Studies in humans and macaques have demonstrated that CD8+ T cells responses are associated with the initial control of HIV or SIV replication. Natural killer (NK) cells also influence virus control and survival. These antiviral activities are dependent on major histocompatibility complex (MHC) molecules and specific MHC genotypes have been associated with lower viral loads and slower disease progression in humans and macaques. However, the exact correlates of protection remain unidentified and the immune responses required for an effective vaccine need to be defined. Crucial information can be obtained from animal models such as the infection of Asian macaques with SIV or SHIV viruses. Three macaque species are used to mimic HIV infection in pathogenesis and vaccine studies: rhesus (Macaca mulatta), pig-tailed (M. nemestrina) and cynomolgus (M. fascicularis) macaques. Pig-tailed macaques possess unique susceptibility and disease development characteristics that make this species particularly informative for AIDS research (high level of cellular activation, rapid disease development, susceptibility to various SIV strains). Our research is focused on exploring how the host genetic background of macaques affects their innate and adaptive immune response to SIV and SHIV infection. Specifically, our research concentrated on characterizing the NK cell capacity to detect infected cells. Previously we identified a subset of macaque NK cells capable of recognizing specific macaque MHC class I alleles (Mane-A1*082/A1*084) by a specific killer cell immunoglobulin receptor (KIR) expressed at the cell surface. Engagement of specific MHC alleles by a KIR3L allele, KIR049-4, results in inhibition of NK cell functions. We have characterized the specificity of the KIR3DL allele (KIR049-4) by screening its binding properties against a panel of MHC class I tetramers generated in collaboration with Dr. David Price (Cardiff University, UK). The panel consists in different macaque and human alleles (Mane-A, Mamu-A, Mamu-B, HLA-A and HLA-B) loaded with viral peptides derived from lentiviruses (SIV, HIV) and herpes viruses (EBV, CMV). We demonstrated that the KIR3DL receptor KIR049-4 has a broad reactivity to macaque and human MHC alleles harboring Bw4, Bw6 and non-Bw4/Bw6 epitope at the end of their alpha 1 helix. This observation contrasts with the specificity of human KIR3DL1 receptors, which is limited to Bw4 bearing MHC alleles. Furthermore, we showed that the nature of the peptide loaded in the MHC class I groove affected drastically the strength of the interaction and that some viral peptides exhibited antagonist properties against KIR049-4 binding. A second receptor responsible for NK cell reactivity with tetramer was identified after cloning multiple KIR alleles from positive animals, expressing these receptors in 721.221 cells and screening with tetramer binding assays. Two KIR3DL allelic variants (KIR033-1, KIR059-5) were isolated from distinct animals harboring primary NK cells subsets binding HLA-B*44 tetramers refolded with an HIV peptide. Further characterization of their MHC specificity using multiple tetramers demonstrated a distinct and narrower reactivity compared to the initial receptor KIR049-4. The presence of a basic amino acid at position 8 in the peptide appears to be a critical requirement for this new KIR3DL receptor, whereas acidic amino acids prevent the KIR MHC interaction. A third KIR3DL receptor (KIR033-7) interacts specifically with Mamu-A1*002 molecules loaded with various peptides. This receptor is an orthologue of the KIR3DL5 receptor identified in rhesus macaques, demonstrating the common evolution of KIR receptors in different macaque species. Further characterization of chimeric receptors between KIR049-4, KIR033-1/59-5 will shed light on the structural elements contributing to the peptide selectivity in KIR/MHC interaction. We have also expanded the characterization of the macaque NK cell pool by analyzing their reactivity to a large panel of MHC class I tetramers. Studying a cohort of pig-tailed macaques with 22 tetramers from human and pig-tailed/rhesus macaque origin, we identified 10 distinct patterns of reactivity corresponding to the expression of distinct polymorphic receptors by macaque NK cells. Each animal had a least one subset of NK cell identified by our tetramer panel, some animals harboring up to five distinct specificities. The prevalence of each receptor varied between 23% and 90% among the cohort. Moreover, the frequency of each NK cell subset was highly variable between animals. Coverage of the NK cell pool by the 10 specificities differed between animals: Complete coverage was obtained for some animals exhibiting multiple reactivity patterns, while coverage was partial in other individuals. This later observation suggests that the NK cell repertoire has a broad recognition spectrum that we have not completely probed yet. Indeed macaques express a larger variety of KIR molecules than found in humans. In addition to the receptors with 3 and 2 Ig domains observed in humans, macaques also possess receptors with 0 or 1 Ig domain. We detected the presence of these new receptors in multiple animals and cloned several allelic variants, demonstrating that they are encoded by polymorphic loci. Further, we expressed representative KIR0DL and KIR1DL molecules in 721.221 cells and detected their presence at the cell surface. This new observation suggests that the macaque-specific KIR1D and KIR0D molecules are truly functional NK cell receptors and not dysfunctional remains of KIR evolution in macaques. The exact nature of their function is unclear at this time and will need further investigations. We also investigated how macaque activating KIR molecules interact with adaptor molecules to activate NK cells. Using a macaque KIR3DS allele (KIR033-5), we showed in collaboration with the group of Dr. Daniel McVicar (NCI, Frederick) that KIR3DS molecules associate with DAP12, but in contrast to human KIR3DS molecules, this association was constitutive and did not dependent of a charged amino acid in the transmembrane region. Furthermore, the macaque KIR033-5 molecules associate with the FceRI-g adaptor molecule in stimulation-dependent manner. This association was dependent on the presence of an arginine residue in KIR033-5 transmembrane domain. This observation indicates that the regulation of macaque NK cell activation is more complex than described in humans. To improve our understanding of the KIR-MHC binding properties, we performed a comparative study of human and non-human primate MHC class I alleles for KIR binding in collaboration with the teams of Profs. Jamie Rossjohn (Monach University, Australia), Andrew Brooks (University of Melbourne, Australia) and David Price. This collaboration resulted in the tridimensional structure of a human KIR3DL molecule in interaction with HLA-B*57:01 molecules. This structure identified 17 amino acids in the MHC class I molecule that contact 21 amino acids in the KIR3DL1 molecule. Phylogenetic analyses of human, chimpanzee, gorilla, rhesus and pig-tailed macaque MHC class I molecules, based on the entire extra cellular region, resulted in clustering of MHC alleles based on their locus. The analysis focused on the amino acid positions serving as point of contacts for KIR resulted in clustering of MHC alleles based on their HLA-KIR binding properties. The greater diversity of macaque MHC alleles was associated with additional MHC clusters, that were not associated with human or Apes molecules, suggesting that macaques may harbor KIR molecules with MHC binding properties not present in humans or great Apes. Our characterization of KIR receptors and the NK cell repertoire in macaques represent a major step forward in elucidating the role of NK cells in the SIV-macaque model.
