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 experimental 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 macaques (Macaca mulatta), pig-tailed macaques (M. nemestrina) and cynomolgus monkeys (M. fascicularis). 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 this year concentrated on characterizing the NK cell capacity to detect infected cells. Previously, we have 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 activation, degranulation and cytokine production (TNFa). We have characterized further the specificity of the KIR3DL allele (KIR049-4) by screening its binding properties against a panel of 21 MHC class I tetramers generated in collaboration with Dr David Price (Cardiff University). The panel consists in 12 different macaque and human alleles (Mane-A, HLA-A and HLA-B) loaded with viral peptides derived from lentiviruses (SIV, HIV) and herperviruses (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. The receptor responsible for a second NK cell reactivity 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. For instance, KIR049-4 recognizes HLA-B*2705 loaded with a HIV Gag peptide but is unable to bind to HLA-B*44 loaded with a HIV RT peptide. In contrast, the newly identified receptors react completely in the opposite way. Both MHC alleles harbor a Bw4 epitope with a threonine in position 80. KIR049-4 and KIR033-1 alleles are also able to recognize HLA-B*5701, an MHC allele carrying a Bw4 epitope with an isoleucine in position 80. However, variations in the peptide at position 8 appear to have opposite effect for these receptors. While the interaction between KIR049-4 and HLA-B*5701 is strong when peptide position 8 is occupied by an small or acid amino acid (proline, alanine, glutamic acid) and the weakest with a basic amino acid (arginine), KIR033-1 has the strongest interaction with an arginine and looses the ability to bind HLA-B*5701 when peptide position 8 is occupied by a glutamic acid. Further characterization of chimeric receptors between KIR049-4 and 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 primary macaque NK cell subsets 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 nine distinct patterns of reactivity corresponding to the expression of distinct polymorphic receptors at the surface of 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 (range 1 to 59% of all NK cells). Coverage of the NK cell pool by the nine 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. In humans, the expression of KIR molecules is stochastic, resulting in the presence of NK cells subsets expressing a variable number of KIR alleles at their cell surface. We investigated the presence of 1, 2 or 3 distinct KIR receptors present at the surface of macaque NK cells by performing co-staining experiments. NK cell expressing multiple KIR molecules were more prevalent than expected by the product of individual frequencies. These results are in agreement with studies in humans indicating a preferential co-expression of multiple KIR receptors, and illustrate further that the regulation of KIR expressions is very similar in humans and macaques. 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 (University of Cardiff, UK). 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. However, the analysis focused on the amino acid positions serving as point of contacts for KIR, as determined by the tri-dimensional structure of the interaction between KIR3DL1 and HLA-B*5701 recently resolved by our Australian colleagues, resulted in clustering of MHC alleles based on their HLA-KIR binding properties. Furthermore 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 diversity of the NK cell repertoire in macaques represent a major step forward in elucidating the role of NK cells in the SIV-macaque model of infection.
