Knowledge of the MHC class I ligands of rhesus macaque killer-cell Ig-like receptors (KIRs) is fundamental to understanding the role of natural killer (NK) cells in this species as a nonhuman primate model for infectious diseases, transplantation and reproductive biology. We previously identified Mamu-AG as a ligand for KIR3DL05. Mamu-AG is a nonclassical MHC class I molecule that is expressed at the maternal-fetal interface of the placenta in rhesus macaques similar to HLA-G in humans. Although Mamu-AG and HLA-G share similar molecular features, including limited polymorphism and a short cytoplasmic tail, Mamu-AG is considerably more polymorphic. To determine which allotypes of Mamu-AG serve as ligands for KIR3DL05, we tested reporter cell lines expressing five different alleles of KIR3DL05 (KIR3DL05*001, KIR3DL05*004, KIR3DL05*005, KIR3DL05*008 and KIR3DL05*X) for responses to target cells expressing eight different alleles of Mamu-AG. All five allotypes of KIR3DL05 responded to Mamu-AG2*01:01, two exhibited dominant responses to Mamu-AG1*05:01, and three had low but detectable responses to Mamu-AG3*03:01, -AG3*03:02, -AG3*03:03 and -AG3*03:04. Since KIR3DL05*X is the product of recombination between KIR3DL05 and KIR3DS02, we also tested an allotype of KIR3DS02 (KIR3DS02*004) and found that this activating KIR also recognizes Mamu-AG2*01:01. Additional analysis of Mamu-AG variants with single amino acid substitutions identified residues in the α1-domain essential for recognition by KIR3DL05. These results reveal variation in KIR3DL05 and KIR3DS02 responses to Mamu-AG and define Mamu-AG polymorphisms that differentially affect KIR recognition.

Definition of the MHC class I ligands of rhesus macaque KIRs is fundamental to NK cell biology in this species as an animal model for infectious diseases, reproductive biology and transplantation. To provide a more complete foundation for studying NK cell responses in this species, rhesus macaque KIRs representing common allotypes of lineage II KIR genes were tested for interactions with MHC class I molecules representing diverse Mamu-A, -B, -E, -F, -I and -AG alleles. KIR-MHC class I interactions were identified by co-incubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR-Ig Fc domain fusion proteins. Ligands for 10 KIRs of previously unknown specificity were identified that fell into two general categories; interactions with multiple Mamu-Bw4 molecules (KIR3DL04*001, KIR3DL10*002, KIR3DL11*001 and KIR3DS04*003) or with Mamu-A-related molecules (KIR3DL07*004, KIR3DS01*003, KIR3DS02*004, KIR3DS06*002, KIR3DSw07*001 and KIR3DSw09*003). Among the latter group, three KIRs (KIR3DL07*004, KIR3DS02*004 and KIR3DSw09*003) also interacted with Mamu-B*045:03, a hybrid molecule with Mamu-A α1-domain sequences. Both groups include inhibitory and activating receptors that vary in the specificity and strength of their MHC class I interactions. However, whereas the majority of KIRs that recognize Mamu-Bw4 ligands are inhibitory, most of the KIRs that recognize Mamu-A-related ligands are activating. These findings more than double the number of rhesus macaque KIRs with defined MHC class I ligands and reveal an overlapping, but nonredundant, pattern of ligand recognition that supports extensive functional diversification of these receptors.