Specific interactions between killer cell Ig-like receptors (KIRs) and MHC class I ligands have not been described in rhesus macaques despite their importance in biomedical research. Using KIR–Fc fusion proteins, we detected specific interactions for three inhibitory KIRs (3DLW03, 3DL05, 3DL11) and one activating KIR (3DS05). As ligands we identified Macaca mulatta MHC (Mamu)-A1– and Mamu-A3–encoded allotypes, among them Mamu-A1*001:01, which is well known for association with slow progression to AIDS in the rhesus macaque experimental SIV infection model. Interactions with Mamu-B or Mamu-I molecules were not found. KIR3DLW03 and KIR3DL05 differ in their binding sites to their shared ligand Mamu-A1*001:01, with 3DLW03 depending on presence of the α1 domain, whereas 3DL05 depends on both the α1 and α2 domains. Fine-mapping studies revealed that binding of KIR3DLW03 is influenced by presence of the complete Bw4 epitope (positions 77, 80–83), whereas that of KIR3DL05 is mainly influenced by amino acid position 77 of Bw4 and positions 80–83 of Bw6. Our findings allowed the successful prediction of a further ligand of KIR3DL05, Mamu-A1*002:01. These functional differences of rhesus macaque KIR3DL molecules are in line with the known genetic diversification of lineage II KIRs in macaques.

Killer cell immunoglobulin-like receptors (KIR) regulate the activity of natural killer (NK) cells and have been shown to be associated with susceptibility to a number of human infectious diseases. Here, we analyzed NK cell function and genetic associations in a cohort of 52 rhesus macaques experimentally infected with SIVmac and subsequently stratified into high viral load (HVL) and low viral load (LVL) plasma viral loads at set point. This stratification coincided with fast (HVL) and slow (LVL) disease progression indicated by the disease course and critical clinical parameters including CD4+ T cell counts. HVL animals revealed sustained proliferation of NK cells but distinct loss of peripheral blood NK cell numbers and lytic function. Genetic analyses revealed that KIR genes 3DL05, 3DS05, and 3DL10 as well as 3DSW08, 3DLW03, and 3DSW09 are correlated, most likely due to underlying haplotypes. SIV-infection outcome associated with presence of transcripts for two inhibitory KIR genes (KIR3DL02, KIR3DL10) and three activating KIR genes (KIR3DSW08, KIR3DS02, KIR3DS05). Presence of KIR3DL02 and KIR3DSW08 was associated with LVL outcome, whereas presence of KIR3DS02 was associated with HVL outcome. Furthermore, we identified epistasis between KIR and MHC class I alleles as the transcript presence of the correlated genes KIR3DL05, KIR3DS05, and KIR3DL10 increased HVL risk when Mamu-B*012 transcripts were also present or when Mamu-A1*001 transcripts were absent. These genetic associations were mirrored by changes in the numbers, the level of proliferation, and lytic capabilities of NK cells as well as overall survival time and gastro-intestinal tissue viral load.

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-012-0640-2) contains supplementary material, which is available to authorized users.

Human killer cell immunoglobulin-like receptors (KIR) recognize A3/11, Bw4, C1 and C2 epitopes carried by mutually exclusive subsets of HLA-A, B, and C allotypes. Chimpanzee and orangutan have counterparts to HLA-A, B, and C, and KIR that recognize the A3/11, Bw4, C1 and C2 epitopes, either individually or in combination. Because rhesus macaque has counterparts of HLA-A and B, but not HLA-C, we expected that rhesus KIR would better recognize HLA-A and B, than HLA-C. Comparison of the interactions of nine rhesus KIR3D with 95 HLA isoforms, showed the KIR have broad specificity for HLA-A, B, and C, but vary in avidity. Considering both the strength and breadth of reaction, HLA-C was the major target for rhesus KIR, followed by HLA-B, then HLA-A. Strong reactions with HLA-A were restricted to the minority of allotypes carrying the Bw4 epitope, whereas strong reactions with HLA-B partitioned between allotypes having and lacking Bw4. Contrasting to HLA-A and B, every HLA-C allotype bound to the nine rhesus KIR. Sequence comparison of high- and low-binding HLA allotypes revealed the importance of polymorphism in the helix of the α1 domain and the peptide-binding pockets. At peptide position 9, nonpolar residues favor binding to rhesus KIR, whereas charged residues do not. Contrary to expectation, rhesus KIR bind more effectively to HLA-C, than to HLA-A and B. This property is consistent with MHC-C having evolved in hominids to be a generally superior ligand for KIR than MHC-A and MHC-B.

The expression of killer cell immunoglobulin-like receptors (KIR) on lymphocytes of rhesus macaques and other Old World monkeys was unknown so far. We used our recently established monoclonal anti-rhesus macaque KIR antibodies in multicolour flow cytometry for phenotypic characterization of KIR protein expression on natural killer (NK) cells and T cell subsets of rhesus macaques, cynomolgus macaques, hamadryas baboons, and African green monkeys. Similar to human KIR, we found clonal expression patterns of KIR on NK and T cell subsets in rhesus macaques and differences between individuals using pan-KIR3D antibody 1C7 and antibodies specific for single KIR. Similar results were obtained with lymphocytes from the other studied species. Notably, African green monkeys show only a low frequency of KIR3D expressed on CD8+ αβT cells. Contrasting human NK cells are KIR-positive CD56bright NK cells and frequencies of KIR-expressing NK cells that are independent of the presence of their cognate MHC class I ligands in rhesus macaques. Interestingly, the frequency of KIR-expressing cells and the expression strength of KIR3D are correlated in γδ T cells of rhesus macaques and CD8+ αβT cells of baboons.

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histocompatibility complex (MHC) class I molecules and a second group that do not recognise these ligands. The second group comprises, besides others, the natural cytotoxicity receptors that are potent activating receptors linked to ITAM (immunoreceptor tyrosine-based activating motif) bearing adaptor molecules. NK cells are also able to detect antibody-coated cells by CD16 (FcγRIIIA) and induce antibodydependent cell cytotoxicity (ADCC) and cytokine production (Perussia et al., 1983). The and belong to the Ig superfamily (Colonna and Samaridis, 1995;D'Andrea et al., 1995; Wagtmann et al., 1995). KIRs are regulatory molecules mainly expressed by NK cells but also by CD8 + αβ T cells and γδ T cells (Moretta et al., 1990; Snyder et al., 2004). The human KIR genes are located in a dense gene cluster on chromosome 19q13.4 and are part of the leucocyte receptor complex (LRC), which in addition to KIR codes also for other Ig-like receptors (Wende et al., 1999). Human KIR haplotypes differ by presence and absence of KIR genes and by allelic variability. To clearly distinguish between these two types of genetic variability this is also referred to as diversity (presence/absence of genes) and polymorphism (alleles). The diversity of KIR genes is based on expansions/contractions and recombination of KIR genes. Therefore, KIR molecules with similar ligand specificities but with different signalling pathways have evolved (e.g. inhibitory KIR2DL1 and activating KIR2DS1). Polymorphism is often accompanied StructureofKIRmoleculesThe nomenclature of KIR molecules and their genes are based on the structure of the corresponding protein. The abbreviation "KIR" is followed by the number of Ig domains ("D") followed by a letter describing the length of the cytoplasmic tail "S" (shortactivating) and "L" (long -inhibitory). The last number stands for the gene that is coding for the KIR molecule. All KIR molecules originate from a long-tailed 3D KIR (Sambrook et al., 2006). This KIR is organised in 9 exons that correspond to the functional areas of the KIR molecule (Martin et al., 2000). Exons 1 and 2 code for the signal sequence and the following exons (exon 3-5) for the Ig domains (D0, D1 and D2).Human KIR molecules occur mostly with two Ig domains (KIR2D) that can be classified into two groups (Figure2). KIR molecules containing the D1 and D2 domain are called type 1 KIRs and have the same genomic arrangement as 3D KIRs. They possess the D0 domain encoding exon 3 that behaves like a "pseudo-exon" whereby the D0 domain is missing in type 1 KIRs (Vilches and Parham, 2002). Type 2 KIRs such as KIR2DL4 and KIR2DL5 have the D0 and D2 domain. However, due to deletion of exon 4, the D1 domain is absent (Selvakumar et al., 1997;Vilches et al., 2000). Crystal structures for KIR2DL1, KIR2DL2 and very recent for KIR3DL1 in complex with their MHC class I ligands showed the three-dimensional structure of those molecules and provided information about their ligand-binding characteristics (Boyington et al., 2000; Fan et al., 2001; Vivian et al....