Delineation of determinants on HLA-B7 and HLA-B27 that are necessary for cytolytic T cell recognition by using inter- and intra-domain recombinants

We have used bulk culture HLA-B7 and HLA-B27 specific CTL lines derived from 11 donors, and a series of rHLA-B7/HLA-B27 genes transfected into and expressed on the surface of the murine cell P815, to determine the amino acid residues on these HLA class I molecules that are critical for allospecific CTL recognition. The results obtained indicate that for four of six HLA-B7-specific CTL lines the alpha-1 domain for CTL recognition. Furthermore, we found that residues 77 and/or 80 had a critical effect on recognition for all of the CTL lines tested. The region 97-156 in the alpha-2 domain was also important for some of these CTL lines. Furthermore, by using five bulk culture HLA-B27-specific CTL lines we were able to show that residues 77 and/or 80 and residue 152 are also essential for recognition of HLA-B27 by HLA-B27-specific CTL. The strong influence exerted by these residues is discussed in terms of the three-dimensional structure of class I molecules. Finally, a selection was regularly observed in the bulk cultures such that the CTL that were preferentially influenced by either the alpha-1 or the alpha-2 domain were lost after 4 to 7 wk of culture resulting in CTL cell lines which were extremely sensitive to sequence modifications of HLA-B7 or HLA-B27. The possible reasons for this selection, which we have previously observed with both anti-HLA-A2 and anti-HLA-A3 cell lines and is therefore not unique to HLA-B7 or HLA-B27, are discussed.     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.     

Murine H-2Dd-reactive monoclonal antibodies recognize shared antigenic determinant(s) on human HLA-B7 or HLA-B27 molecules or both

We have evaluated the serological relationships between the murine H-2Dd and human HLA molecules using four H-2Dd-reactive monoclonal antibodies (mAbs) produced in the A.BY (KbIbDb) anti-A.TL (KsIkDd) combination. In the mouse, these reagents exhibited three distinct reactivity patterns: Dd, Ks, and H-2u (mAb 81.L); Dd, H-2p, and H-2u (mAb 81.R); and Dd, Kd, H-2p, H-2u, and H-2v (mAbs 97.G and 97.H). Sequential immunoprecipitation and cross-competitive mAb binding experiments revealed that these mAbs recognized determinants in two spatially distinct polymorphic domains on the H-2Dd molecule of B10.A(5R) cells (defined by mAbs 81.L and 81.R, 97.H, and 97.G, respectively). MAbs 81.R, 97.G, and 97.H, but not 81.L, also defined an HLA-linked polymorphism in the human, the main characteristics of which can be summarized as follows: (i) on B lymphoblastoid cell lines, mAbs 81.R and 97.H bound to cells expressing the HLA-B7, HL-B27 or Bw40 cross-reacting specificities, (ii) on peripheral blood lymphocyte (PBL) panel mAb 81.R exerted C dependent cytotoxicity to 118 of 400 cells tested, including almost all HLA-B7 or HLA-B27 cells or both (r: 0.952), (iii) the expression of the 81.R cross-reacting determinant segregated in an informative family with the parental haplotype carrying the HLA-B7 allele, and (iv) mAbs 81.R, 97.G, and 97.H recognized topologically related determinants on the same class I molecule(s) of the human B lymphoblastoid cells JY (HLA-A2,2, -B7,7). These data support the view that some, but not all H-2Dd allotopes have been conserved throughout evolution and are associated in the human with the HLA-B7, -B27 cross-reacting specificities.     

Polymorphic sites away from the Bw4 epitope that affect interaction of Bw4+ HLA-B with KIR3DL1

KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81, 82, and 83 in the alpha(1) helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and 81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that polymorphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the alpha(1) domain, 116 in the alpha(2) domain, and 194 in the alpha(3) domain. Lesser contributions were made by additional positions in the alpha(2) domain. These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in the peptides bound, which alter the conformation of the Bw4 epitope.     

Allodeterminants and evolution of a novel HLA-B5 CREG antigen, HLA-B SNA.

A novel HLA-B5 CREG gene, HLA-B SNA was cloned and the primary structure was determined. The sequence data showed that HLA-B SNA was identical to HLA-B51 except the alpha 1 domain in which one amino acid substitution at residue 74 and 5 amino acid substitutions associated with the Bw4/Bw6 epitopes were observed between these Ag. The comparison with other HLA-B locus genes suggested that HLA-B SNA evolved from HLA-B51 by gene exchange or recombination at the exon 2 between HLA-B51 and B8. A total of 10 of 14 HLA-B51-specific CTL clones showed significantly weak or no recognition of HLA-B SNA Ag. They also gave the same degree of a lysis of Hmy2CIR cells expressing the HLA-B35/51 chimeric Ag composed of the alpha 1 domain of HLA-B35 and other domains of HLA-B51 as that of Hmy2CIR cells expressing the HLA-B SNA Ag. These results demonstrated that amino acid substitutions within positions 77-83 associated with the HLA-Bw4/Bw6 epitopes have an influence on recognition of the HLA-B SNA antigen by HLA-B51-specific CTL.     

The primary structure of HLA-A32 suggests a region involved in formation of the Bw4/Bw6 epitopes

All HLA-B locus molecules have either the Bw4 or Bw6 epitopes. In addition, the Bw4 epitope is found on HLA-Aw23, Aw24, and A32, and Bw6 is also found on HLA-Cw3. The structural basis for these determinants and the evolution of their distribution among products of the HLA-B locus has been a long standing puzzle. To identify residues that may be involved in these determinants, we have cloned a gene for A32 and sequenced the protein encoding exons. Comparison of the predicted protein sequence with other HLA-A,B,C sequences identified residues 79 through 83 of the alpha 1 domain as having a pattern of polymorphic substitution that correlates with the presence and absence of the Bw4 and Bw6 epitopes.     

Transfection of murine LMTK− cells with purified HLA class I genes

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2K ^d, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the 137 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.Abbreviations used in this paper BSA bovine serum albumin - FCS fetal calf serum - mAb monoclonal antibody - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline     

HLA-Bw22: a family of molecules with identity to HLA-B7 in the alpha 1-helix.

Various HLA-B molecules exhibit serologic cross-reactions with HLA-B7, including HLA-B27, B40, Bw42, and Bw22. Of this group, primary structures for the three serologic subdivisions of HLA-Bw22, HLA-Bw54, Bw55, and Bw56, have yet to be determined. Here, we describe the nucleotide sequences of five distinctive HLA-Bw22 alleles isolated from cells of different ethnic origins typed either for Bw54, Bw55, or Bw56. Heterogeneity in molecules typed as Bw55 and Bw56 was defined. The five HLA-Bw22 alleles form a closely related family that appears to have evolved by a series of simple gene conversion events, all of which alter the antigen recognition site of the encoded proteins. Of note, HLA-Bw54 is the product of a gene conversion between HLA-B and C alleles. All the Bw22 alleles encode an alpha 1-helix identical in amino acid sequence to that of HLA-B7 and Bw42, a feature almost certainly responsible for the serologic cross-reactivity of these molecules. Shared substitutions in the alpha 1-helix can also explain the cross-reactivity of Bw22 and B7 with B27. Patterns of amino acid substitution in the alpha 2-domain of Bw22 heavy chains correlate with certain antibody and T cell cross-reactivities, thereby implicating particular amino acids in their target epitopes.     

Structure and diversity of HLA-B27-specific T cell epitopes. Analysis with site-directed mutants mimicking HLA-B27 subtype polymorphism.

HLA-B27 subtype polymorphism is amenable to differential recognition by CTL. Site-directed mutagenesis was used to construct a series of HLA-B27 mutants reproducing most of the changes occurring in the natural subtypes. The reactivity of 21 anti-HLA-B27 CTL clones was examined with these mutants to address three issues concerning the alloreactive response against HLA-B27: 1) diversity of clonotypic specificities, 2) structural features of the epitopes recognized by these clones, and 3) role of individual positions in the differential recognition of HLA-B27 subtypes. Virtually all CTL clones displayed unique reaction patterns with the mutants, indicating a corresponding diversity of epitopes. However, these share some molecular features, such as certain amino acid residues and related locations. Individual mutations induced complex effects on multiple B27-specific CTL epitopes, revealing some of their very precise stereochemical constrains. An important feature of HLA-B27 subtype polymorphism is that every individual change was relevant, altering recognition by many CTL clones. Although the specific set affected by each mutation was partially different, the global number of clones affected by most changes was very similar. This suggests that the antigenic profile of any given subtype is not dominated by one particular change but is uniquely defined by its corresponding set of changes. An exception was the change at position 152, which totally abrogated recognition by all 20 anti-B*2705 CTL clones. This effect decisively influences the profound differences in T cell recognition between B*2705 and the two subtypes, B*2704 and B*2706, carrying this change. The results are compatible with the idea that HLA-B27 allorecognition may involve multiple peptides bound to the alloantigen on the cell surface.     

In vitro mutagenesis of HLA-B27. Amino acid substitutions at position 67 disrupt anti-B27 monoclonal antibody binding in direct relation to the size of the substituted side chain.

The class I MHC molecule HLA-B27 bears an unpaired Cys residue at position 67, which is predicted to face the Ag binding pocket, based on the x-ray crystallographic model of HLA-A2. To investigate the potential of this residue in the antigenic structure of HLA-B27, a panel of 11 mutant HLA-B27 genes has been created, each bearing a separate amino acid substitution at position 67. The genes were transfected into mouse L cells and the resulting cells analyzed by cytofluorography with a panel of antibodies reactive with the wild-type B27 molecule. Although previous studies had indicated that all mAb that bound the B27 molecule on human lymphocytes bound comparably to L cells transfected with the wild-type B27 gene in the absence of h beta 2-m (human beta 2-microglobulin), the first of the mutant B27 genes was found to express several mAb epitopes in the presence but not in the absence of a h beta 2-m gene. Therefore, subsequent analysis of the B27 mutant panel was conducted in L cells coexpressing the h beta 2-m gene. Under these circumstances, all of the mutants bound the monomorphic anti-class I HLA mAb W6/32 and B.9.12.1, as well as the broadly polymorphic mAb B.1.23.2. Binding to the mutant transfectants of three anti-B27 mAb that cross-react with HLA-B7, ME1, GS145.2, and GSP5.3, was directly proportional to the size of the substituted amino acid side chain. The binding of another anti-B27 mAb, B27M2, that recognizes a B27 determinant that includes the region of amino acids 77-81, was not affected by the Cys67- greater than Tyr67 substitution. Rabbit antibodies to a synthetic peptide composed of B27 amino acids 61-84 bound to both the wild-type B27 and to the Tyr67 mutant. This binding, but not the binding of ME1 or B27M2, was inhibited by the synthetic peptide. These data are interpreted as suggesting that the large amino acid substitutions at position 67 induce a limited conformational change that disrupts the epitopes of the three anti-B27, B7 mAb, that are themselves at least partially conformational. The potential implications of these findings for the role of HLA-B27 in disease pathogenesis are discussed.     

The complete primary structure of HLA-Bw58

Serological studies indicate that HLA-B17 molecules are unusually cross-reactive with products of the HLA-A locus. In particular, a mouse monoclonal antibody MA2.1 defines an epitope that is shared by HLA-A2 and the two subtypes (Bw57 and Bw58) of B17. To investigate these relationships at the structural level, we have isolated a gene coding for Bw58 from the WT49 B cell line. The gene was transfected into mouse L cells and its protein product was characterized with a panel of monoclonal anti-HLA antibodies. The nucleotide sequence of 3520 base pairs of DNA encompassing the seven exons coding for Bw58 and associated introns was determined. The deduced protein sequences for Bw58 and eight other HLA-A,B,C molecules were compared. In the first polymorphic domain (alpha 1), Bw58 is unusual in that it is as homologous to HLA-A locus products as to HLA-B locus products. In the second polymorphic domain (alpha 2), Bw58 has greater homology to B locus products. In the alpha 1 domain of Bw58, small segments of amino acid and nucleotide sequence homology with A2 (residues 62-65) and with Aw24 (residues 75-83) are found in the major region of polymorphic diversity (residues 62-83). These similarities provide structural correlates for the serological relationships between Bw58 and A locus molecules, with residues 62-65 possibly being involved in the MA2.1 epitope. From comparisons of four HLA-A and four HLA-B sequences, there is a difference in the patterns of variation for A and B locus molecules. For B locus molecules there is greater variation in the alpha 1 domain than in the alpha 2 domain. For A locus molecules, variation in the two domains is similar and like that for B locus alpha 2 domains. In comparison to other HLA-A,B,C genes, novel inverted repeat sequences were found in the nucleotide sequence of HLA-Bw58. These sequences flank the putative RNA splicing sites at the 3' end of the exons encoding the alpha 2 and alpha 3 protein domains.     

Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.     

Interesting relations in the HLA system

There exists no absolute binding between the antigens HLA-Cw 2, Cw 3 and Cw 4, on the one hand, and HLA-B 27, HLA-B 15 and HLA-Bw 35, on the other hand. Even if 91% of human beings with HLA Cw 4 will simultaneously have the antigen HLA-Bw 35, another antigen as HLA-B 27 or HLA-B 15 can be identified in approximately 55% of individuals with HLA-Cw 2 and Cw 3. In this connection, the joint presence of some pairs of cross-reacting HLA antigens (A 2 and A 28, B 5 and Bw 35, B 7 and B 27, B 8 and B 14, B 12 and Bw 2) could be proved and their frequency be determined. 2 cases of a simultaneous presence of two subtypes of HLA-A 10 antigen, A 25 and A 26, could be found in family examinations. Moreover, two atypical bindings of anti-HLA-Bw 4 and anti-HLA-Bw 6 cytotoxins with HLA antigens could be identified: 7,49% of HLA-Bw 35 positive lymphocytes no positive response with anti-HLA-B 4 and 1,69% of HLA-B 12 with anti-HLA Bw 6. The importance of the findings for determining HLA in practice is discussed.     

Immunochemical variants of HLA-B27

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.     

A transposable epitope of HLA-137, B40 molecules

The monoclonal antibody M1340.2 defines a novel subtype of HLA-1340 that is expressed by the Sweig cell line. This molecule, called HLA-B40^*, lacks an antigenic determinant that is common to HLA-137 and the HLA-Bw60 subtype of HLA-1340. Genes encoding HLA-B40^* and HLA-BW60 have now been isolated and the amino acid sequences of these proteins compared with other HLA-13 locus molecules. These results show that HLA-B40^* is a unique protein which differs from HLA-BW60 by eight amino acid substitutions. Comparison of the sequences for HLA-B40^*, -Bw60, and -B7 localizes the MB40.2 epitope to a cluster of three substitutions at positions 177, 178, and 180 at the end of the ₂ domain. Gene conversion or reciprocal recombination are postulated to have transferred this cluster of substitutions, and their associated epitope, during the evolution of HLA-B locus genes. The epitope may consist of an a helical segment which is exclusively found on MB40.2-positive molecules.     

A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain.

There are six known HLA-B alleles that share the HLA-B27 allospecificity, yet differ by one to six amino acid substitutions. Each of these B27 alleles can be readily assigned by one of the six representative IEF patterns. Two unrelated individuals, LH and HS, express B27 Ag that appear to be identical by IEF, but an HLA-B27 alloreactive CTL clone I-73 was found to react differently with these cells, suggesting these B27 molecules are not identical. We sequenced polymerase chain reaction-amplified B27 cDNA clones obtained from HS and compared its deduced amino acid sequence (B27-HS) with the B27 sequence of LH (B27-LH) which was previously designated the B*2701 allele. B27-HS and B27-LH differ by eight amino acids; three in alpha 1 domain and five in alpha 2 domain. These amino acid substitutions of B27-HS altered T cell recognition but not the B27 serologic epitope or IEF pattern. B27-HS differs from the six known B27 alleles by five to eight amino acid substitutions, and thus it represents the seventh allele of the HLA-B27 Ag family. This novel B27 allele might have been derived from a gene conversion event. Previously, two amino acid residues at positions 70 and 97 were suggested to be specific for B27 Ag family. B27-HS now reveals that Lys at position 70 is specific for B27 but Asn at position 97 is not. We propose that the region around position 70 might be crucial in determining the B27 serologic epitope and possibly in peptide Ag binding. This study also demonstrates that class I molecules of the same Ag specificity sharing an indistinguishable IEF pattern are not necessarily identical, and indicates that only the definitive determination of primary structure would identify all the class I alleles that are functionally relevant in regard to alloreactivity, T cell restriction, and disease association.     

External quality assessment of flow cytometric HLA-B27 typing

A biannual external quality assurance (EQA) scheme for flow cytometric typing of the HLA-B27 antigen is operational in The Netherlands and Belgium since 1995. We report here on the results of the first seven send-outs to which 36 to 47 laboratories participated. With the send-out, four specimens from blood bank donors, who had been typed for HLA Class I antigens by complement-dependent cytotoxicity, were distributed. Subtyping of the HLA-B27 allele was performed by PCR-SSP. Ten samples were HLA-B27(pos) (all HLA-B*2705) and 18 were HLA-B27(neg). For flow cytometry, the most widely monoclonal antibody (MoAb) used was FD705, followed by GS145.2 and ABC-m3. The majority of laboratories used more than 1 anti-HLA-B27 MoAb for typing. The HLA-B27(pos) samples were correctly classified as positive by the large majority of participants (median 95%; range 85% to 100% per send out); some participants considered further typing necessary and misclassification as negative was only sporadically seen. The classification of HLA-B27(neg) samples as negative was less straightforward. Ten samples were correctly classified as such by 97% (82% to 100%) of the participants, whereas 64% (range 53% to 70%) of the participants classified the remaining eight samples as HLA-B27(neg). There was no significant prevalence of a particular HLA-B allele among these eight "poor concordancy" samples as compared to the ten "good concordancy" samples. Inspection of the reactivity patterns of the individual MoAb with HLA-B27(neg) samples revealed that ABC-m3 showed very little cross-reactivity apart from its well-known cross-reactivity with HLA-B7, whereas the cross-reactivity patterns of GS145.2 and FD705 were more extensive. The small sample size (n = 18) and the distribution of HLA-B alleles other than HLA-B27 did not allow assignment of specificities to these cross-reactions. Finally, we showed that standardized interpretation of the combined results of two anti-HLA-B27 MoAb reduced the frequency of false-positive conclusions on HLA-B27(neg) samples. In this series, the lowest frequency of false-positive assignments was observed with the combination of the FD705 and ABC-m3 MoAb.     

Rhesus macaque KIR bind human MHC class I with broad specificity and recognize HLA-C more effectively than HLA-A and HLA-B

Human killer cell immunoglobulin-like receptors (KIR) recognize A3/11, Bw4, C1, and C2 epitopes carried by mutually exclusive subsets of human leukocyte antigen (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 major histocompatibility complex (MHC)-C having evolved in hominids to be a generally superior ligand for KIR than MHC-A and MHC-B.     

Peptide binding to HLA-A2 and HLA-B27 isolated from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy chain/beta 2-microglobulin complexes requires specific peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.