EVIDENCE OF ONLY ONE H-2D/L - RELATED GLYCOPROTEIN IN H-2dm1 MUTANT CELLS

Analyses of the H-2D/L-related glycoproteins from dm1 mutant cell extracts by sequential immunoprecipitation, by SDS gel electrophoresis and by tryptic peptide mapping indicate that dm1 cells express only a single glycoprotein with H-2D/L-related determinants. In contrast to the four H-2D/L-related antigens identified for the parental d haplotype viz. H-2D^d, H-2M^d, H-2L^d and H-2R^d, separate and distinguishable “H-2D^dm1”, “H-2M^dm1”, “H-2L^dm1” and “H-2R^dm1” glycoprotein counterparts are apparently lacking in the dm1 mutant haplotype. Only a single H-2D/L-related glycoprotein is identified in dm1 extracts by standard serological methods and this glycoprotein is designated H-2D/L^dm1 because of its H-2D^d/H-2L^d hybrid characteristics, as recently shown by Burnside and colleagues (1984). Thus, the seemingly complex phenotype of the dm1 mutant appears to originate primarily from one molecule having properties of two (or more) molecules of the parental haplotype.     

Structural studies on H-2Db and H-2Kd molecules indicate that murine major histocompatibility b and d haplotype alloantigens share structural features

Structural properties of the H-2D^b and H-2K^d murine major histocompatibility complex (MHC) antigens were examined by radiochemical methods. Radiolabelled preparations of the H-2D^b and H-2K^d antigens were obtained by indirect immune precipitation of NP-40 lysates of the lymphoid tumor cell lines EL-4 (H-2^b) and C14 (H-2^d), respectively. After preparation of the 37,000 molecular weight papain fragment the antigens were cleaved with CNBr. The H-2K^d antigen yielded four major CNBr fragments whereas the H-2D^b molecule provided six. These CNBr fragments were subjected to partial NH₂-terminal amino acid sequence analysis and aligned by homology to the H-2K^b glycoprotein. Comparison of the structural properties of the H-2K^d and H-2D^b molecules with previously published data on the other known major transplantation antigens of the b and d haplotypes (H-2K^b, H-2D^d and H-2L^d) reveal a marked structural similarity. First, the data show that certain methionine residues have been highly conserved and that cleavage by CNBr at these positions provides an initial strategy for the study of these molecules. Secondly, disulfide-linked peptides obtained after CNBr cleavage could be aligned and the data suggest the presence of disulfide bridges in homologous positions. Third, after CNBr cleavage both the H-2K^d and H-2D^b molecules yielded two glycopeptides which were homologous to glycopeptides from the H-2K^b molecule. Fourth, overall homology for a limited number of comparable positions is about 81% between the H-2K^b and H-2K^d gene products and 88% between the H-2K^b and H-2D^b gene products.     

α1 / α2 domains of H-2Dd,but not H-2Ld,induce “missing self” reactivity in vivo – No effect of H-2Ld on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2D^d on C57BL / 6 (B6) background conveys NK cell-mediated “missing self” reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2D^d / L^d MHC class I transgenes, and show that the α1 / α2 domains of H-2D^d were necessary and sufficient to induce “missing self” recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the α1 / α2 domains of H-2L^d induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the α1 / α2 domains of H-2L^d on NK cell specificity was surprising, considering that both H-2L^d and H-2D^d have been reported to interact with Ly49G2. Therefore, the role of H-2L^d for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2D^d, but not H-2L^d, abolished killing by sorted Ly49G2⁺ NK cells, indicating that H-2L^d does not inhibit NK cells via the Ly49G2 receptor.     

Lack of F1 anti-parental resistance in H-2b/d F1 hybrids devoid of β2-microglobulin

F₁ hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F₁ hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H-2^b/d) devoid of β₂-microglobulin (β₂m) are incapable of rejecting β₂m?/? parental C57BL/6 cells (H-2^b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F₁ mice of the H-2^b/d haplotype, requires expression of MHC class I molecules complexed with β₂m.     

Isolation,characterization and amino acid sequence studies of the cyanogen bromide fragments of the H-2 Dd glycoprotein

The papain-solubilized fragment of the H-2D^d antigen, representing the NH₂-terminal 80% of the native glycoprotein, can be fragmented into five polypeptides using CNBr cleavage. These fragments have been isolated, characterized and subjected to amino acid sequence analysis using radiochemical microtechniques. It was possible to align these CNBr fragments by comparison of their NH₂-terminal amino acid sequence with the known amino acid sequence of the H-2 K^b molecule. Thus, the NH₂-terminal fragment, D, contains 23 residues and is followed by a glycopeptide, fragment C, of 75 residues. Following fragment C are three fragments held together by disulfide bonds: fragment b 4, which has 40 amino acid residues, a second glycopeptide, fragment b 2, of 90 residues, and fragment b 3, a peptide of approximately 55 residues which terminates at the site of papain cleavage. The complete amino acid sequence of the NH₂-terminal CNBr fragment, D, has been determined, and this is presented here along with sequence data for the NH₂-terminal 30 residues of each of the other four CNBr fragments of the papain fragment of H-2D^d. A total of 146 positions have been examined in the approximately 280-residue H-2D molecule, and a total of 127 residues have been positively determined. Most of the unassigned positions may be tentatively assigned as Asp or Asn, the only amino acids so far not incorporated in radiolabeled form by our metabolic labeling procedures into the H-2 D^d molecule. Comparison of the sequence data obtained for the H-2 D^d molecule to data available for other histocompatibility antigens revealed homologies of approximately 90% with H-2 K^b and H-2 L^d, and approximately 75% with HLA-B 7 and HLA-A2.     

THE GENETIC REGULATION OF NK-ACTIVITY: STUDIES ON CONGENIC MICE WITH B10 OR AKR BACKGROUND

Mouse strains differ widely in their natural killer(NK)-cell activity. In the (A X B6) X A backcross, high reactivity was linked to H-2^b, although non-H-2-linked genes were also demonstrated (Petranyi et al., 1975). Harmon et al. (1977) demonstrated an H-2D^d-associated reactivity gene (1977). In the present study, we have tested eleven B10 congenic strains for NK activity. The H-2D^d strains B10.A, B10.T(6R), B10.S(7R), B10.HTT and B10.D2 were more highly reactive than B10, B10.S, B10.G, B10.A(2R) and B10.BR, which do not carry the d allele at the H-2D locus. While this confirms the H-2D^d association of a reactivity gene, an exception was found in the B10.A(5R) strain that was low reactive in spite of the fact that it carries H-2D^d. This suggests the possibility that the H-2D^d-associated gene is outside H-2, to the right of Tla. The AKR.H-2^b congenic line had the same low activity as the AKR.H-2^k strain; both were much lower than B6. This suggests either one of two possibilities: the H-2^b-linked reactivity gene is relatively distant from the H-2 complex, although localized on chromosome 17 or alternatively, if localized within or in the close neighbourhood of H-2, it requires non-H-2 genes for full expression. Previously, we have shown that the B6 X DBA/2 F₁ hybrid was more highly reactive than either one of its two parental strains (Klein et al., 1978). A similar complementation effect is described in the present paper for the B10.D2 congenic strain. The high reactivity of this line can be due to the combined effect of an H-2^b-linked gene from DBA/2 and the non-H-2 background of B10 or, alternatively, the former, together with an H-2^b-linked gene from B6 that lies well outside outside the H-2 locus itself.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

Amino acid sequence of the cytoplasmic segment of the h-2kd (h2.31) murine major histocompatibility antigen

The primary structure of the carboxy-terminal portion of the H-2K_d murine major histocompatibility antigen has been determined using radiosequencing methodology. The two peptides encompassing the entire cytoplasmic portion of the H-2K^d molecule were isolated from cyanogen bromide digests of the detergent solubilized molecule. These two peptides are not present in CNBr digests of papain-solubilized H-2K^d. Alignment of the two CNBr peptides was deduced from tryptic overlap peptides derived from the whole molecule. Alignment with the corresponding region of the H-2K^b antigen shows 90% homology and supports the assignment of this segment of H-2K^d as the C-terminal. The sequence obtained in this study is (Met)-Arg-Arg-Asn-Thr-Gly-Gly-Lys-Gly-Val-Asn-Tyr-Ala-Leu-Ala-Pro-Gly-Ser-Gln-Thr-Ser-Asp- Leu-Ser-Leu-Pro-Asp-Pro-Lys-Val-Met-Val-His-Asp-Pro-His-Ser-Leu-Ala. These data allow extensive comparisons with the protein sequences deduced from the 3' ends of H-2^d haplotype cDNA and genomic clones as well as with the homologous regions of H-2K^b and H-2D^b.     

Structure of murine major histocompatibility complex alloantigens: Identification of cysteine residues involved in two intrachain disulfide bonds of H-2Kb

Two disulfide bonds are present in the antigenic portion of H-2K^b. Four of the five cysteine residues have been shown to be involved in these intrachain linkages. Isolation of the disulfide-bonded residues was accomplished by trypsin digestion of H-2K^b_pap∥ and subsequent gel filtration of the digested material. Reduction, alkylation and further separation of each of the pairs of disulfide-bonded peptides resulted in the separation of individual cysteine-containing peptides. Sequence analysis of each of these separated peptides indicated that Cys 101 is linked to Cys 164, and Cys 203 is linked to Cys 259. Cys 121 is not involved in intramolecular disulfide linkages.The disulfide bonds found in H-2K^b are homologous to those found in HLA-B7 and, most probably, in H-2D^b, H-2K^d, H-2D^d and H-2L^d. This pattern of disulfide linkages suggest that this feature is common to all major histocompatibility complex class I antigens.     

MHC Class I Phenotype and Function of Human β2-Microglobulin Transgenic Murine Lymphocytes

Lymphoid cells from β₂-microglobulin (β₂m) knockout mice transgenic for human (h) β₂m (C57BL/10 mβ₂m^?/hβ₂m⁺) were compared with normal mice for their binding to exogenously added hβ₂m, binding to a H-2D^b peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous hβ₂m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/hβ₂m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous hβ₂m to MHC-I, in particular, to the H-2D^b molecule through an exchange with endogenous mouse β₂m. In contrast to normal H-2D^b molecules, hybrid H-2D^b expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added hβ₂m suggesting that a stable fraction of hybrid H-2D^b molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/hβ₂m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.     

Polymorphism of a Qa-1-associated antigen defined by cytotoxic T cells. I. Qed-1a and Qed-1d

Tla^d mice have a distinct Qed-1 allele, Qed-1^d. Its product is detected by cytotoxic T cells raised in C57BW6 (H-2^b, Tla^b) mice against cells from a new recombinant, B6-TL.123⁺ (H-2^b, Tla^d/b). Qed-1^d is also found on cells from B10.M, A.CA and B10.STC90 mice. It cross-reacts weakly with Qed-1^b. (C57BL/6 X BALB/c) F₁ anti-B10. A (SR) effectors discriminate Qed-1^a and Qed-1^d, while C3H/HeJ anti-B10. BR effectors cross-react extensively. CB6F₁ anti-5R effector cells also discriminate between the Qed-1 antigens of B6-Tla^a (H-2^b, Tla^a) and those of B10. BR and other H-2k, Tla^a strains.     

POLYGENIC INFLUENCES ON THE LENGTH OF OESTROUS CYCLES IN INBRED MICE INVOLVE MHC ALLELES*

Genetic influences on female reproductive cycles were analysed in histocompatibilitycongenic strains of mice. Oestrous cycles of young, virgin mice of inbred-congenic strains, hybrid crosses (F₁, and parental-hybrid backcrosses (F₂) were monitored for 3 months. Oestrous cycles were categorized by length (inter-oestrous interval): 4,5,6, or 7–14 days. Mice with the following H-2 haplotypes had a greater proportion of 5-day oestrous cycles: H-2^b, H-2^r, H-2^h2, H-2^h4, and H-2ⁱ⁵. In contrast, the H-2^k and H-2^d haplotypes had mostly 4-day oestrous cycles. Influences of H-2 haplotype were seen on two genetic backgrounds, C57BL/10Sn and C3H. Non-H-2 alleles were also implied by different patterns of cycles between strains with the same H-2^b haplotype: C57BL/10Sn with predominantly 5-day cycles vs. C57BL/6J with a mix of 4- and 5-day cycles. The genetic basis for strain differences was investigation in F₁ hybrids and their backcrosses. F₁ hybrids of an H-2^b (C57BL/10Sn; 5-day cycles) and an H-2^k (B10.BR; 4-day cycles) strain had mostly 5-day cycles, indicating dominance of an H-2^b allele(s). However, F₁ hybrids from the reciprocal B6 × B10 cross (both H-2^b) also display a preponderance of 5-day cycles, indicating dominance of a non-H-2 autosomal allele from the C57BL/10Sn strain. Among F₂ mice, a ‘4-day’ phenotype segregated with homozygosity for the k haplotype (P < 0.05, χ²). These findings demonstrate the influence of genetic differences at the major histocompatibility complex on oestrous cycles.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO COLLAGEN: I. QUANTITATIVE DETERMINATION OF RESPONSE LEVELS BY MULTIPLE I-REGION GENES

The antibody response in mice to type I calf skin collagen is quantitatively determined by genes which map to the I region of the H-2 histocompatibility complex. The use of H-2 recombinant B10 congenic strains of mice reveals that a gene in the IA subregion and a gene to the right of the IA subregion affect responsivenss. To examine complementation patterns in the antibody response to collagen, five B10 congenic strains, each bearing an independent H-2 haplotype, were intercrossed to obtain nine hybrid strains heterozygous at the H-2 locus. In five combinations heterozygous progeny produced significantly greater antibody responses than those observed for the homozygous parental strains. Two low responder haplotypes, H-2^k and H-2^d, were shown to be qualitatively different. Mice of these haplotypes show a different dose-response pattern and a different phenotypic pattern of inheritance with respect to the high responder H-2^b haplotype. Complementation effects found in F₁ hybrid mice derived from H-2 recombinant parental strains indicate that high responsiveness, controlled by an IA^b subregion gene, can be influenced by an interaction between an IA^k subregion gene and an IC^d subregion gene on different chromosomes. These data are consistent with the possibility that there exist two or more I region genes that have distinct functions and can interact to affect the levels of immune responsiveness.     

Evidence for three carbohydrate prosthetic groups on mouse histocompatibility antigens H-2Kd and H-2Db

Previous studies have determined that H-2K^b molecules have two glycosyl units which are attached at asparagine-86 and asparagine-176. Investigations concerning the number and location of carbohydrate groups in the H-2D^b and,H-2K^d antigens were carried out on [³H]glucosamine-labelled material isolated from EL-4 (H-2^b) and C14 (H-2^d) tumor cells, respectively. The present study, employing CNBr cleavage, indicates that H-2K^d and H-2D^b have, in addition to those reported for H-2K^b, a third glycosyl unit located between residues 229 and the papain cleavage site near residue 281.     

Functional cell surface expression by a recombinant single-chain class I major histocompatibility complex molecule with a cis-active β2-microglobulin domain

As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2D^d class I MHC molecule (SCβD^dm) which has β₂-microglobulin (β₂m) covalently linked to the amino terminus of a full-length H-2D^d heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2D^d-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SCβD^dm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27° or 37 °C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SCβD^dm can be stably expressed in β₂m-negative cells, showing that the single-chain molecule uses its own β₂m domain to achieve correct folding and surface expression. Furthermore, the β₂m domain of SCβD^dm, unlike transfected free β₂m, does not rescue surface expression of endogenous class I MHC in the β₂m-negative cells. This strict cis activity of the β₂m domain of SCβD^dm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.     

H - 2 ANTIGENS OF CERTAIN t AND NON-t MICE REACT WITH THE SAME MONOCLONAL ANTIBODY

Monoclonal antibody 212.i.4.2 mediated complement-dependent lysis of spleen and lymph node cells carrying the t^w1, t^w12, t^w71, t⁶, t^w73, and t^Lub1 haplotypes, while cells from mice carrying 11 other t haplotypes were not lysed. The antibody also detected an epitope controlled by genes in the H-2D^d region of non-t mice. A molecule of 46,000 molecular weight was immunoprecipitated by 212.i.4.2 from detergent extracts of ¹²⁵I-labelled spleen cells of +/t^w12 and B10.D2 mice. The H-2^dm2 mutation did not alter the expression of the epitope recognized by 212.i.4.2. However, the H-2^dm1 mutation decreased the reactivity of lymphoid cells with the antibody in cytotoxicity tests, and 212.i.4.2 immunoprecipitated little or no protein from extracts of B10.D2(R106) spleen cells which carry the H-2^dm1 mutation.     

GENETIC ANALYSIS OF H-2 LINKED GENE(S) AFFECTING EARLY MOUSE EMBRYO DEVELOPMENT

The number of cells in preimplantation mouse embryos of different H-2 haplotypes was analysed. It was found that embryos of the H-2^k haplotype have fewer cells per embryo than those of the H-2^b haplotype. By analysing reciprocal congenic pairs of mice it was demonstrated unequivocally that slow development is linked to the H-2^k haplotype and fast development to the H-2^b haplotype. The gene(s) in the H-2 complex which influence early mouse embryo development have been named Ped: preimplantation embryo development. Analysis of F₁ hybrid embryos showed that fast development is dominant. Reciprocal F₁ crosses yielded identical results, which indicated there was no apparent effect of the maternal egg cytoplasm on Ped gene expression. Analysis of F₂ and back-cross embryos was consistent with the interpretation that there is a major gene located in the H-2 complex (Ped), which is modified by environment and genetic background, that influences early mouse embryo development.     

The presence of a common idiotype in anti-H-2 immune sera as detected by anti-idiotype to a monoclonal anti-H-2 antibody

To study the idiotypes (Id) of the humoral response to H-2L^d antigens, xenogeneic antisera were produced to three independent monoclonal antibodies, recognizing L^d determinants H-2.64 or 65. The reactivity of each of these anti-Id was specific for the immunizing antibody, no cross-reactions among the three anti-L hybridomas being detectable. Antisera produced in BALB/c H-2^dm2 mice hyperimmunized with BALB/c tissue were examined for the presence of cross-reactive Id (Id^CR). When the ability of these anti-Id to inhibit the binding of alloantibodies to L^d splenic antigens was tested, a broadly shared Id was detected by one anti-Id (anti-23-10-1S). The shared Id represented between one-fourth and one-half of the total anti-L^d humoral response and was found in every BALB/c H-2^dm2 anti-BALB/c serum tested. The Id^CR was limited to those alloantibodies reacting with the determinant H-2.65 which corresponds with the serologic reactivity of monoclonal antibody 23-10-1S. The other two anti-Id did not detect components of the anti-L^d humoral response, even though one of these was made against monoclonal antibody 30-5-7S which also detects serologic specificity H-2.65. The detection of a Id^CR in anti-L^d responses stands in contrast to our previous failure to detect Id^CR in anti-H-2K^k responses. Among the possible reasons for this contrast are (a) the fact that 23-10-1S is an IgM antibody, whereas the anti-H-2K^k antibodies studies were IgG; (b) the chance occurrence of anti-H-2 hybridomas with dominant Id and (c) the possibility that the anti-L^d antibody repertoire may be less heterogeneous than that directed to K^k antigens.     

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft

The MHC class I molecule H-2D^d (D^d) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered D^d molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the D^b molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (D^dS73WD156Y) impaired the protective effect of the D^d molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A⁺ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the α helix of the α2 domain, facing the solvent and without direct contact with the peptide (D^dA150S) had no effect. D^d recognition by Ly-49A⁺ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the D^d molecule that make the trimolecular complex less permissive for inhibition of Ly-49A⁺ NK cells.