• A bird's eye view of NK cell receptor interactions with their MHC class I ligands.

      Saunders, Philippa M.; Vivian, Julian P.; O'Connor, Geraldine M.; Sullivan, Lucy C.; Pymm, Phillip; Rossjohn, Jamie; Brooks, Andrew G. (2015-08-18)
      The surveillance of target cells by natural killer (NK) cells utilizes an ensemble of inhibitory and activating receptors, many of which interact with major histocompatibility complex (MHC) class I molecules. NK cell recognition of MHC class I proteins is important developmentally for the acquisition of full NK cell effector capacity and during target cell recognition, where the engagement of inhibitory receptors and MHC class I molecules attenuates NK cell activation. Human NK cells have evolved two broad strategies for recognition of human leukocyte antigen (HLA) class I molecules: (i) direct recognition of polymorphic classical HLA class I proteins by diverse receptor families such as the killer cell immunoglobulin-like receptors (KIRs), and (ii) indirect recognition of conserved sets of HLA class I-derived peptides displayed on the non-classical HLA-E for recognition by CD94-NKG2 receptors. In this review, we assess the structural basis for the interaction between these NK receptors and their HLA class I ligands and, using the suite of published KIR and CD94-NKG2 ternary complexes, highlight the features that allow NK cells to orchestrate the recognition of a range of different HLA class I proteins.
    • Characterization of a weakly expressed KIR2DL1 variant reveals a novel upstream promoter that controls KIR expression

      Wright, Paul W.; Li, Honchuan; Huehn, Andrew; O'Connor, Geraldine M.; Cooley, Sarah; Miller, Jeffrey S.; Anderson, Stephen K.; Basic Science Program, Leidos Biomedical Research Inc., Lab of Experimental Immunology, Frederick National Lab, Frederick, MD, USA. Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA. Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, USA. (Nature Publishing Group, 2014-07-03)
      Members of the human KIR (killer cell immunoglobulin-like receptor) class I major histocompatibility complex receptor gene family contain multiple promoters that determine the variegated expression of KIR on natural killer cells. In order to identify novel genetic alterations associated with decreased KIR expression, a group of donors was characterized for KIR gene content, transcripts and protein expression. An individual with a single copy of the KIR2DL1 gene but a very low level of gene expression was identified. The low expression phenotype was associated with a single-nucleotide polymorphism (SNP) that created a binding site for the inhibitory ZEB1 (Zinc finger E-box-binding homeobox 1) transcription factor adjacent to a c-Myc binding site previously implicated in distal promoter activity. Individuals possessing this SNP had a substantial decrease in distal KIR2DL1 transcripts initiating from a novel intermediate promoter located 230 bp upstream of the proximal promoter start site. Surprisingly, there was no decrease in transcription from the KIR2DL1 proximal promoter. Reduced intermediate promoter activity revealed the existence of alternatively spliced KIR2DL1 transcripts containing premature termination codons that initiated from the proximal KIR2DL1 promoter. Altogether, these results indicate that distal transcripts are necessary for KIR2DL1 protein expression and are required for proper processing of sense transcripts from the bidirectional proximal promoter.
    • Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells

      Townsley, Elizabeth; O'Connor, Geraldine M.; Cosgrove, Cormac; Woda, Marcia; Co, Mary; Thomas, Stephen J.; Kalayanarooj, Siripen; Yoon, In-Kyu; Nisalak, Ananda; Srikiatkhachorn, Anon; et al. (2015-10-06)
      Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.
    • The interaction of KIR3DL1*001 with HLA class I molecules is dependent upon molecular microarchitecture within the Bw4 epitope

      Saunders, Philippa M.; Vivian, Julian P.; Baschuk, Nikola; Beddoe, Travis; Widjaja, Jacqueline M.; O’Connor, Geraldine M.; Hitchen, Corinne; Pymm, Phillip; Andrews, Daniel M.; Gras, Stephanie; et al. (2015-01-02)
      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 alpha1 helix that is less permissive for KIR3DL1 binding.
    • Killer cell Immunoglobulin-like Receptor 3DL1 polymorphism defines distinct hierarchies of HLA class I recognition

      Saunders, Philippa M.; Pymm, Phillip; Pietra, Gabriella; Hughes, Victoria A.; Hitchen, Corinne; O'Connor, Geraldine M.; Loiacono, Fabrizio; Widjaja, Jacqueline M.; Price, David A.; Falco, Michela; et al. (Rockefeller University Press, 2016-04-04)
      NK cells play a key role in immunity, but how HLA-I and KIR3DL1 polymorphism impacts on 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 recognising the widest array of HLA-I ligands. These differences were further reflected in unctional studies utilising 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.
    • Mutational and Structural Analysis of KIR3DL1 Reveals a Lineage-Defining Allotypic Dimorphism That Impacts Both HLA and Peptide Sensitivity

      O'Connor, Geraldine M.; Vivian, Julian P.; Widjaja, Jacqueline M.; Bridgeman, John S.; Gostick, Emma; Lafont, Bernard A.; Anderson, Stephen K.; Price, David A.; Brooks, Andrew G.; Rossjohn, Jamie; et al. (American Association of Immunologists, 2014-03-15)
      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.
    • Pemphigus is associated with KIR3DL2 expression levels and provides evidence that KIR3DL2 may bind HLA-A3 and A11 in vivo.

      Augusto, Danillo G.; O'Connor, Geraldine M.; Lobo-Alves, Sara C.; Bass, Sara; Martin, Maureen P.; Carrington, Mary; McVicar, Daniel W.; Petzl-Erler, Maria L.; Departamento de Genética, Universidade Federal do Paraná, Curitiba, PR, Brazil; Ragon Institute of MGH, MIT and Harvard, Boston, MA, USA; Leidos Biomedical Research, Cancer and Inflammation Program, Laboratory for Experimental Immunology, Frederick National Laboratory for Cancer Research, Frederick, MD, USA; Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA. (Wiley, 2015-05-06)
      Although HLA-A3 and A11 have been reported to be ligands for KIR3DL2, evidence for any in vivo relevance of this interaction is still missing. To explore the functional importance of KIR3DL2 allelic variation, we analyzed the autoimmune disease pemphigus foliaceus, previously associated (lower risk) with activating KIR genes. KIR3DL2*001 was increased in patients (odds ratio (OR) = 2.04; p = 0.007). The risk was higher for the presence of both KIR3DL2*001 and HLA-A3 or A11 (OR = 3.76, p = 0.013), providing the first evidence that HLA-A3 and A11 may interact with KIR3DL2 in vivo. The nonsynonymous single nucleotide polymorphism 1190T (rs3745902) was associated with protection (OR = 0.52, p = 0.018). This SNP results in a threonine-to-methionine substitution. Individuals who have methionine in this position exhibit a lower percentage of KIR3DL2-positive natural killer (NK) cells and also lower intensity of KIR3DL2 on expressing natural killer cells; additionally, we show that the expression of KIR3DL2 is independent of other killer cell immunoglobulin-like receptors. Pemphigus foliaceus is a very unique complex disease strongly associated with immune-related genes. It is the only autoimmune disease known to be endemic, showing a strong correlation with environmental factors. Our data demonstrate that this relatively unknown autoimmune disease may facilitate understanding of the molecular mechanisms of KIR3DL2 ligand recognition.
    • Peptide-Dependent Recognition of HLA-B*57:01 by KIR3DS1

      O'Connor, Geraldine M.; Vivian, Julian P.; Gostick, Emma; Pymm, Phillip; Lafont, Bernard A.; Price, David A.; Rossjohn, Jamie; Brooks, Andrew G.; McVicar, Daniel W.; Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia. Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, Wales, United Kingdom. Non-Human Primate Immunogenetics and Cellular Immunology Unit, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, Wales, United Kingdom Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria, Australia Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, Wales, United Kingdom. Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia. (American Society for Microbiology, 2015-04-21)
      Killer cell immunoglobulin-like receptors (KIRs) play an important role in the activation of natural killer (NK) cells, which in turn contribute to the effective immune control of many viral infections. In the context of HIV infection, the closely related KIR3DL1 and KIR3DS1 molecules, in particular, have been associated with disease outcome. Inhibitory signals via KIR3DL1 are disrupted by downregulation of HLA class I ligands on the infected cell surface and can also be impacted by changes in the presented peptide repertoire. In contrast, the activatory ligands for KIR3DS1 remain obscure. We used a structure-driven approach to define the characteristics of HLA class I-restricted peptides that interact with KIR3DL1 and KIR3DS1. In the case of HLAB*57:01, we used this knowledge to identify bona fide HIV-derived peptide epitopes with similar properties. Two such peptides facilitated productive interactions between HLA-B*57:01 and KIR3DS1. These data reveal the presence of KIR3DS1 ligands within the HIV-specific peptide repertoire presented by a protective HLA class I allotype, thereby enhancing our mechanistic understanding of the processes that enable NK cells to impact disease outcome.