Mutational and Structural Analysis of KIR3DL1 Reveals a Lineage-Defining Allotypic Dimorphism That Impacts Both HLA and Peptide Sensitivity

Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01-restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation

Recognition of the Major Histocompatibility Complex (MHC) class Ib molecule H2-Q10 by the natural killer cell receptor Ly49C

Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 μm) than that observed between Ly49C and MHC-Ia (H-2Kb/H-2Dd, both ∼1 μm), and this recognition could be prevented by cis interactions with H-2K in situ. To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated β2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2Kb. Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2Kb possess similar energetic footprints focused around residues located within the Ly49C β4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules

Mutational and Structural Analysis of KIR3DL1 Reveals a Lineage-Defining Allotypic Dimorphism That Impacts Both HLA and Peptide Sensitivity

Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01–restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation

Killer cell immunoglobulin-like receptor 3DL1-mediated recognition of human leukocyte antigen B [Letter]

Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses1. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0–D1–D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies1, 2, 3. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards β2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an ‘innate HLA sensor’ domain. Second, whereas the D2–HLA-B*5701 interface exhibited a high degree of complementarity, the D1–pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C4, 5 and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1–pHLA-B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species

The Evolutionary Genetics and Emergence of Avian Influenza Viruses in Wild Birds

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient “genome constellations,” continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses

Killer cell immunoglobulin-like receptor 3DL1 polymorphism defines distinct hierarchies of HLA class I recognition

Natural killer (NK) cells play a key role in immunity, but how HLA class I (HLA-I) and killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1) polymorphism impacts disease outcome remains unclear. KIR3DL1 (*001/*005/*015) tetramers were screened for reactivity against a panel of HLA-I molecules. This revealed different and distinct hierarchies of specificity for each KIR3DL1 allotype, with KIR3DL1*005 recognizing the widest array of HLA-I ligands. These differences were further reflected in functional studies using NK clones expressing these specific KIR3DL1 allotypes. Unexpectedly, the Ile/Thr80 dimorphism in the Bw4-motif did not categorically define strong/weak KIR3DL1 recognition. Although the KIR3DL1*001, *005, and *015 polymorphisms are remote from the KIR3DL1-HLA-I interface, the structures of these three KIR3DL1-HLA-I complexes showed that the broader HLA-I specificity of KIR3DL1*005 correlated with an altered KIR3DL1*005 interdomain positioning and increased mobility within its ligand-binding site. Collectively, we provide a generic framework for understanding the impact of KIR3DL1 polymorphism on the recognition of HLA-I allomorphs

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

The role of the HLA class I α2 helix in determining ligand hierarchy for the killer cell Ig-like receptor 3DL1

HLA class I molecules that represent ligands for the inhibitory killer cell Ig-like receptor (KIR) 3DL1 found on NK cells are categorically defined as those HLA-A and HLA-B allotypes containing the Bw4 motif, yet KIR3DL1 demonstrates hierarchical recognition of these HLA-Bw4 ligands. To better understand the molecular basis underpinning differential KIR3DL1 recognition, the HLA-ABw4 family of allotypes were investigated. Transfected human 721.221 cells expressing HLA-A*32:01 strongly inhibited primary human KIR3DL1+ NK cells, whereas HLA-A*24:02 and HLA-A*23:01 displayed intermediate potency and HLA-A*25:01 failed to inhibit activation of KIR3DL1+ NK cells. Structural studies demonstrated that recognition of HLA-A*24:02 by KIR3DL1 used identical contacts as the potent HLA-B*57:01 ligand. Namely, the D1–D2 domains of KIR3DL1 were placed over the α1 helix and α2 helix of the HLA-A*24:02 binding cleft, respectively, whereas the D0 domain contacted the side of the HLA-A*24:02 molecule. Nevertheless, functional analyses showed KIR3DL1 recognition of HLA-A*24:02 was more sensitive to substitutions within the α2 helix of HLA-A*24:02, including residues Ile142 and Lys144. Furthermore, the presence of Thr149 in the α2 helix of HLA-A*25:01 abrogated KIR3DL1+ NK inhibition. Together, these data demonstrate a role for the HLA class I α2 helix in determining the hierarchy of KIR3DL1 ligands. Thus, recognition of HLA class I is dependent on a complex interplay between the peptide repertoire, polymorphisms within and proximal to the Bw4 motif, and the α2 helix. Collectively, the data furthers our understanding of KIR3DL1 ligands and will inform genetic association and immunogenetics studies examining the role of KIR3DL1 in disease settings

The presence of HLA-E-Restricted, CMV-Specific CD8+ T Cells in the blood of lung transplant recipients correlates with chronic allograft rejection

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8+ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation

The Interaction of KIR3DL1*001 with HLA Class I Molecules Is Dependent upon Molecular Microarchitecture within the Bw4 Epitope

The killer cell Ig-like receptor 3DL1 (KIR3DL1) inhibits activation of NK cells upon interaction with HLA class I molecules such as HLA-B*57:01, which contains the Bw4 epitope spanning residues 77-83 (e.g., NLRIALR), and not with HLA allomorphs that possess the Bw6 motif (e.g., HLA-B*08:01), which differ at residues 77, 80, 81, 82, and 83. Although Bw4 residues Ile(80) and Arg(83) directly interact with KIR3DL1*001, their precise role in determining KIR3DL1-HLA-Bw4 specificity remains unclear. Recognition of HLA-B*57:01 by either KIR3DL1(+) NK cells or the NK cell line YTS transfected with KIR3DL1*001 was impaired by mutation of residues 80 and 83 of HLA-B*57:01 to the corresponding amino acids within the Bw6 motif. Conversely, the simultaneous introduction of three Bw4 residues at positions 80, 82, and 83 into HLA-B*08:01 conferred an interaction with KIR3DL1*001. Structural analysis of HLA-B*57:01, HLA-B*08:01, and mutants of each bearing substitutions at positions 80 and 83 revealed that Ile(80) and Arg(83) within the Bw4 motif constrain the conformation of Glu(76), primarily through a salt bridge between Arg(83) and Glu(76). This salt bridge was absent in HLA-Bw6 molecules as well as position 83 mutants of HLA-B*57:01. Mutation of the Bw4 residue Ile(80) also disrupted this salt bridge, providing further insight into the role that position 80 plays in mediating KIR3DL1 recognition. Thus, the strict conformation of HLA-Bw4 allotypes, held in place by the Glu(76)-Arg(83) interaction, facilitates KIR3DL1 binding, whereas Bw6 allotypes present a platform on the α1 helix that is less permissive for KIR3DL1 binding