLA-C1 ligand to unusually high frequency in the Japanese, who provide a particularly informative population for investigating the mechanisms by which KIR and HLA polymorphism influence NK cell repertoire and function. HLA class I ligands increase the frequencies of NK cells expressing cognate KIR, an effect modified by gene dose, KIR polymorphism, and the presence of other cognate ligand-receptor pairs. The five common Japanese KIR3DLI allotypes have distinguishable inhibitory capacity, frequency of cellular expression, and level of cell surface expression as measured by antibody binding. Although KIR haplotypes encoding 3DL1*001 or 3DL1*005, the strongest inhibitors, have no activating KIR, the dominant haplotype encodes a moderate inhibitor, 3DL1*01502, plus functional forms of the activating receptors 2DL4 and 2DS4. In the population, certain combinations of KIR and HLA class I ligand are overrepresented or underrepresented in women, but not men, and thus influence female fitness and survival. These findings show how KIR-HLA interactions shape the genetic and phenotypic KIR repertoires for both individual humans and the population.

Assessing the Response of Human NK Cell Subsets to Infection by Clinically Isolated Virus Strains.

Natural killer (NK) cells play a critical role in defending against virus infections.Investigating human NK cell antiviral functions is of prime importance; however, there are challenges such as the human-specific nature of many viruses and differences in NK cell surface markers between humans and rodents. Research on the antivirus response of human NK cells must therefore be carefully planned around species tropism of the viruses of interest and the specific biological questions to be answered. The initial site of many virus infections is a mucosal/epithelial surface. In this context, a clinical virus infection at the ocular surface enables direct analyses on the mechanisms and consequences of infection and immune reactions in situ over the course of disease. For example, the site of infection of a clinical infection in the conjunctiva and cornea can be directly observed in real-time, utilizing split-lamp microscopy, and specimens are readily accessed with minimally invasive techniques.In this chapter, we describe protocols for investigating NK cell responses using clinically isolated viruses in co-culture assays. We also describe procedures for ex vivo analysis of conjunctiva-derived NK cells in adenovirus infection.

Practical Considerations and Workflow in Utilizing KIR Genotyping in Transplantation Medicine.

This chapter is intended to serve as a practical guide for establishing a workflow using sequence-specific polymorphism PCR (SSP-PCR) for killer cell immunoglobulin-like receptor (KIR) genotyping in a clinical setting, especially in allogeneic hematopoietic stem cell transplantation (HSCT). As clinical evidence accumulates on the application of KIR and HLA genetics to guide donor selection in HSCT, there is an increasing need for KIR genotyping in clinical settings, and thus medical institutes may need to build this capability. Among the various KIR genotyping approaches now available, SSP-PCR methods are well-established and are the most cost-effective and will likely be the method of choice especially when expenses will be passed on to the patient. The protocol described in this chapter developed by Vilches et al. features small amplicon PCR and is suitable for KIR genotyping using FFPE-derived DNA as well as DNA extracted from blood samples. Setting up a laboratory workflow for in-house KIR genotyping is relatively straightforward; in this chapter, considerations for KIR genotyping to guide clinical decisions are discussed.In HSCT, a main objective of KIR genotyping is to apply the genetic analysis to predict donor and recipient combinations that have the most potential to produce NK cell alloresponses either through the missing-self mechanism or by action associated with activating KIR. The desired effects are reduction in acute GVHD and relapse rates and enhancement of overall survival. The information herein may also be useful to clinical laboratories considering the application of KIR genotyping in areas such as solid organ transplantation, NK cell-based treatment in other forms of cancer and autoimmune diseases, humanized antibody treatment, regenerative medicine, and reproductive medicine. Some background knowledge on KIR genetics will be necessary in managing a KIR genotyping platform. This chapter aims to address the main difficulties often encountered by physicians in understanding the KIR system, such as basic aspects of the nomenclature of KIR genes and haplotypes, genotypes, and determining presence/absence of KIR ligands in the patient and donor from the extensively diversified HLA class I allotypes. In describing the workflow, emphasis has been placed on the processes after genotype PCR and gel image acquisition: haplotype inference, generating B content scores, deduction of KIR ligands from HLA typing results, and the emerging algorithms for donor selection based on KIR and HLA genetics.

Decoding the diversity of killer immunoglobulin-like receptors by deep sequencing and a high-resolution imputation method.

The killer cell immunoglobulin-like receptor (KIR) recognizes human leukocyte antigen (HLA) class I molecules and modulates the function of natural killer cells. Despite its role in immunity, the complex genomic structure has limited a deep understanding of the KIR genomic landscape. Here we conduct deep sequencing of 16 KIR genes in 1,173 individuals. We devise a bioinformatics pipeline incorporating copy number estimation and insertion or deletion (indel) calling for high-resolution KIR genotyping. We define 118 alleles in 13 genes and demonstrate a linkage disequilibrium structure within and across KIR centromeric and telomeric regions. We construct a KIR imputation reference panel (nreference = 689, imputation accuracy = 99.7%), apply it to biobank genotype (ntotal = 169,907), and perform phenome-wide association studies of 85 traits. We observe a dearth of genome-wide significant associations, even in immune traits implicated previously to be associated with KIR (the smallest p = 1.5 × 10-4). Our pipeline presents a broadly applicable framework to evaluate innate immunity in large-scale datasets.

Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

Human cytomegalovirus (HCMV) infections develop into CMV diseases that result in various forms of manifestations in local organs. CMV-retinitis is a form of CMV disease that develops in immunocompromised hosts with CMV-viremia after viruses in the peripheral circulation have entered the eye. In the HCMV genome, extensive diversification of the UL40 gene has produced peptide sequences that modulate NK cell effector functions when loaded onto HLA-E and are subsequently recognized by the NKG2A and NKG2C receptors. Notably, some HCMV strains carry UL40 genes that encode peptide sequences identical to the signal peptide sequences of specific HLA-A and HLA-C allotypes, which enables these CMV strains to escape HLA-E-restricted CD8+T cell responses. Variations in UL40 sequences have been studied mainly in the peripheral blood of CMV-viremia cases. In this study, we sought to investigate how ocular CMV disease develops from CMV infections. CMV gene sequences were compared between the intraocular fluids and peripheral blood of 77 clinical cases. UL40 signal peptide sequences were more diverse, and multiple sequences were typically present in CMV-viremia blood compared to intraocular fluid. Significantly stronger NK cell suppression was induced by UL40-derived peptides from intraocular HCMV compared to those identified only in peripheral blood. HCMV present in intraocular fluids were limited to those carrying a UL40 peptide sequence corresponding to the leader peptide sequence of the host's HLA class I, while UL40-derived peptides from HCMV found only in the peripheral blood were disparate from any HLA class I allotype. Overall, our analyses of CMV-retinitis inferred that specific HCMV strains with UL40 signal sequences matching the host's HLA signal peptide sequences were those that crossed the blood-ocular barrier to enter the intraocular space. UL40 peptide repertoires were the same in the intraocular fluids of all ocular CMV diseases, regardless of host immune status, implying that virus type is likely to be a common determinant in ocular CMV disease development. We thus propose a mechanism for ocular CMV disease development, in which particular HCMV types in the blood exploit peripheral and central HLA-E-mediated tolerance mechanisms and, thus, escape the antivirus responses of both innate and adaptive immunity.

Corrigendum: Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

[This corrects the article DOI: 10.3389/fimmu.2022.1008220.].

Dimorphic motifs in D0 and D1+D2 domains of killer cell Ig-like receptor 3DL1 combine to form receptors with high, moderate, and no avidity for the complex of a peptide derived from HIV and HLA-A*2402.

Comparison of mutant killer cell Ig-like receptor (KIR) 3DL1*015 substituted at natural positions of variation showed that tryptophan/leucine dimorphism at position 283 uniquely changes receptor conformation and can strongly influence binding of the A24nef tetramer. Dimorphic motifs at positions 2, 47, and 54 in D0 and 182 and 283 in D1+D2 distinguish the two 3DL1 lineages, typified by 3DL1*005 and 3DL1*015. The interlineage recombinant, KIR3DL1*001, combines D0 of 3DL1*005 with D1+D2 of 3DL1*015 and binds A24nef more strongly than either parent. In contrast, the reciprocal recombinant with D0 from 3DL1*015 and D1+D2 from 3DL1*005 cannot bind A24nef. Thus, D0 polymorphism directly affects the avidity of the KIR3DL1 ligand binding site. From these observations, multiple sequence alignment, and homology modeling, we constructed structural models for KIR3DL1 and its complex with A24nef. In these models, D0, D1, and D2 come together to form a binding surface for A24nef, which is contacted by all three Ig-like domains. A central pocket binds arginine 83, the only Bw4 motif residue essential for KIR3DL1 interaction, similar to the binding of lysine 80 in HLA-C by KIR2DL1. Central to this interaction is a salt bridge between arginine 83 of Bw4 and glutamate 282 of 3DL1, which juxtaposes the functionally influential dimorphism at position 283. Further 3DL1 mutants were tested and shown to have A24nef-binding properties consistent with the models. A24nef was not bound by KIR3DS1, the activating counterpart of KIR3DL1. Moreover, introducing any one of three residues specific to KIR3DS1, serine 163, arginine 166, or leucine 199, into 3DL1*015, abrogated A24nef binding.

MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response.

Variegated expression of 6 inhibitory HLA class I-specific receptors on primary NK cells was studied using high-dimension flow cytometry in 58 humans to understand the structure and function of NK-cell repertoires. Sixty-four subsets expressing all possible receptor com-binations were present in each repertoire, and the frequency of receptor-null cells varied among the donors. Enhancement in missing-self response between NK subsets varied substantially where subset responses were defined by donor KIR/HLA allotypes, reflecting the differences in interaction between inhibitory receptors and their ligands. This contrasted to the enhancement conferred by NKG2A, which was constant and of intermediate strength. We infer a mechanism that modulates frequencies of the NK subsets displaying diverse levels of missing-self response, a system that reduces the presence of KIR-expressing subsets that display either too strong or too weak a response and effectively replaces them with NKG2A-expressing cells in the repertoire. Through this high-resolution analysis of inhibitory receptor expression, 5 types of NK-cell repertoire were defined by their content of NKG2A(+)/NKG2A(-) cells, frequency of receptor-null cells, and degree of KIR receptor coexpression. The analyses provide new perspective on how personalized human NK-cell repertoires are structured.

HLA-A allele associations with viral MER9-LTR nucleotide sequences at two distinct loci within the MHC alpha block.

The study of the association of the Human Leukocyte Antigen (HLA) alleles and polymorphic retrotransposons such as Alu, HERV, and LTR at various loci within the Major Histocompatibility Complex allows for a better identification and stratification of disease associations and the origins of HLA haplotypes in different populations. This paper provides sequence and association data on two structurally polymorphic MER9-LTR retrotransposons that are located 54 kb apart and in close proximity to the multiallelic HLA-A gene involved in the regulation of the human immune system. Direct DNA sequencing and analysis of the PCR products identified DNA nucleotide variations between the MER9-LTR sequences at the two loci and their associations with HLA-A alleles as potential haplotype and evolutionary markers. All MER9-LTR sequences were haplotypic when associated with common HLA-A alleles. The number of SNP loci was 2.5 times greater for the solo LTR at the AK locus, which is located closer to the HLA-A gene than the solo or 3' LTR at the HG locus. Our study shows that the nucleotide variations of the MER9-LTR DNA sequences are additional informative markers in fine mapping HLA-A genomic haplotypes for future population, evolutionary, and disease studies.

Kinetics of Tear Fluid Proteins after Endothelial Keratoplasty and Predictive Factors for Recovery from Corneal Haze.

Endothelial keratoplasty (EK) is less invasive with faster recovery as compared to conventional penetrating keratoplasty, however, it relies on the clarity of the host corneal stroma. Corneal transplantation involves the induction of immune tolerance for allogeneic tissues as well as the corneal wound healing process, in which coordinated interactions between cytokines and growth factors are critical. In this study, we profiled the expression of 51 soluble factors in the tear fluid over the course of EK and have provided evidence of dynamic changes in cytokine expression in the ipsilateral and contralateral eyes. Cluster analyses classified the cytokine expression kinetics into five groups. Group 1 proteins included TGF-b1, IL-1b, and innate proinflammatory cytokines, which bilaterally increased after surgery, despite the use of topical corticosteroid in the transplanted eyes. Local corticosteroids suppressed cytokines involved in adaptive immunity in the transplanted eyes but not in the contralateral eyes. We found tear protein expression at baseline and one week post-surgery to be a potential predictive biomarker of delayed recovery after EK in terms of the corneal haze and visual acuity. Furthermore, Group 1 tear proteins were most associated with persistent corneal haze pre-surgery as well as visual acuity at one month-post transplant.

NK Cell Alloreactivity against KIR-Ligand-Mismatched HLA-Haploidentical Tissue Derived from HLA Haplotype-Homozygous iPSCs.

HLA haplotype-homozygous (HLA-homo) induced pluripotent stem cells (iPSCs) are being prepared to be used for allogeneic transplantation of regenerated tissue into recipients carrying an identical haplotype in one of the alleles (HLA-hetero). However, it remains unaddressed whether natural killer (NK) cells respond to these regenerated cells. HLA-C allotypes, known to serve as major ligands for inhibitory receptors of NK cells, can be classified into group 1 (C1) and group 2 (C2), based on their binding specificities. We found that the T cells and vascular endothelial cells regenerated from HLA-homo-C1/C1 iPSCs were killed by specific NK cell subsets from a putative HLA-hetero-C1/C2 recipient. Such cytotoxicity was canceled when target cells were regenerated from iPSCs transduced with the C2 gene identical to the recipient. These results clarify that NK cells can kill regenerated cells by sensing the lack of HLA-C expression and further provide the basis for an approach to prevent such NK cell-mediated rejection responses.

Variation within the human killer cell immunoglobulin-like receptor (KIR) gene family.

The killer cell immunoglobulin-like receptors (KIR) form a family of highly homologous immune receptors that regulate the response of natural killer (NK) cells and some T cells. The genetics of the human KIR family is reviewed in this article. In human populations, diversity in KIR genotype arises from variations in gene content and allelic polymorphism. Comparisons of 81 human KIR sequences reveal past events ofgene duplication and recombination, and indicate that individual KIR genes have diversified from the influence of natural selection. Comparison and compilation of population studies reveal extensive KIR genotype variability within human populations and among them. Genomic analysis shows the KIR genes to be close to each other and separated by homologous sequences that promote haplotype diversification through assymetric recombination. In contrast, homologous recombination appears favored at a unique sequence in the center of the KIR locus, and much haplotypic diversity can be explained by recombination between a limited number of gene-content motifs in the centromeric and telomeric halves of the locus. The importance of NK cells for early defenses against infection suggests that human KIR genotype diversity is the accumulated consequence of a history of numerous and successive selective episodes by different pathogens on human NK-cell responses.

Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3.

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the "cross-reactions" were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor's interactions with HLA-C.

Episodes of natural selection shaped the interactions of IgA-Fc with FcalphaRI and bacterial decoy proteins.

FcalphaRI, a receptor for IgA-Fc, recruits myeloid cells to attack IgA-coated pathogens. By competing with FcalphaRI for IgA, bacterial decoys, like SSL7 of Staphylococcus aureus, subvert this defense. We examined how pathogen selection has driven the diversification and coevolution of IgA and FcalphaRI. In higher primates, the IgA binding site of FcalphaRI diversified under positive selection, a strong episode occurring in hominoid ancestors about the time of the IgA gene duplication. The differential binding of SSL7 to IgA-Fc of different species correlates with substitution at seven positions in IgA-Fc, two of which were positively selected in higher primates. Two others, which reduce SSL7 binding, emerged during episodes of positive selection in the rabbit and rodent lineages. The FcalphaRI-IgA interaction evolves episodically under two types of positive selection: pressure from pathogen decoys selects for IgA escape variants which, in turn, selects for FcalphaRI variants to keep up with the novel IgA. When FcalphaRI cannot keep up, its function is lost and the gene becomes susceptible to elimination, as occurred in the mouse genome, either by chance or selection on one of the many linked, variable immune system genes. A cluster of positively selected residues presents a putative binding site for unknown IgA-binding factors.

Cutting Edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B.

Although it is clear that KIR3DL1 recognizes Bw4(+) HLA-B, the role of Bw4(+) HLA-A allotypes as KIR3DL1 ligands is controversial. We therefore examined the binding of tetrameric HLA-A and -B complexes, including HLA*2402, a common Bw4(+) HLA-A allotype, to KIR3DL1*001, *005, *007, and *1502 allotypes. Only Bw4(+) tetramers bound KIR3DL1. Three of four HLA-A*2402 tetramers bound one or more KIR3DL1 allotypes and all four KIR3DL1 allotypes bound to one or more HLA-A*2402 tetramers, but with different binding specificities. Only KIR3DL1*005 bound both HLA-A*2402 and HLA-B*5703 tetramers. HLA-A*2402-expressing target cells were resistant to lysis by NK cells expressing KIR3DL1*001 or *005. This study shows that HLA-A*2402 is a ligand for KIR3DL1 and demonstrates how the binding of KIR3DL1 to Bw4(+) ligands depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism.

Single-cell analysis of the human NK cell response to missing self and its inhibition by HLA class I.

Natural killer (NK) cells activate quickly in response to pathogens, tumors, and allogeneic hematopoietic cell transplants. Modulating the NK cell response are clonally distributed NK cell receptors that survey cells for change in the expression of major histocompatibility complex (MHC) class I and structurally related ligands. Here the enzyme-linked immunospot (ELISPOT) assay, intracellular cytokine staining (ICS), and short-term culture were used to quantify the response of bulk NK cell populations from human donors to HLA class I-deficient 221 cells and to 221 cells transfected with single HLA class I allotypes. NK cells in cultures containing interleukin-2 (IL-2) or IL-12 exhibited specificities of HLA class I-mediated inhibition that correlated well with those previously defined using NK cell clones in long-term culture and with the frequencies of cells expressing particular inhibitory HLA class I receptors. Culture with IL-12, but not IL-2, gave an increased frequency of cells expressing CD94: NKG2A but no change in killer immunoglobulin-like receptor (KIR) expression. For some heterozygote combinations of KIR3DL1 alleles, ICS can be used to compare the functional properties of the 2 allotypes. Thus, both the low-expressing KIR3DL1*005 and the high-expressing KIR3DL1*002 gave similar inhibitory response on challenge with an HLA-B*5801 ligand. The single-cell assays developed here should facilitate future population study and clinical analysis of human NK cell regulation by MHC class I.