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In the treatment of acute myelogenous leukemia with allogeneic hematopoietic cell transplantation, we previously demonstrated that there is a greater protection from relapse of leukemia when the hematopoietic cell transplantation donor has either the Cen B / B KIR genotype or a genotype having two or more KIR B gene segments. In those earlier analyses, KIR genotyping could only be assessed at the low resolution of gene presence or absence. To give the analysis greater depth, we developed high-resolution KIR sequence-based typing that defines all the KIR alleles and distinguishes the expressed alleles from those that are not expressed. We now describe and analyze high-resolution KIR genotypes for 890 donors of this human transplant cohort. Cen B01 and Cen B02 are the common CenB haplotypes, with Cen B02 having evolved from Cen B01 by deletion of the KIR2DL5 , 2DS3/5 , 2DP1 , and 2DL1 genes. We observed a consistent trend for Cen B02 to provide stronger protection against relapse than Cen B01 This correlation indicates that protection depends on the donor having inhibitory KIR2DL2 and/or activating KIR2DS2, and is enhanced by the donor lacking inhibitory KIR2DL1, 2DL3, and 3DL1. High-resolution KIR typing has allowed us to compare the strength of the interactions between the recipient's HLA class I and the KIR expressed by the donor-derived NK cells and T cells, but no clinically significant interactions were observed. The trend observed between donor Cen B02 and reduced relapse of leukemia points to the value of studying ever larger transplant cohorts., (Copyright © 2021 by The American Association of Immunologists, Inc.) more...

Immune dysfunction plays a role in the development of Parkinson disease (PD). NK cells regulate immune functions and are modulated by killer cell immunoglobulin-like receptors (KIR). KIR are expressed on the surface of NK cells and interact with HLA class I ligands on the surface of all nucleated cells. We investigated KIR -allelic polymorphism to interrogate the role of NK cells in PD. We sequenced KIR genes from 1314 PD patients and 1978 controls using next-generation methods and identified KIR genotypes using custom bioinformatics. We examined associations of KIR with PD susceptibility and disease features, including age at disease onset and clinical symptoms. We identified two KIR3DL1 alleles encoding highly expressed inhibitory receptors associated with protection from PD clinical features in the presence of their cognate ligand: KIR3DL1*015 /HLA-Bw4 from rigidity ( p c HLA-Bw4i from gait difficulties (p KIR3DL1*002/ HLA-Bw4i from gait difficulties (p c = 0.05, OR = 0.62, 95% CI 0.44-0.88), as well as composite symptoms associated with more severe disease. We also developed a KIR3DL1/HLA interaction strength metric and found that weak KIR3DL1/HLA interactions were associated with rigidity ( p c = 0.05, OR = 9.73, 95% CI 2.13-172.5). Highly expressed KIR3DL1 variants protect against more debilitating symptoms of PD, strongly implying a role of NK cells in PD progression and manifestation., (Copyright © 2020 by The American Association of Immunologists, Inc.) more...

Behçet disease is a chronic, relapsing-remitting autoinflammatory syndrome with a strong HLA-B*51 association. In this paper, we describe a human cohort of 267 individuals with Behçet disease and 445 matched controls from a tertiary referral center in the U.K. HLA-B*51 was confirmed as a genetic risk factor in this group ( p = 0.0006, Bonferroni-Dunn correction for multiple testing [ Pc ] = 0.0192, odds ratio [OR] 1.92, 95% confidence interval [CI] 1.33-2.76). KIR3DL1/S1 allele-level analysis indicated that low-expressing KIR3DL1/S1 alleles in combination with KIR3DS1 increased the risk of developing Behçet disease (KIR3DL1 LOW /KIR3DS1: p = 0.0004, Pc = 0.0040, OR 2.47, 95% CI 1.43-4.25), whereas high-expressing KIR3DL1/S1 alleles in combination with a null-expressing KIR3DL1 reduced the risk of disease (KIR3DL1 HIGH /KIR3DL1 NULL : p = 0.0035, Pc = 0.0350, OR 0.53, 95% CI 0.33-0.87). Behçet disease can manifest as a purely mucocutaneous disease or can involve other organ systems such as the eyes. In the U.K. cohort studied in this study, KIR3DL1 LOW /KIR3DS1 increased the risk of ophthalmic disease ( p = 1.2 × 10 -5 , OR 3.92, 95% CI 2.06-7.47), whereas KIR3DL1 HIGH /KIR3DL1 NULL reduced the risk of having purely mucocutaneous disease ( p = 0.0048, OR 0.45, 95% CI 0.25-0.81). To our knowledge, this is the first analysis of KIR3DL1/S1 allelic variation in Behçet disease and may provide insight into the pathogenic role of HLA-B*51 and its interaction with KIR3DL1/S1., (Copyright © 2019 by The American Association of Immunologists, Inc.) more...

HLA class I and KIR sequences were determined for Dogon, Fulani, and Baka populations of western Africa, Mbuti of central Africa, and Datooga, Iraqw, and Hadza of eastern Africa. Study of 162 individuals identified 134 HLA class I alleles (41 HLA-A , 60 HLA-B , and 33 HLA-C ). Common to all populations are three HLA-C alleles ( C1 + C*07:01 , C1 + C*07:02 , and C2 + C*06:02 ) but no HLA-A or -B Unexpectedly, no novel HLA class I was identified in these previously unstudied and anthropologically distinctive populations. In contrast, of 227 KIR detected, 22 are present in all seven populations and 28 are novel. A high diversity of HLA A-C-B haplotypes was observed. In six populations, most haplotypes are represented just once. But in the Hadza, a majority of haplotypes occur more than once, with 2 having high frequencies and 10 having intermediate frequencies. The centromeric ( cen ) part of the KIR locus exhibits an even balance between cenA and cenB in all seven populations. The telomeric ( tel ) part has an even balance of telA to telB in East Africa, but this changes across the continent to where telB is vestigial in West Africa. All four KIR ligands (A3/11, Bw4, C1, and C2) are present in six of the populations. HLA haplotypes of the Iraqw and Hadza encode two KIR ligands, whereas the other populations have an even balance between haplotypes encoding one and two KIR ligands. Individuals in these African populations have a mean of 6.8-8.4 different interactions between KIR and HLA class I, compared with 2.9-6.5 for non-Africans., (Copyright © 2019 by The American Association of Immunologists, Inc.) more...

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., (Copyright © 2018 by The American Association of Immunologists, Inc.) more...

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., (Copyright © 2018 by The American Association of Immunologists, Inc.) more...

The extent of NK cell activity during the innate immune response affects downstream immune functions and, ultimately, the outcome of infectious or malignant disease. However, the mechanisms that terminate human NK cell responses have yet to be defined. When activation receptors expressed on NK cell surfaces bind to ligands on diseased cells, they initiate a signal that is propagated by a number of intracellular kinases, including Zap70 and Syk, eventually leading to NK cell activation. We assayed Zap70 and Syk content in NK cells from healthy human donors and identified a subset of NK cells with unusually low levels of these two kinases. We found that this Zap70 low Syk low subset consisted of NK cells expressing a range of surface markers, including CD56 hi and CD56 low NK cells. Upon in vitro stimulation with target cells, Zap70 low Syk low NK cells failed to produce IFN-γ and lysed target cells at one third the capacity of Zap70 hi Syk hi NK cells. We determined two independent in vitro conditions that induce the Zap70 low Syk low phenotype in NK cells: continuous stimulation with activation beads and DNA damage. The expression of inhibitory receptors, including NKG2A and inhibitory killer Ig-like receptors (KIRs), was negatively correlated with the Zap70 low Syk low phenotype. Moreover, expression of multiple KIRs reduced the likelihood of Zap70 downregulation during continuous activation, regardless of whether NK cells had been educated through KIR-HLA interactions in vivo. Our findings show that human NK cells are able to terminate their functional activity without the aid of other immune cells through the downregulation of activation kinases., (Copyright © 2018 by The American Association of Immunologists, Inc.) more...

Fast-evolving MHC class I polymorphism serves to diversify NK cell and CD8 T cell responses in individuals, families, and populations. Because only chimpanzee and bonobo have strict orthologs of all HLA class I , their study gives unique perspectives on the human condition. We defined polymorphism of Papa-B , the bonobo ortholog of HLA-B , for six wild bonobo populations. Sequences for Papa-B exon 2 and 3 were determined from the genomic DNA in 255 fecal samples, minimally representing 110 individuals. Twenty-two Papa-B alleles were defined, each encoding a different Papa-B protein. No Papa-B is identical to any chimpanzee Patr-B, human HLA-B, or gorilla Gogo-B. Phylogenetic analysis identified a clade of MHC-B, defined by residues 45-74 of the α 1 domain, which is broadly conserved among bonobo, chimpanzee, and gorilla. Bonobo populations have 3-14 Papa-B allotypes. Three Papa-B are in all populations, and they are each of a different functional type: allotypes having the Bw4 epitope recognized by killer cell Ig-like receptors of NK cells, allotypes having the C1 epitope also recognized by killer cell Ig-like receptors, and allotypes having neither epitope. For population Malebo, these three Papa-B are the only Papa-B allotypes. Although small in number, their sequence divergence is such that the nucleotide diversity (mean proportional distance) of Papa-B in Malebo is greater than in the other populations and is also greater than expected for random combinations of three Papa-B Overall, Papa-B has substantially less diversity than Patr-B in chimpanzee subspecies and HLA-B in indigenous human populations, consistent with bonobo having experienced narrower population bottlenecks., (Copyright © 2017 by The American Association of Immunologists, Inc.) more...

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., (Copyright © 2017 by The American Association of Immunologists, Inc.) more...

KIR2DP1 is an inactive member of the human lineage III KIR family, which includes all HLA-C-specific receptor genes. The lethal, and only, defect in KIR2DP1 is a nucleotide deletion in codon 88. Fixed in modern humans, the deletion is also in archaic human genomes. KIR2DP1 is polymorphic, with dimorphism at specificity-determining position 44. By repairing the deletion, we resurrected 11 alleles of KIR2DP1 F , the functional antecedent of KIR2DP1 We demonstrate how K44-KIR2DP1 F with lysine 44 recognized C1 + HLA-C, whereas T44-KIR2DP1 F recognized C2 + HLA-C. Dimorphisms at 12 other KIR2DP1 F residues modulate receptor avidity or signaling. KIR2DP1 and KIR2DL1 are neighbors in the centromeric KIR region and are in tight linkage disequilibrium. Like KIR2DL1 , KIR2DP1 contributed to CenA and CenB KIR haplotype differences. Encoded on CenA , C1-specific K44-KIR2DP1 F were stronger receptors than the attenuated C2-specific T44-KIR2DP1 F encoded on CenB The last common ancestor of humans and chimpanzees had diverse lineage III KIR that passed on to chimpanzees but not to humans. Early humans inherited activating KIR2DS4 and an inhibitory lineage III KIR , likely encoding a C1-specific receptor. The latter spawned the modern family of HLA-C receptors. KIR2DP1 F has properties consistent with KIR2DP1 F having been the founder gene. The first KIR2DP1 F alleles encoded K44-C1 receptors; subsequently KIR2DP1 F alleles encoding T44-C2 receptors evolved. The emergence of dedicated KIR2DL2/3 and KIR2DL1 genes encoding C1 and C2 receptors, respectively, could have led to obsolescence of KIR2DP1 F Alternatively, pathogen subversion caused its demise. Preservation of KIR2DP1 F functional polymorphism was a side effect of fixation of the deletion in KIR2DP1 F by micro gene conversion., (Copyright © 2017 by The American Association of Immunologists, Inc.) more...

MHC haplotypes of humans and the African great ape species have one copy of the MHC-A, -B, and -C genes. In contrast, MHC haplotypes of orangutans, the Asian great ape species, exhibit variation in the number of gene copies. An in-depth analysis of the MHC class I gene repertoire in the two orangutan species, Pongo abelii and Pongo pygmaeus, is presented in this article. This analysis involved Sanger and next-generation sequencing methodologies, revealing diverse and complicated transcription profiles for orangutan MHC-A, -B, and -C. Thirty-five previously unreported MHC class I alleles are described. The data demonstrate that each orangutan MHC haplotype has one copy of the MHC-A gene, and that the MHC-B region has been subject to duplication, giving rise to at least three MHC-B genes. The MHC-B*03 and -B*08 lineages of alleles each account for a separate MHC-B gene. All MHC-B*08 allotypes have the C1-epitope motif recognized by killer cell Ig-like receptor. At least one other MHC-B gene is present, pointing to MHC-B alleles that are not B*03 or B*08. The MHC-C gene is present only on some haplotypes, and each MHC-C allotype has the C1-epitope. The transcription profiles demonstrate that MHC-A alleles are highly transcribed, whereas MHC-C alleles, when present, are transcribed at very low levels. The MHC-B alleles are transcribed to a variable extent and over a wide range. For those orangutan MHC class I allotypes that are detected by human monoclonal anti-HLA class I Abs, the level of cell-surface expression of proteins correlates with the level of transcription of the allele., (Copyright © 2016 by The American Association of Immunologists, Inc.) more...

Mass cytometry was used to investigate the effect of CMV reactivation on lymphocyte reconstitution in hematopoietic cell transplant patients. For eight transplant recipients (four CMV negative and four CMV positive), we studied PBMCs obtained 6 mo after unrelated donor hematopoietic cell transplantation (HCT). Forty cell-surface markers, distinguishing all major leukocyte populations in PBMC, were analyzed with mass cytometry. This group included 34 NK cell markers. Compared with healthy controls, transplant recipients had higher HLA-C expression on CD56(-)CD16(+) NK cells, B cells, CD33(bright) myeloid cells, and CD4CD8 T cells. The increase in HLA-C expression was greater for CMV-positive HCT recipients than for CMV negative recipients. Present in CMV-positive HCT recipients, but not in CMV-negative HCT recipients or controls, is a population of killer cell Ig-like receptor (KIR)-expressing CD8 T cells not previously described. These CD8 T cells coexpress CD56, CD57, and NKG2C. The HCT recipients also have a population of CD57(+)NKG2A(+) NK cells that preferentially express KIR2DL1. An inverse correlation was observed between the frequencies of CD57(+)NKG2C(+) NK cells and CD57(+)NKG2A(+) NK cells. Although CD57(+)NKG2A(+) NK cells are less abundant in CMV-positive recipients, their phenotype is of a more activated cell than the CD57(+)NKG2A(+) NK cells of controls and CMV-negative HCT recipients. These data demonstrate that HCT and CMV reactivation are associated with an increased expression of HLA-C. This could influence NK cell education during lymphocyte reconstitution. The increased inhibitory KIR expression by proliferating CMV-specific CD8 T cells suggests regulatory interactions between HLA-C and KIR might promote Graft-versus-Leukemia effects following transplantation., (Copyright © 2015 by The American Association of Immunologists, Inc.) more...

Chimpanzees have orthologs of the six fixed, functional human MHC class I genes. But, in addition, the chimpanzee has a seventh functional gene, Patr-AL, which is not polymorphic but contributes substantially to population diversity by its presence on only 50% of MHC haplotypes. The ancestral AL gene emerged long before the separation of human and chimpanzee ancestors and then subsequently and specifically lost function during human evolution, but was maintained in chimpanzees. Patr-AL is an alloantigen that participates in negative and positive selection of the T cell repertoire. The three-dimensional structure and the peptide-binding repertoire of Patr-AL and HLA-A*02 are surprisingly similar. In contrast, the expression of these two molecules is very different, as shown using specific mAbs and polyclonal Abs made against Patr-AL. Peripheral blood cells and B cell lines express low levels of Patr-AL at the cell surface. Higher levels are seen for 221-cell transfectants expressing Patr-AL, but in these cells a large majority of Patr-AL molecules are retained in the early compartments of the secretory pathway: mainly the endoplasmic reticulum, but also cis-Golgi. Replacing the cytoplasmic tail of Patr-AL with that of HLA-A*02 increased the cell-surface expression of Patr-AL substantially. Four substitutions distinguish the Patr-AL and HLA-A*02 cytoplasmic tails. Systematic mutagenesis showed that each substitution contributes changes in cell-surface expression. The combination of residues present in Patr-AL appears unique, but each individual residue is present in other primate MHC class I molecules, notably MHC-E, the most ancient of the functional human MHC class I molecules., (Copyright © 2015 by The American Association of Immunologists, Inc.) more...

The human killer cell Ig-like receptor (KIR) locus comprises two groups of KIR haplotypes, termed A and B. These are present in all human populations but with different relative frequencies, suggesting they have different functional properties that underlie their balancing selection. We studied the genomic organization and functional properties of the alleles of the inhibitory and activating HLA-C receptors encoded by KIR haplotypes. Because every HLA-C allotype functions as a ligand for KIR, the interactions between KIR and HLA-C dominate the HLA class I-mediated regulation of human NK cells. The C2 epitope is recognized by inhibitory KIR2DL1 and activating KIR2DS1, whereas the C1 epitope is recognized by inhibitory KIR2DL2 and KIR2DL3. This study shows that the KIR2DL1, KIR2DS1, and KIR2DL2/3 alleles form distinctive phylogenetic clades that associate with specific KIR haplotypes. KIR A haplotypes are characterized by KIR2DL1 alleles that encode strong inhibitory C2 receptors and KIR2DL3 alleles encoding weak inhibitory C1 receptors. In striking contrast, KIR B haplotypes are characterized by KIR2DL1 alleles that encode weak inhibitory C2 receptors and KIR2DL2 alleles encoding strong inhibitory C1 receptors. The wide-ranging properties of KIR allotypes arise from substitutions throughout the KIR molecule. Such substitutions can influence cell surface expression, as well as the avidity and specificity for HLA-C ligands. Consistent with the crucial role of inhibitory HLA-C receptors in self-recognition, as well as NK cell education and response, most KIR haplotypes have both a functional C1 and C2 receptor, despite the considerable variation that occurs in ligand recognition and surface expression., (Copyright © 2015 by The American Association of Immunologists, Inc.) more...

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig-like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle., (Copyright © 2014 The Authors.) more...

NK cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non-Ag-specific features termed "altered self" through combinatorial signals from activating and inhibitory receptors. These NKRs are also expressed on CD4(+) and CD8(+) T cells, B cells, and monocytes, although a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKRs are more likely to be expressed on non-NK cells, especially CD8(+) T cells. Mature NK and CD8(+) T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, whereas CD8(+) T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor leukocyte Ig-like receptor subfamily b member 1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of "altered self" recognition potential among leukocyte lineages. This likely reduces Ag specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection., (Copyright © 2014 by The American Association of Immunologists, Inc.) more...

Killer cell Ig-like receptors (KIRs) interact with HLA class I ligands to regulate NK cell development and function. These interactions affect the outcome of unrelated donor hematopoietic cell transplantation (HCT). We have shown previously that donors with KIR B versus KIR A haplotypes improve the clinical outcome for patients with acute myelogenous leukemia by reducing the incidence of leukemic relapse and improving leukemia-free survival (LFS). Both centromeric and telomeric KIR B genes contribute to the effect, but the centromeric genes are dominant. They include the genes encoding inhibitory KIRs that are specific for the C1 and C2 epitopes of HLA-C. We used an expanded cohort of 1532 T cell-replete transplants to examine the interaction between donor KIR B genes and recipient class I HLA KIR ligands. The relapse protection associated with donor KIR B is enhanced in recipients who have one or two C1-bearing HLA-C allotypes, compared with C2 homozygous recipients, with no effect due to donor HLA. The protective interaction between donors with two or more, versus none or one, KIR B motifs and recipient C1 was specific to transplants with class I mismatch at HLA-C (RR of leukemia-free survival, 0.57 [0.40-0.79]; p = 0.001) irrespective of the KIR ligand mismatch status of the transplant. The survival advantage and relapse protection in C1/x recipients compared with C2/C2 recipients was similar irrespective of the particular donor KIR B genes. Understanding the interactions between donor KIR and recipient HLA class I can be used to inform donor selection to improve outcome of unrelated donor hematopoietic cell transplantation for acute myelogenous leukemia. more...

Through recognition of HLA class I, killer cell Ig-like receptors (KIR) modulate NK cell functions in human immunity and reproduction. Although a minority of HLA-A and -B allotypes are KIR ligands, HLA-C allotypes dominate this regulation, because they all carry either the C1 epitope recognized by KIR2DL2/3 or the C2 epitope recognized by KIR2DL1. The C1 epitope and C1-specific KIR evolved first, followed several million years later by the C2 epitope and C2-specific KIR. Strong, varying selection pressure on NK cell functions drove the diversification and divergence of hominid KIR, with six positions in the HLA class I binding site of KIR being targets for positive diversifying selection. Introducing each naturally occurring residue at these positions into KIR2DL1 and KIR2DL3 produced 38 point mutants that were tested for binding to 95 HLA- A, -B, and -C allotypes. Modulating specificity for HLA-C is position 44, whereas positions 71 and 131 control cross-reactivity with HLA-A*11:02. Dominating avidity modulation is position 70, with lesser contributions from positions 68 and 182. KIR2DL3 has lower avidity and broader specificity than KIR2DL1. Mutation could increase the avidity and change the specificity of KIR2DL3, whereas KIR2DL1 specificity was resistant to mutation, and its avidity could only be lowered. The contrasting inflexibility of KIR2DL1 and adaptability of KIR2DL3 fit with C2-specific KIR having evolved from C1-specific KIR, and not vice versa. Substitutions restricted to activating KIR all reduced the avidity of KIR2DL1 and KIR2DL3, further evidence that activating KIR function often becomes subject to selective attenuation. more...

Variegated expression of variable NK cell receptors for polymorphic MHC class I broadens the range of an individual's NK cell response and the capacity for populations and species to survive disease epidemics and population bottlenecks. On evolutionary time scales, this component of immunity is exceptionally dynamic, unstable, and short-lived, being dependent on coevolution of ligands and receptors subject to varying, competing selection pressures. Consequently these systems of variable NK cell receptors are largely species specific and have recruited different classes of glycoprotein, even within the primate order of mammals. Such disparity helps to explain substantial differences in NK cell biology between humans and animal models, for which the population genetics is largely ignored. KIR3DL1/S1, which recognizes the Bw4 epitope of HLA-A and -B and is the most extensively studied of the variable NK cell receptors, exemplifies how variation in all possible parameters of function is recruited to diversify the human NK cell response. more...

Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ∼50% of chimpanzee MHC haplotypes. Comparing Patr-AL(+) and Patr-AL(-) haplotypes showed Patr-AL defines a unique 125-kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5' part of human pseudogene HLA-Y, carried by ∼10% of HLA haplotypes. Thus, the AL gene alternatively evolved in these closely related species to become classical, nonclassical, and nonfunctional. Although differing by 30 aa substitutions in the peptide-binding α(1) and α(2) domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable with that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α(2) helix. Stimulating PBMCs from Patr-AL(-) chimpanzees with B cells expressing Patr-AL produced potent alloreactive CD8 T cells with specificity for Patr-AL and no cross-reactivity toward other MHC class I molecules, including HLA-A*02. In contrast, PBMCs from Patr-AL(+) chimpanzees are tolerant of Patr-AL. more...

Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4(+) HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-γ. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4(+) HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4(+) HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4(+) HLA-B with the small fraction of cell surface KIR3DL1*004. more...

Modulation of human NK cell function by killer cell Ig-like receptors (KIR) and MHC class I is dominated by the bipartite interactions of inhibitory lineage III KIR with the C1 and C2 epitopes of HLA-C. In comparison, the ligand specificities and functional contributions of the activating lineage III KIR remain poorly understood. Using a robust, sensitive assay of KIR binding and a representative panel of 95 HLA class I targets, we show that KIR2DS1 binds C2 with ~50% the avidity of KIR2DL1, whereas KIR2DS2, KIR2DS3, and KIR2DS5 have no detectable avidity for C1, C2, or any other HLA class I epitope. In contrast, the chimpanzee has activating C1- and C2-specific lineage III KIR with strong avidity, comparable to those of their paired inhibitory receptors. One variant of chimpanzee Pt-KIR3DS2, the activating C2-specific receptor, has the same avidity for C2 as does inhibitory Pt-KIR3DL4, and a second variant has ~73% the avidity. Chimpanzee Pt-KIR3DS6, the activating C1-specific receptor, has avidity for C1 that is ~70% that of inhibitory Pt-KIR2DL6. In both humans and chimpanzees we observe an evolutionary trend toward reducing the avidity of the activating C1- and C2-specific receptors through selective acquisition of attenuating substitutions. However, the extent of attenuation has been extreme in humans, as exemplified by KIR2DS2, an activating C1-specific receptor that has lost all detectable avidity for HLA class I. Supporting such elimination of activating C1-specific receptors as a uniquely human phenomenon is the presence of a high-avidity activating C1-specific receptor (Gg-KIR2DSa) in gorilla. more...

Interactions between HLA class I and killer cell Ig-like receptors (KIRs) diversify human NK cell responses. Dominant KIR ligands are the C1 and C2 epitopes of MHC-C, a young locus restricted to humans and great apes. C1- and C1-specific KIRs evolved first, being present in orangutan and functionally like their human counterparts. Orangutans lack C2 and C2-specific KIRs, but have a unique C1+C2-specific KIR that binds equally to C1 and C2. A receptor with this specificity likely provided the mechanism by which C2-KIR interaction evolved from C1-KIR while avoiding a nonfunctional intermediate, that is, either orphan receptor or ligand. Orangutan inhibitory MHC-C-reactive KIRs pair with activating receptors of identical avidity and specificity, contrasting with the selective attenuation of human activating KIRs. The orangutan C1-specific KIR reacts or cross-reacts with all four polymorphic epitopes (C1, C2, Bw4, and A3/11) recognized by human KIRs, revealing their structural commonality. Saturation mutagenesis at specificity-determining position 44 demonstrates that KIRs are inherently restricted to binding just these four epitopes, either individually or in combination. This restriction frees most HLA-A and HLA-B variants to be dedicated TCR ligands, not subject to conflicting pressures from the NK cell and T cell arms of the immune response. more...

The killer cell Ig-like receptors (KIRs) of NK cells recognize MHC class I ligands and function in placental reproduction and immune defense against pathogens. During the evolution of monkeys, great apes, and humans, an ancestral KIR3DL gene expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterizing the KIR locus are three framework regions, defining two intervals of variable gene content. By analysis of four KIR haplotypes from two species of gibbon, we find that the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional genes. Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 5' end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC-C-driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organization of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species. more...

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. more...

IFN-gamma emanating from NK cells is an important component of innate defense against infection. In this study, we demonstrate that, following in vitro stimulation of human peripheral blood NK cells with a variety of microbial ligands, CD56(dim) as well as CD56(bright) NK cells contribute to the overall NK cell IFN-gamma response with, for most cell donors, IFN-gamma(+) CD56(dim) NK cells outnumbering IFN-gamma(+) CD56(bright) NK cells. We also observe that the magnitude of the human NK IFN-gamma response to microbial ligands varies between individuals; that the antimicrobial response of CD56(bright), but not CD56(dim), NK cells is highly correlated with that of myeloid accessory cells; and that the ratio of IFN-gamma(+) CD56(dim) to IFN-gamma(+) CD56(bright) NK cells following microbial stimulation differs between individuals but remains constant for a given donor over time. Furthermore, ratios of IFN-gamma(+) CD56(dim) to IFN-gamma(+) CD56(bright) NK cells for different microbial stimuli are highly correlated and the relative response of CD56(dim) and CD56(bright) NK cells is highly significantly associated with killer Ig-like receptor (KIR) genotype. These data reveal an influence of KIR genotype, possibly mediated via NK cell education, on the ability of NK cells to respond to nonviral infections and have implications for genetic regulation of susceptibility to infection in humans. more...

Ly49 lectin-like receptors and killer cell Ig-like receptors (KIR) are structurally unrelated cell surface glycoproteins that evolved independently to function as diverse NK cell receptors for MHC class I molecules. Comparison of primates and various domesticated animals has shown that species have either a diverse Ly49 or KIR gene family, but not both. In four pinniped species of wild marine carnivore, three seals and one sea lion, we find that Ly49 and KIR are each represented by single, orthologous genes that exhibit little polymorphism and are transcribed to express cell surface protein. Pinnipeds are therefore species in which neither Ly49 nor KIR are polygenic, but retain the ancestral single-copy state. Whereas pinniped Ly49 has been subject to purifying selection, we find evidence for positive selection on KIR3DL during pinniped evolution. This selection, which focused on the D0 domain and the stem, points to the functionality of the KIR and most likely led to the sea lion's loss of D0. In contrast to the dynamic and rapid evolution of the KIR and Ly49 genes in other species, the pinniped KIR and Ly49 have been remarkably stable during the >33 million years since the last common ancestor of seals and sea lions. These results demonstrate that long-term survival of placental mammal species need not require a diverse system of either Ly49 or KIR NK cell receptors. more...

Humans and chimpanzees have orthologous MHC class I, but few orthologous killer cell Ig-like receptors (KIR). Most divergent are lineage III KIR, which in humans include the inhibitory KIR2DL1 and 2DL2/3 specific for HLA-C. Six lineage III chimpanzee KIR were identified as candidate inhibitory MHC-C receptors and studied using cytolytic assays, to assess the capacity of a defined KIR to function with a defined MHC class I allotype, and direct binding assays with KIR-Fc fusion proteins. Pt-KIR2DL6 and 2DL8 were demonstrated to be inhibitory C1 receptors with a specificity and specificity-determining residue (lysine 44) like KIR2DL3. Analogously, Pt-KIR2DL7 is like KIR2DL1, an inhibitory C2 receptor having methionine 44. Pt-KIR3DL4 and 3DL5 are unusual lineage III KIR with D0 domains, which are also inhibitory C2 receptors with methionine 44. Removal of D0 from KIR3DL, or its addition to KIR2DL, had no effect on KIR function. Pt-KIR2DL9, a fourth inhibitory C2 receptor, has glutamate 44, a previously uncharacterized specificity-determining residue that is absent from human KIR. Reconstruction of the ancestral hominoid KIR sequence shows it encoded lysine 44, indicating that KIR having methionine 44 and glutamate 44 subsequently evolved by independent point substitutions. Thus, MHC-C2-specific KIR have evolved independently on at least two occasions. None of the six chimpanzee KIR studied resembles KIR2DL2, which interacts strongly with C1 and cross-reacts with C2. Whereas human HLA-B allotypes that have functional C1 epitopes are either rare (HLA-B*73) or geographically localized (HLA-B*46), some 25% of Patr-B allotypes have the C1 epitope and are functional KIR ligands. more...

KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81, 82, and 83 in the alpha(1) helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and 81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that polymorphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the alpha(1) domain, 116 in the alpha(2) domain, and 194 in the alpha(3) domain. Lesser contributions were made by additional positions in the alpha(2) domain. These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in the peptides bound, which alter the conformation of the Bw4 epitope. more...

KIR3DL1 shows extensive polymorphism, and its variation has functional significance in terms of cell-surface expression levels and inhibitory capacity. We characterized nine KIR3DL1 alleles (*022, *028, *029, *033, *035, *051, *052, *053, and *054), four of which were identified for the first time in this study, and compared them to known alleles in phylogenetic analysis. Blood was available from eight individuals with these alleles, and cell-surface expression on NK cells could be determined for six of them using the KIR3DL1-specific Ab DX9. Four of the alleles were expressed at clearly detectable levels, and two others showed exceptionally low levels of expression. Site-directed mutagenesis demonstrated that single amino acid changes can result in either diminished or enhanced DX9 staining compared with the respective related KIR3DL1 allotypes. These results raise the possibility that KIR3DL1 evolution maintains variation in KIR3DL1 cell-surface expression levels, potentially due to the effect of such variation on functional capacity. more...

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. more...

Orangutan (Pongo pygmaeus) MHC-C appears less evolved than human HLA-C: Popy-C is not fixed and its alleles encode only one (C1) of the two motifs for killer cell Ig-like receptor (KIR) ligands. To assess the structure and complexity of the orangutan KIR locus, the complete nucleotide sequence of an orangutan KIR haplotype was determined. The PopyKIR locus is flanked by LILR and FCAR and consists of seven genes and pseudogenes, two novel and five corresponding to known cDNA. Distinguishing all KIRs in this rapidly evolving KIR locus from the KIR3DX1 gene is an LTR33A/MLT1D element in intron 3. These two forms of KIR represent lineages that originated by duplication of a common ancestor. The conserved, framework regions of primate KIR loci comprise the 5' part of a lineage V KIR, the 3' part of a pseudogene, the complete 2DL4 gene, and the 3' part of a lineage II KIR. Although previously defined PopyKIR2DL4 alleles contain premature termination codons, the sequenced haplotype's PopyKIR2DL4 allele encodes a full-length protein. A model for KIR evolution is proposed. Distinguishing the orangutan KIR haplotype from the proposed common ancestor of primate KIR haplotypes is an increased number to give three lineage III KIR genes in the centromeric part of the locus, the site for most human lineage III genes encoding HLA-C specific KIR. Thus, expansion of lineage III KIR is associated with emergence of MHC-C. more...

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. more...

At an early phase of viral infection, contact and cooperation between dendritic cells (DCs) and NK cells activates innate immunity, and also influences recruitment, when needed, of adaptive immunity. Influenza, an adaptable fast-evolving virus, annually causes acute, widespread infections that challenge the innate and adaptive immunity of humanity. In this study, we dissect and define the molecular mechanisms by which influenza-infected, human DCs activate resting, autologous NK cells. Three events in NK cell activation showed different requirements for soluble mediators made by infected DCs and for signals arising from contact with infected DCs. IFN-alpha was mainly responsible for enhanced NK cytolysis and also important for CD69 up-regulation, whereas IL-12 was necessary for enhancing IFN-gamma production. Increased CD69 expression and IFN-gamma production, but not increased cytolysis, required recognition of influenza-infected DCs by two NK cell receptors: NKG2D and NKp46. Abs specific for these receptors or their known ligands (UL16-binding proteins 1-3 class I-like molecules for NKG2D and influenza hemagglutinin for NKp46) inhibited CD69 expression and IFN-gamma production. Activation of NK cells by influenza-infected DCs and polyinosinic:polycytidylic acid (poly(I:C))-treated DCs was distinguished. Poly(I:C)-treated DCs did not express the UL16-binding protein 3 ligand for NKG2D, and in the absence of the influenza hemagglutinin there was no involvement of NKp46. more...

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. more...

NK cells express both inhibitory and activatory receptors that allow them to recognize target cells through HLA class I Ag expression. KIR3DL1 is a receptor that recognizes the HLA-Bw4 public epitope of HLA-B alleles. We demonstrate that polymorphism within the KIR3DL1 receptor has functional consequences in terms of NK cell recognition of target. Inhibitory alleles of KIR3DL1 differ in their ability to recognize HLA-Bw4 ligand, and a consistent hierarchy of ligand reactivity can be defined. KIR3DS1, which segregates as an allele of KIR3DL1, has a short cytoplasmic tail characteristic of activatory receptors. Because it is very similar to KIR3DL1 in the extracellular domains, it has been assumed that KIR3DS1 will recognize a HLA-Bw4 ligand. In this study, we demonstrate that KIR3DS1 is expressed as a protein at the cell surface of NK cells, where it is recognized by the Z27 Ab. Using this Ab, we found that KIR3DS1 is expressed on a higher percentage of NK cells in KIR3DS1 homozygous compared with heterozygous donors. In contrast to the inhibitory KIR3DL1 allotypes, KIR3DS1 did not recognize HLA-Bw4 on EBV-transformed cell lines. more...

Signals transduced by inhibitory receptors that recognize self-MHC class I molecules prevent NK cells from being activated by autologous healthy target cells. In order for NK cells to be activated upon contact with an infected cell, the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. By studying liver-derived NK cells, we show that only a subpopulation of NK cells expressing high levels of the inhibitory receptor NKG2A are able to lyse autologous vaccinia-infected targets, and that this is due to selective down-regulation of HLA-E. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors in humans generates a repertoire of NK cells with different antiviral potentials. more...

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I receptors expressed by NK cells and several T cell subpopulations. Factors contributing to human KIR haplotype diversity are differences in gene number, gene content, and allelic polymorphism. Whereas functional and clinical consequences of the first two factors are established, knowledge of the effects of KIR gene polymorphism is limited to special cases in which signaling function is reversed or cell surface expression lost. In this study we use retrovirally transduced human cell lines to show that 3DL1*002 is a stronger inhibitory receptor for HLA-Bw4 ligands than 3DL1*007. Analysis of mutant 3DL1*002 and 3DL1*007 molecules demonstrates that residue 238 in the D2 domain and 320 in the transmembrane region contribute to the difference in receptor strength. Neither position 238 nor 320 is predicted to interact directly with HLA-Bw4 ligand. This study also revealed that KIR3DL1 and LILRB1 both contribute to developing an inhibitory response to HLA-Bw4 ligands. more...

Beta2-microglobulin (beta2m) associates with MHC and related class I H chains to form cell surface glycoproteins that mediate a variety of functions in defense. In humans, monomorphism of a single beta2m gene contrasts with the diversity and polymorphism of the class I H chain genes, and a similar picture was seen in almost all other species examined. In this regard, rainbow trout (Oncorhynchus mykiss) appeared unusual: trout beta2m genes gave a complicated and polymorphic pattern in Southern blots, and a minimum of 10 different mRNA encoding two distinct types of beta2m were expressed by a single fish. Characterization of genomic clones from the same fish now shows that the rainbow trout beta2m locus consists of two expressed genes and one partial gene that are closely linked. Four copies of the locus were identified and allelic variants of each gene defined, largely through comparison of the noncoding regions. A dramatic variation in the lengths of introns is caused by variable repetitive elements and accounts for the complex pattern seen in Southern blots. By comparison to noncoding sequences, the coding regions are conserved but the three loci differ within a cluster of codons that encode residues of beta2m that do not interact with class I H chains. Additional diversity in the trout beta2m genes appears to be due to somatic mutation that might be facilitated by the abundance of repetitive DNA elements within the 12 beta2m genes of an individual rainbow trout. more...

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I-specific receptors, which regulate NK cell responses and are also expressed on subpopulations of T cells. KIR haplotypes vary in gene content, which, in combination with allelic polymorphism, extensively diversifies the KIR genotype both within and between human populations. Species comparison indicates that formation of new KIR genes and loss of old ones are frequent events, so that few genes are conserved even between closely related species. In this regard, the hominoids define a time frame that is particularly informative for understanding the processes of KIR evolution and its potential impact on killer cell biology. KIR cDNA were characterized from PBMC of three gorillas, and genomic DNA were characterized for six additional individuals. Eleven gorilla KIR genes were defined. With attainment of these data, a set of 75 KIR sequences representing five hominoid species was assembled, which also included rhesus monkey, cattle, and rodent KIR. Searching this data set for recombination events, and phylogenetic analysis using Bayesian methods, demonstrated that new KIR were usually the result of recombination between loci in which complete protein domains were shuffled. Further phylogenetic analysis of the KIR sequences after removal of confounding recombined segments showed that only two KIR genes, KIR2DL4 and KIR2DL5, have been preserved throughout hominoid evolution, and one of them, KIR2DL4, is also common to rhesus monkey and hominoids. Other KIR genes represent recombinant forms present in a minority of species, often only one, as exemplified by 8 of the 11 gorilla KIR genes. more...

KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function. more...

Human NK cells are the earliest source of the protective cytokine IFN-gamma when PBMC from nonimmune donors are exposed to Plasmodium falciparum-infected RBC (iRBC) in vitro. In this study, we show that human NK cells form stable conjugates with iRBC but not with uninfected RBC and that induction of IFN-gamma synthesis is dependent on direct contact between the NK cell and the iRBC. NK cells respond to iRBC only in the presence of a source of IL-12/IL-18 and the subset of NK cells that preferentially respond to iRBC express high levels of the lectin-like receptor CD94/NKG2A. There is heterogeneity between donors in their ability to respond to iRBC. DNA analysis has revealed considerable heterogeneity of killer Ig-like receptor (KIR) genotype among the donor population and has identified 21 new KIR allelic variants in the donors of African and Asian descent. Importantly, we find evidence for significant associations between KIR genotype and NK responsiveness to iRBC. This emphasizes the need for large-scale population-based studies to address associations between KIR genotype and susceptibility to malaria. more...

Through differential killer cell Ig-like receptor (KIR) and CD94:NKG2 gene expression, human NK cells generate diverse repertoires, each cell having an inhibitory receptor for autologous HLA class I. Using a new method for measuring repertoire difference that integrates multiple flow cytometry parameters, we found individual repertoire stability, but population variability. Correlating repertoire differences with KIR and HLA genotype for 85 sibling pairs reveals the dominant influence of KIR genotype; HLA genotype having a subtle, modulating effect on relative KIR expression frequencies. HLA and/or KIR genotype also influences CD94:NKG2A expression. After HLA-matched stem cell transplantation, KIR repertoires either recapitulated that of the donor or were generally depressed for KIR expression. Human NK cell repertoires are defined by combinations of variable KIR and HLA class I genes and conserved CD94:NKG2 genes. more...

CD94, NKG2, Ly49, and killer cell Ig-like receptor (KIR) expressed by orangutan peripheral blood cells were examined by cloning and sequencing cDNA from a panel of individuals. Orthologs of human CD94, NKG2A, D, and F were defined. NKG2C and E are represented by one gene, Popy-NKG2CE, that is equidistant from the two human genes. Several Popy-CD94, NKG2A, and NKG2CE alleles were defined. Popy-Ly49L is expressed in cultured NK cells and has a sequence consistent with it encoding a functional receptor. Orangutan KIR corresponding to the three KIR lineages expressed in humans and chimpanzees were defined. Popy-KIR2DL4 of lineage I is the only ortholog of a human or chimpanzee KIR, but in all individuals examined, the transcripts of this gene produced premature termination, either in the D2 domain or at the beginning of the cytoplasmic domain. Ten Popy-KIR3DL and one Popy-KIR3DS of lineage II are all closely related, but represent the products of at least two genes. The two Popy-KIR2DL and four Popy-KIR2DS of lineage III also represent two genes, both being more related to KIR2DS4 than to other human and chimpanzee KIR of lineage III. The Popy-KIR2D include ones predicted to be specific for the C1 epitope of MHC-C, but none specific for C2. This correlates with the observation that all orangutan MHC-C allotypes examined have the C1 motif. more...

Killer Ig-like receptor (KIR) genes are a multigene family on human chromosome 19. KIR genes occur in various combinations on different haplotypes. Additionally, KIR genes are polymorphic. To examine how allelic polymorphism diversifies KIR haplotypes with similar or identical combinations of KIR genes, we devised methods for discriminating alleles of KIR2DL1, -2DL3, -3DL1, and -3DL2. These methods were applied to 143 individuals from 34 families to define 98 independent KIR haplotypes at the allele level. Three novel 3DL2 alleles and a chimeric 3DL1/3DL2 sequence were also identified. Among the A group haplotypes were 22 different combinations of 2DL1, 2DL3, 3DL1, and 3DL2 alleles. Among the B group haplotypes that were unambiguously determined were 15 distinct haplotypes involving 9 different combinations of KIR genes. A and B haplotypes both exhibit strong linkage disequilibrium (LD) between 2DL1 and 2DL3 alleles, and between 3DL1 and 3DL2 alleles. In contrast, there was little LD between the 2DL1/2DL3 and 3DL1/3DL2 pairs that define the two halves of the KIR gene complex. The synergistic combination of allelic polymorphism and variable gene content individualize KIR genotype to an extent where unrelated individuals almost always have different KIR types. This level of diversity likely reflects strong pressure from pathogens on the human NK cell response. more...

To assess polymorphism and variation in human and chimpanzee NK complex genes, we determined the coding-region sequences for CD94 and NKG2A, C, D, E, and F from several human (Homo sapiens) donors and common chimpanzees (Pan troglodytes). CD94 is highly conserved, while the NKG2 genes exhibit some polymorphism. For all the genes, alternative mRNA splicing variants were frequent among the clones obtained by RT-PCR. Alternative splicing acts similarly in human and chimpanzee to produce the CD94B variant from the CD94 gene and the NKG2B variant from the NKG2A gene. Whereas single chimpanzee orthologs for CD94, NKG2A, NKG2E, and NKG2F were identified, two chimpanzee paralogs of the human NKG2C gene were defined. The chimpanzee Pt-NKG2CI gene encodes a protein similar to human NKG2C, whereas in the chimpanzee Pt-NKG2CII gene the translation frame changes near the beginning of the carbohydrate recognition domain, causing premature termination. Analysis of a panel of chimpanzee NK cell clones showed that Pt-NKG2CI and Pt-NKG2CII are independently and clonally expressed. Pt-NKG2CI and Pt-NKG2CII are equally diverged from human NKG2C, indicating that they arose by gene duplication subsequent to the divergence of chimpanzee and human ancestors. Genomic DNA from 80 individuals representing six primate species were typed for the presence of CD94 and NKG2. Each species gave distinctive typing patterns, with NKG2A and CD94 being most conserved. Seven different NK complex genotypes within the panel of 48 common chimpanzees were due to differences in Pt-NKG2C and Pt-NKG2D genes. more...

HLA class I alleles containing premature termination codons (PTCs) are increasingly being found. To understand their effects on MHC class I expression, HLA-A*2402 mutants containing PTCs were transfected into class I-deficient cells, and expression of HLA-A mRNA and protein was determined. In exons 2, 3, and 4, and in the 5' part of exon 5, PTCs reduced mRNA levels by up to 90%, whereas in the 3' part of exon 5 and in exons 6 and 7 they had little effect. Transition in the extent of nonsense-mediated mRNA decay occurred within a 48-nt segment of exon 5, placed 58 nt upstream from the exon 5/exon 6 junction. This transition did not conform to the positional rule obeyed by other genes, which predicted it to be approximately 50-55 nt upstream of the exon 7/exon 8 junction and thus placing it in exon 6. Mutants containing extra gene segments showed the difference is caused by the small size of exons 5 and 6, which renders them invisible to the surveillance machinery. For the protein, a transition from secretion to membrane association occurs within a 26-nt segment of exon 5, 17 nt upstream of the exon 5/exon 6 junction. Premature termination in exon 5 can produce secreted and membrane-associated HLA-A variants expressed at high levels. more...

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family. more...