Human Killer Cell Ig-like Receptor Research Articles

KIR3DL3-HHLA2 is a human immunosuppressive pathway and a therapeutic target.

The B7 family ligand HERV-H LTR-associating protein 2 (HHLA2) is an attractive target for cancer immunotherapy because of its coinhibitory function, overexpression in human cancers, and association with poor prognoses. However, the knowledge of the HHLA2 pathway is incomplete. HHLA2 has an established positive receptor transmembrane and immunoglobulin (Ig) domain containing 2 (TMIGD2) but a poorly characterized negative receptor human killer cell Ig-like receptor, three Ig domains, and long cytoplasmic tail (KIR3DL3). Here, KIR3DL3 and TMIGD2 simultaneously bound to different sites of HHLA2. KIR3DL3 was mainly expressed on CD56dim NK and terminally differentiated effector memory CD8+ T (CD8+ TEMRA) cells. KIR3DL3+ CD8+ TEMRA acquired an NK-like phenotype and function. HHLA2 engagement recruited KIR3DL3 to the immunological synapse and coinhibited CD8+ T and NK cell function and killing, inducing immune-evasive HHLA2+ tumors. KIR3DL3 recruited SHP-1 and SHP-2 to attenuate Vav1, ERK1/2, AKT, and NF-κB signaling. HHLA2+ tumors from human kidney, lung, gallbladder, and stomach were infiltrated by KIR3DL3+ immune cells. KIR3DL3 blockade inhibited tumor growth in multiple humanized mouse models. Thus, our findings elucidated the molecular and cellular basis for the inhibitory function of KIR3DL3, demonstrating that the KIR3DL3-HHLA2 pathway is a potential immunotherapeutic target for cancer.

Human and Rhesus Macaque KIR Haplotypes Defined by Their Transcriptomes.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele-associated diseases, as well as the immune response in transplantation and reproduction.

Polymorphic HLA-C Receptors Balance the Functional Characteristics of KIR Haplotypes.

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.

HLA Reduces Killer Cell Ig-like Receptor Expression Level and Frequency in a Humanized Mouse Model

NK cells use NK cell receptors to be able to recognize and eliminate infected, transformed, and allogeneic cells. Human NK cells are prevented from killing autologous healthy cells by virtue of inhibitory NKRs, primarily killer cell Ig-like receptors (KIR) that bind "self" HLA class I molecules. Individual NK cells stably express a selected set of KIR, but it is currently disputed whether the fraction of NK cells expressing a particular inhibitory KIR is influenced by the presence of the corresponding HLA ligand. The extreme polymorphism of the KIR and HLA loci, with wide-ranging affinities for individual KIR and HLA allele combinations, has made this issue particularly hard to tackle. In this study, we used a transgenic mouse model to investigate the effect of HLA on KIR repertoire and function in the absence of genetic variation inside and outside the KIR locus. These H-2K(b-/-) and H-2D(b-/-) mice lacked ligands for inhibitory Ly49 receptors and were transgenic for HLA-Cw3 and a KIR B haplotype. In this reductionist system, the presence of HLA-Cw3 reduced the frequency of KIR2DL2(+) cells, as well as the surface expression levels of KIR2DL2. In addition, in the presence of HLA-Cw3, the frequency of NKG2A(+) cells and the surface expression levels of NKG2A were reduced. In line with these findings, both transgene-encoded KIR and endogenous NKG2A contributed to the rejection of cells lacking HLA-Cw3. These findings support the idea that HLA influences the human KIR repertoire.

Degenerate Recognition of MHC Class I Molecules with Bw4 and Bw6 Motifs by a Killer Cell Ig-like Receptor 3DL Expressed by Macaque NK Cells

The killer cell Ig-like receptors (KIRs) expressed on the surface of NK cells recognize specific MHC class I (MHC-I) molecules and regulate NK cell activities against pathogen-infected cells and neoplasia. In HIV infection, survival is linked to host KIR and MHC-I genotypes. In the SIV macaque model, however, the role of NK cells is unclear due to the lack of information on KIR-MHC interactions. In this study, we describe, to our knowledge, the first in-depth characterization of KIR-MHC interactions in pigtailed macaques (Macaca nemestrina). Initially, we identified three distinct subsets of macaque NK cells that stained ex vivo with macaque MHC-I tetramers loaded with SIV peptides. We then cloned cDNAs corresponding to 15 distinct KIR3D alleles. One of these, KIR049-4, was an inhibitory KIR3DL that bound MHC-I tetramers and prevented activation, degranulation, and cytokine production by macaque NK cells after engagement with specific MHC-I molecules on the surface of target cells. Furthermore, KIR049-4 recognized a broad range of MHC-I molecules carrying not only the Bw4 motif, but also Bw6 and non-Bw4/Bw6 motifs. This degenerate, yet peptide-dependent, MHC reactivity differs markedly from the fine specificity of human KIRs.

Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-Like Receptor Locus

The fast evolving human KIR gene family encodes variable lymphocyte receptors specific for polymorphic HLA class I determinants. Nucleotide sequences for 24 representative human KIR haplotypes were determined. With three previously defined haplotypes, this gave a set of 12 group A and 15 group B haplotypes for assessment of KIR variation. The seven gene-content haplotypes are all combinations of four centromeric and two telomeric motifs. 2DL5, 2DS5 and 2DS3 can be present in centromeric and telomeric locations. With one exception, haplotypes having identical gene content differed in their combinations of KIR alleles. Sequence diversity varied between haplotype groups and between centromeric and telomeric halves of the KIR locus. The most variable A haplotype genes are in the telomeric half, whereas the most variable genes characterizing B haplotypes are in the centromeric half. Of the highly polymorphic genes, only the 3DL3 framework gene exhibits a similar diversity when carried by A and B haplotypes. Phylogenetic analysis and divergence time estimates, point to the centromeric gene-content motifs that distinguish A and B haplotypes having emerged ∼6 million years ago, contemporaneously with the separation of human and chimpanzee ancestors. In contrast, the telomeric motifs that distinguish A and B haplotypes emerged more recently, ∼1.7 million years ago, before the emergence of Homo sapiens. Thus the centromeric and telomeric motifs that typify A and B haplotypes have likely been present throughout human evolution. The results suggest the common ancestor of A and B haplotypes combined a B-like centromeric region with an A-like telomeric region.

Coevolution of Killer Cell Ig-Like Receptors with HLA-C To Become the Major Variable Regulators of Human NK Cells

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.

Chimpanzees Use More Varied Receptors and Ligands Than Humans for Inhibitory Killer Cell Ig-Like Receptor Recognition of the MHC-C1 and MHC-C2 Epitopes

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.

Protein Kinase C Regulates Expression and Function of Inhibitory Killer Cell Ig-Like Receptors in NK Cells

The inhibitory killer cell Ig-like receptors (KIR) negatively regulate NK cell cytotoxicity by activating the Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 following ligation with MHC class I molecules expressed on normal cells. This requires tyrosine phosphorylation of KIR on ITIMs in the cytoplasmic domain. Surprisingly, we have found that KIR3DL1 is strongly and constitutively phosphorylated on serine and weakly on threonine residues. In this study, we have mapped constitutive phosphorylation sites for casein kinases, protein kinase C, and an unidentified kinase on the KIR cytoplasmic domain. Three of these phosphorylation sites are highly conserved in human inhibitory KIR. Functional studies of the wild-type receptor and serine/threonine mutants indicated that phosphorylation of Ser(394) by protein kinase C slightly suppresses KIR3DL1 inhibitory function, and reduces receptor internalization and turnover. Our results provide evidence that serine/threonine phosphorylation is an important regulatory mechanism of KIR function.

KIR3DL1 Polymorphisms That Affect NK Cell Inhibition by HLA-Bw4 Ligand

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.

Complete elucidation of a minimal class I MHC natural killer cell receptor haplotype

The BALB/c inbred mouse is widely used in models of infectious disease, transplantation, and cancer. The differences in the immune responses of BALB/c compared to C57BL/6 mice are especially valuable for the identification of immune regulation genes. One striking immune variance between these mice is in the function of natural killer (NK) cells, and there is strong evidence implicating differential expression of Ly49 genes. In this study, the complete BALB/c Ly49 gene cluster has been sequenced and found to contain six functional genes and two pseudogenes. Compared to C57BL/6 mice, there is a 200 kb region absent in the BALB/c cluster including a complete lack of Ly49h-related genes, which explains the increased susceptibility of BALB/c to cytomegalovirus infection. In addition, there is no BALB/c Ly49d allele, explaining the inability of BALB/c NK cells to kill certain tumor cells. The Ly49 region has now been sequenced in three different inbred mouse strains, and comparisons indicate that the evolution of each haplotype is not straightforward and has involved large-scale deletions/insertions, gene recombination, and unequal crossing over between divergent haplotypes. This study confirms that relatively small murine class I MHC receptor haplotypes exist, analogous to observations made of human killer cell Ig-like receptor gene haplotypes.

Cutting Edge: KIR2DL4 Transduces Signals into Human NK Cells through Association with the Fc Receptor γ Protein

KIR2DL4 (2DL4, CD158d), a member of the human killer cell Ig-like receptor (KIR) family, triggers potent IFN-gamma responses but weak cytotoxicity in resting NK cells. 2DL4 mRNA has been detected in most NK cell clones from most humans examined, but surface protein expression is detectable only on CD56(high) NK cells from certain donors. The receptor possesses a transmembrane arginine residue, suggesting association with a signaling accessory protein that has remained elusive. We provide biochemical and functional evidence that FcepsilonRI-gamma (gamma) associates with 2DL4 to promote surface expression and provide signal transducing function. Weak cytolytic responses triggered through 2DL4 may result from low stoichiometric association with gamma. Selective association with gamma distinguishes 2DL4 from all other activating forms of the KIR family, which alternatively associate with DNAX-activating protein (DAP)12.

Specific amplification of cDNA ends (SPACE): A new tool for the analysis of rare transcripts and its application for the promoter analysis of killer cell receptor genes

The expression control of activating and inhibitory killer cell Ig-like receptors (KIR) on natural killer (NK) cells is highly relevant for the initiation of NK cell mediated cytolysis and cytokine secretion. Transcription start points of nine human KIR genes from two Caucasian donors and the NK cell line NK3.3 were investigated. To overcome sensitivity problems due to the low abundance of the respective transcripts, a novel protocol, specific amplification of cDNA ends (SPACE) with superior specificity and sensitivity was applied. A total of 235 individual SPACE clones resulting from different KIR genes were analysed and revealed a series of transcription start sites tightly clustered between 10 and 60 bp upstream of the start codon. The comparison of the adjacent putative promoter region of the human, chimpanzee and macaque KIR genes revealed a very high conservation for almost all of the KIR family members. An inter-gene and inter-species comparative approach revealed transcription factor binding sites at regions of maximal homology for all primate KIR genes analysed.

The silent KIR3DP1 gene (CD158c) is transcribed and might encode a secreted receptor in a minority of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes are duplicated

Killer-cell Ig-like receptors (KIR) are structurally and functionally diverse, and enable human NK cells to survey the expression of individual HLA class I molecules, often altered in infections and tumors. Multiple events of non-reciprocal recombination have contributed to the rapid diversification of KIR. We show that approximately 4.5% of the individuals of a Caucasoid population bear a recombinant allele of KIR3DP1, officially designed KIR3DP1*004, that associates tightly with gene duplications of KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1. The KIR3DP1 gene is normally silent, but the recombinant allele carries a novel promoter sequence and, as a consequence, is transcribed in all tested individuals. Messenger RNA of KIR3DP1*004 is made up of six exons; of these, exons 1-5 are similar to, and spliced like, those encoding the leader peptide and Ig-domains of KIR3D. By contrast, exon 6 is homologous to no other human KIR sequence, but only to possible homologs in chimpanzees and rhesus macaques, and encodes a short hydrophilic tail. The putative KIR3DP1*004 product, like those of the related genes LAIR-2 and LILRA3/ILT6/LIR4, is predicted to be secreted to the extracellular medium rather than anchored to the cell membrane.

NK Cell Receptors of the Orangutan (Pongo pygmaeus): A Pivotal Species for Tracking the Coevolution of Killer Cell Ig-Like Receptors with MHC-C

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.

A BAC Contig Map of the Ly49 Gene Cluster in 129 Mice Reveals Extensive Differences in Gene Content Relative to C57BL/6 Mice

The murine Ly49 gene family is functionally analogous to the human killer cell Ig-like receptor (KIR) family of class I major histocompatibility complex (MHC) receptors. The number of KIR genes varies dramatically between individuals; however, the organization of the Ly49 genes has only been determined for the C57BL/6 (B6) mouse. The organization of the 129 Ly49 loci was determined from a BAC contig map by PCR and Southern blot analysis. In addition to the 10 Ly49 genes known from previous studies of the 129/J strain, 8 new genes were localized to the 129 Ly49 cluster. A gene order of Ly49q1, e, (v, q2), e/c2, l/r, s, t, e/c1, r, u, u/i, i1, g, p/d, (i2, p), and o was determined. The 129 Ly49 gene cluster is predicted to span ∼600 kb. These results indicate that Ly49 gene numbers can be significantly different between inbred mouse strains, analogous to the haplotype differences observed in the human KIR genes.

The ever-expanding Ly49 gene family: repertoire and signaling.

The mouse lectin-related Ly49 family and the human killer cell Ig-like receptor (KIR) family represent structurally distinct, yet functionally analogous, class I MHC receptors that are expressed on natural killer cells and some T cells. The functional similarity of these two families has been borne out by the demonstration of identical signal transduction pathways associated with each receptor family. The Ly49 family therefore provides a useful model system to study the role of this dass of receptors in the regulation of the immune system. Recent data relating to the Ly49 repertoire in several mouse strains has revealed an additional evolutionary parallel between KIR and Ly49 receptor families. There is now an appreciation of the variation in the number and type of Ly49s expressed in different mouse strains, similar to the previously demonstrated differences in the number of KIR genes found in humans. This review summarizes the current members of the Ly49 gene family, their MHC class I recognition and associated signal transduction pathways.

Diversity of the killer cell Ig-like receptors of rhesus monkeys.

Because the killer cell Ig-like receptors (KIRs) have only been characterized in humans and chimpanzees, we do not have a full understanding of their evolutionary history. Therefore, cDNAs encoding the KIR molecules of five rhesus monkeys were characterized, and were found to differ from the KIR molecules identified in humans and chimpanzees. Whereas only one KIR2DL4 molecule is detected in humans and chimpanzees, two distinct KIR2DL4 homologues were identified in the monkeys. Although the two human KIR3DL molecules are limited in their polymorphism, the KIR3DL homologues in the monkeys were highly polymorphic. Up to five KIR3DL homologues were identified in each monkey that was studied, and eleven distinct KIR3DL molecules were detected in the five rhesus monkeys. Two novel families of KIR molecules were identified in the rhesus monkeys, KIR3DH and KIR1D. The KIR3DH molecules have three Ig domains, transmembrane domains homologous to KIR2DL4 molecules that contain an arginine, and short cytoplasmic domains. With these features, the KIR3DH molecules resemble the activating forms of the human KIR molecules. The KIR1D molecule encodes only one complete Ig domain before a frame-shift in the second Ig domain occurs, leading to early termination of the molecule. Multiple splice variants of KIR1D exist that encode at least one Ig domain, as well as transmembrane and cytoplasmic domains. The extensive diversity of the rhesus monkey KIR3DL homologues and the novel KIR3DH and KIR1D molecules suggests that the KIR family of molecules has evolved rapidly during the evolution of primates.

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes.

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.