ABSTRACT
Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the beta4-beta5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49G(B6) and a Ly-49G(B6)-like receptor, Ly-49M(nonobese diabetic), but not Ly-49G(BALB/c). By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the beta4-beta5 loop and the beta1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49G(B6) ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the beta4-beta5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Ly/immunology , Epitope Mapping , Killer Cells, Natural/immunology , Lymphocyte Subsets/immunology , Amino Acid Sequence , Animals , Antigens, Ly/chemistry , Antigens, Ly/genetics , COS Cells , Chlorocebus aethiops , Flow Cytometry , Genes, MHC Class I/immunology , Killer Cells, Natural/cytology , Lectins, C-Type , Lymphocyte Subsets/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Polymorphism, Genetic , Protein Structure, Tertiary , Receptors, NK Cell Lectin-Like , Sequence Alignment , Species SpecificityABSTRACT
Mouse Ly-49 receptors are known to recognize xenogeneic ligands from hamster and rat. However, until now, there has been no description of a specific rat xenogeneic ligand for any mouse Ly-49 receptor. In this report, we identify RT1-A1c, a rat classical class I MHC molecule, as a ligand for the Ly-49G(BALB/c) inhibitory receptor and the closely related activating receptor, Ly-49W. Xenogeneic class I recognition of targets from PVG but not DA strain rats was mapped to the classical region of the RT1c haplotype by using Con A blasts from RT1c/RT1av1 intra-MHC recombinant rats as targets for RNK-16 cells expressing either Ly-49W or Ly-49G(BALB/c) receptors. Individual expression of class I molecules from PVG and DA rat strains in YB2/0 target cells demonstrate the xenogeneic recognition to be allele specific, because other class I molecules of the RT1c haplotype, RT1-A2c and RT1-U2c, and a classical class I molecule encoded by the RT1av1 haplotype, RT1-Aa, are not recognized by Ly-49W and -G(BALB/c). Furthermore, specificity for RT1-Ac can be transferred from Ly-49W to Ly-49P, which is normally unable to recognize RT1-Ac, by substitution of three residues shared by Ly-49W and -G(BALB/c) but not Ly-49P. These residues are located in the Ly-49 beta4-beta5 loop, which can determine class I allele specificity in mouse Ly-49 receptor interactions with mouse class I ligands, suggesting that mouse Ly-49 recognition of rat class I molecules follows similar principles of interaction. These findings have implications for xenotransplantation studies and for discerning Ly-49 recognition motifs present in MHC molecules.
Subject(s)
Antigen Presentation , Antigens, Heterophile/metabolism , Antigens, Ly/metabolism , Cytotoxicity, Immunologic , Histocompatibility Antigens/metabolism , Lymphocyte Activation , Alleles , Animals , Antigen Presentation/genetics , Antigens, Heterophile/genetics , Antigens, Ly/genetics , Antigens, Ly/physiology , Cell Line, Tumor , Concanavalin A/pharmacology , Cytotoxicity, Immunologic/genetics , Female , Histocompatibility Antigens/genetics , Histocompatibility Antigens Class I/metabolism , Lectins, C-Type , Ligands , Lymphocyte Activation/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Mutagenesis, Site-Directed , Protein Structure, Tertiary/genetics , Rats , Receptors, NK Cell Lectin-Like , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TransfectionABSTRACT
Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2D(d), or H-2D(d) and D(k), respectively. In this report, we demonstrate that specificity for H-2D(k) can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the beta4-beta5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2D(k) recognition while still preserving H-2D(d) recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the beta4-beta5 loop, in determining class I MHC allele specificity.
Subject(s)
Alleles , Amino Acid Substitution/immunology , Antigens, Ly/metabolism , H-2 Antigens/genetics , Lymphocyte Activation/genetics , Receptors, Immunologic/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Antibody-Dependent Cell Cytotoxicity/genetics , Antigens, Ly/genetics , Antigens, Ly/pharmacology , Aspartic Acid/genetics , Cell Line, Tumor , Epitopes/genetics , Epitopes/metabolism , Female , Glycine/genetics , H-2 Antigens/metabolism , Histocompatibility Antigen H-2D , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary/genetics , Rats , Receptors, Immunologic/genetics , Receptors, Immunologic/physiologyABSTRACT
The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2D(d) and D(k) class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes D(d), and not D(k). The specificity for D(k) as well as D(d) of the wild-type Ly-49G(BALB/c) allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49G(BALB/c) allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49G(B6). Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.
