Studies in humans and in macaques have demonstrated that CD8+ T cells responses are associated with the initial control of HIV or SIV replication. Specific major histocompatibility complex (MHC) genotypes have been found to be associated with lower viral loads and slower disease progression. However the exact correlates of protection remain unidentified and cellular immune responses required for an effective vaccine need to be defined. Crucial information can be obtained from experimental models of infection such as infection of Asian macaques with SIV or SHIV viruses, but also from studies of natural hosts of SIVs, such as African green monkeys, which exhibit high viral loads but remain disease-free. Comparison of immune responses induced in these natural hosts and in Asian macaques infected with the same SIV strains would shed light on the protective mechanisms used by African primates to resist the development of immunodeficiency. Three macaque species are used to mimic HIV infection in pathogenesis and vaccine studies, namely rhesus macaques (Macaca mulatta), pig-tailed macaques (M. nemestrina) and cynomolgus monkeys (M. fascicularis). Pig-tailed macaques possess particular susceptibility and disease development characteristics that make this species particularly informative for AIDS research. Specific MHC class I alleles have been associated with slower disease progression and lower viral loads in humans and in macaques. A similar link between MHC genetic background and disease course has not been established for pig-tailed macaques. Furthermore the organization of MHC class I genes in the African green monkeys remains unknown. Given our expertise in macaque immunogenetics, several investigators, including Dr. Hirsch, Dr. Martin and Dr. Roederer at NIAID and Dr. Mattapallil at USUHS, have requested our help in 2009 for MHC genotyping of their rhesus macaques. For most studies, we assessed the presence of specific Mamu alleles (Mamu-A*01, Mamu-B*08, Mamu-B*290101, Mamu-B*7301) by SSP-PCR assays, some of which were developed in our lab. For one specific study, we obtained full length Mamu-A and Mamu-B cDNAs by RT-PCR from each animal before cloning and sequencing. Individual Mamu-A and Mamu-B alleles were identified by comparing each clone to a database of 56 Mamu-A and 157 Mamu-B alleles. This more exhaustive genotyping approach allowed to obtain a broader view of the MHC class I allele repertoire expressed in each animal. In parallel to studies characterizing the immunogenetics of pig-tailed macaques, we are exploring how the host genetic background affects the immune response to SIV and SHIV infection in this species. Previously, we have shown that CD8+ T cells from several SHIV-infected pig-tailed macaques responded to a SIV p27 Gag epitope (HR9 HQAAMQIIR) by producing IFNg and TNFa. These animals had controlled viral replication soon after inoculation and have remained asymptomatic for up to 6 years with undetectable levels of plasma viral RNA. In contrast, other pig-tailed macaques inoculated under similar conditions experienced a complete and irreversible elimination of their CD4+ T cells and had to be euthanized within 6 months post infection. Further analyses demonstrated that CD8+ T cells responses specific to SIV Gag HR9 were restricted by the Mane-A*03 allele, potentially explaining the difference in disease progression between the two groups of monkeys. However, not all infected pig-tailed macaques carrying the Mane-A*03 allele had SIV Gag HR9 specific CD8+ T cells, as detected by intracellular IFNg staining. In 2009, we performed tetramer staining to assess if this lack of detection was due to the absence of SIV Gag HR9 specific CD8+ T lymphocytes, or if such CD8+ T cells were present but not detected because their response was skewed toward expression of other effector molecules. Mane-A*03 tetramers loaded with SIV Gag HR9 peptide or a shorter peptide were generated in collaboration with Dr David Price. In macaques responsive to SIV Gag HR9, the presence of epitope specific CD8+ T cells was confirmed by staining with the Mane-A*03 tetramer loaded with Gag HI8 rather than Gag HR9. This observation suggests that the optimal peptide for Mane-A*03 binding is likely an 8-mer. Staining with SIV Gag HI8 Mane-A*03 and SIV Gag HR9 Mane-A*03 tetramers confirmed the absence of CD8+ T cells able to recognize the epitope in the unresponsive animals. However, a significant subpopulation (6 to 22%) of NK cells was labeled with both tetramers. Further analyses showed that tetramer staining of NK cells was restricted to some animals. All animals with tetramer positive NK cells were SHIV-infected pig-tailed macaques and had background level of staining on their CD8+ T cells. There was no correlation with the presence of Mane-A*03 allele in these animals. A third tetramer, Mane-A*1702 molecules loaded with SIV Gag AF9 peptide, did not bind NK cells in macaques who had Mane-A*03 tetramer positive NK cells, suggesting that Mane-A*03 tetramer binding on NK cells was MHC- and peptide-specific. In addition, the third tetramer, SIV Gag AF9 Mane-A*1702, recognized a significant subset of NK cells (10-15%) in other infected pig-tailed macaques. To investigate the nature of the receptors responsible for this interaction, we initially performed phenotypic analyses of pig-tailed macaque NK cells positive or negative with Mane-A*03 or Mane-A*1702 tetramers. In all animals tested, almost all NK cells, expressed the NK receptors NKG2A, NKG2D, NKp30, NKp46, the adhesion molecule CD2, the IL-2Rb chain, the FcGRIII receptor. In contrast CD56, an adhesion molecule found on human NK cells, was only present to a very small subset of NK cells in all animals. None of these markers appeared to correlate with tetramer binding patterns. These patterns which are observed in a limited number of animals, are restricted to a minor NK cell subsets, and are specific to an MHC allele / peptide complex, could be due however to the presence of polymorphic NK receptors involved in self, non-self and altered self recognition. Primates express such molecules at the NK cell surface as a family of receptors named killer Ig-like receptors (KIR). KIRs comprise activating and inhibitory molecules, which recognized MHC molecules in a peptide dependant manner. KIRs are encoded by a multigenic family and the diversity at the population level is generated by both the number of genes carried on the chromosome and by gene polymorphism. Within an individual, NK cell diversity is also generated by the expression of some but not all KIR genes in a stochastic manner. KIRs have not been described in pig-tailed macaques and we have initiated in 2009 their molecular characterization. For this purpose, we have developed a culture system allowing expansion of pig-tailed macaque NK cells from peripheral blood. After depletion of CD8b+ and gd lymphocytes from PBMC, the remaining cell fraction that includes CD4+ T cells, was cultured in presence of IL-15 and IL-2 and stimulated with MHC deficient 721.221 cells. The cells were maintained in culture for 2 to 5 weeks and NK cells expanded more than 20 fold on average. The proportion of NK cells increased over time representing between 30 and 80% of all cells. Initial cloning experiments have already identified multiple KIR alleles expressed in pig-tailed macaques NK cells. The diversity of receptor comprises KIR molecules containing either 3 Ig domains, 1 Ig domain or no Ig domain. Analysis of their transmembrane and cytoplasmic tail suggests the presence of both inhibitory and activating receptors. Further experiments will be conducted to identify the receptors involved in tetramer binding and characterize the immune response associated with this property.
