A Peptide-Binding Assay for the Disease-Associated HLA-DQ8 Molecule

The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide-binding properties of DQ molecules. Here we report a biochemical peptide-binding assay for the type I diabetes-associated DQ8, i.e. DQ (α1*0301, β1*0302), molecule. Affinity-purified DQ8 molecules were tested in peptide-binding assays using a radiolabelled influenza haemagglutinin (Ha) peptide encompassing positions 255–271(Y) as an indicator peptide. The Ha 255–271(Y) peptide bound to DQ8 in a pH-dependent fashion showing optimal binding around pH 5. The association kinetics were relatively slow and the resulting complexes were heat labile. The specificity of peptide binding to DQ8 was investigated in competitive inhibition experiments with a panel of 43 peptides of different lengths and sequences. The DQ8 molecules showed a different pattern of peptide binding compared to a previously studied DQ2 molecule. Peptides derived from thyroid peroxidase, HLA-DQ(α1*0301), HLA-DQ(α1*0302), retinol receptor and p21ras were among the high-affinity binders, whereas peptides derived from myelin basic protein were among the low-affinity binders. The sequence of the high-affinity peptides conformed with a previously published peptide-binding motif of DQ8.     

The HLA-DQ(α1*0102, β1*0602) heterodimer may confer susceptibility to multiple sclerosis in the absence of the HLA-DR(α1*01, β1*1501) heterodimer

The frequencies of DR2, DQ6-related DRB1, DQA1, DQB1 haplotypes were compared in 181 multiple sclerosis patients and 294 controls in Norway. All individuals carried either DR2 or DQ6, i.e., the DQ(α1*0102, β1*0602) heterodimer. The DR(α1*01,β1*1501) and the DQ(α1*O102,β1*O602) heterodimers were carried by 171 of the patients (94%) and 289 (98%) of the controls. Seven of the patients and one of the controls carried the DQ(α1*0102, β1*0603) heterodimer together with the DR(α1*01, β1* 1501) heterodimer. Two patients carried the DQ(α1*0102, β1*O602) heterodimer in the absence of the DR(α1*O1, β1*1501) heterodimer. The DR(α1*01, β1*1501) heterodimer was not observed in the absence of the DQ(α1*0102, β1*O602) heterodimer or the DQ(α1*O102, β1*0603) heterodimer, neither in the patients nor in therols. Our findings indicate that the genes encoding the DQ(α1*0102,β1*0602) heterodimer may confer susceptibility to developing multiple sclerosis in the absence of the DRB 1* 1501 allele.     

Naturally processed peptides from HLA-DQ7 (α1*0501-β1*0301): influence of both α and β chain polymorphism in the HLA-DQ peptide binding specificity

Self peptides bound to HLA-DQ7 (α1*0501-β1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (α1*0501-β1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (α1*0501-β1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215 – 230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (α1*0501-β1*0301); (2) when either the DQα or DQβ chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the α and β chains. (3) Unexpectedly, the TfR 215 – 230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.     

T cell clones specific for p21 ras-derived peptides: Characterization of their fine specificity and HLA restriction

Peptides corresponding to the mutated regions of the oneoprotein p21 ras are immunogenic and capable of eliciting HLA class II-restricted T cell responses. Here we report studies on the fine specificity of four T lymphocyte clones (TLC) from a single donor, using various truncated peptides derived from the residues 6–19 of p21 ras and a panel of well-characterized HLA homozygous cells as antigen-presenting cells. Putative minimum peptides of nine or ten amino acids could be defined for each TLC. Two of the TLC recognized peptides presented by DR2, and the two others recognized peptides presented by DQ6. Some notable differences in the requirement for certain amino acids were seen between the DR-and DQ-restricted TLC. Thus, Ser at residue 17 was required for stimulation of the DQ6-but not the DR2-restricted TLC. Val at residue 8 was essential for stimulation of all TLC, whereas one of the DR2-restricted TLC also required Val at residue 7. Some peptides which were nonstimulatory were still capable of binding to DQ6 molecules in peptide competition experiments. The results may be of importance for potential immunotherapy of cancer where transforming ras oncoproteins are involved.     

The peptide binding motif of the disease associated HLA-DQ (α 1* 0501, β 1* 0201) molecule

To identify the binding motifs of peptides which bind to the celiac disease and insulin-dependent-diabetes-mellitus (IDDM)-associated DQ2 molecule, peptides were eluted from affinity-purified DQ2 molecules. The eluted peptides were separated by reverse-phase HPLC. Prominent peptide peaks and the remaining pool of peptides were sequenced by Edman degradation. Truncated variants of eight different peptides with a length of 9–19 amino acids were identified; among them class II-associated invariant chain peptides (CLIP) and peptides that stem from HLA class I α, HLA-DQα1*0501, Ig and CD20 molecules. Data from the pool sequencing and the biochemical binding analyses of synthetic variants of an eluted high-affinity ligand (HLA class I α 46–60), indicate that the side chains of amino acid residues at relative position P1 (bulky hydrophobic), P4 (negatively charged or aliphatic), P6 (Pro or negatively charged), P7 (negatively charged) and P9 (bulky hydrophobic) are important for binding of peptides to DQ2. Computer modeling of the DQ2 with variants of the high-affinity ligand in the groove suggests that peptides bind to DQ2 through the primary anchors P1, P7 and P9 and making additional advantageous interactions using the P4 and P6 positions.     

The Interaction between Beta 2-Microglobulin (ßm) and Purified Class-I Major Histocompatibility (MHC) Antigen

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8⁺ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-ß₂m-class-I complexes a biochemical peptidc-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between ß₂m and class I. As a model system human ß₂m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, K_D, is close to 0.4 nw. The rate of association at 37 C is very fasi (the k_a is around 5 × 10⁴/M/s) whereas the dissociation is slow (the k_d is around 8 × 10⁻⁶/s); the ratio of dissociation to association yields a calculated K_D close to the observed value. At 37° C almost all of the purified class I participates in binding of the exogenously offered ß₂m showing that a considerable exchange of the endogenous ß₂m occurs. Finally, it was demonstrated that exogenous ß₂m enhances binding to MHC class-I of short perfectly-matching peplides as well as longer peptides.     

Dermatitis herpetiformis and celiac disease are both primarily associated with the HLA-DQ (α1*0501, (β1*02) or the HLA-DQ (α1*03, (β1*0302) heterodimers

HLA-DRB1, -DQA1, and -DQB1 genomic typing of 50 patients with dermatitis herpetiformis and of 290 healthy blood donors was performed. Genes encoding the DQ (α1*0501, (β1*02) heterodimer were carried by 43 (86%) of the patients and 72 (25%) of the controls. Of die remaining seven patients six (12% of all the patients) carried genes encoding the DQ (α1*03, β1*0302) heterodimer. These HLA associations are very similar to those observed in patients with celiac disease. We thus conclude that dermatitis herpetiformis and celiac disease are associated to the very same HLA-DQαβ heterodimers.     

Autoimmunity versus tolerance: analysis using HLAtransgenic mice

On the basis of our extensive studies on collagen induced arthritis in HLA class II transgenic mice, we proposed a hypothesis to explain role of shared epitope in rheumatoid arthritis (RA) association. According to our hypothesis, complementation between both DQ and DR molecules is required for susceptibility or protection from disease. While certain DQ alleles predispose individuals to RA, DRB1 molecule can modulate disease by shaping T-cell repertoire in the thymus by providing self-peptides and presented by DQ molecules. Using Aβo.DQ8 transgenic mice, we tested ability of peptides derived from HV3 of DR molecules, implicated in RA positively or negatively, to activate T cells. While the peptides derived from RA susceptible DR molecule were poor binders and poor in activating T cells, the peptides derived from RA resistant DR molecules were high affinity binders and efficient T-cell activators. Our experiments suggest that high affinity DR peptides could induce tolerance to autoimmunity while the low affinity peptides could be permissive to autoimmunity. Using peptide from DRB1*0402 molecule, known to be associated with resistance to RA, prior to induction of collagen induced arthritis prevents the onset of disease. Thus, self-peptides derived from HLA molecules could potentially generate tolerance or autoimmunity depending on their binding affinity with HLA molecules.     

Autoimmunity versus tolerance: analysis using transgenic mice

On the basis of our extensive studies on collagen induced arthritis in HLA class II transgenic mice, we proposed a hypothesis to explain role of shared epitope in rheumatoid arthritis (RA) association. According to our hypothesis, complementation between both DQ and DR molecules is required for susceptibility or protection from disease. While certain DQ alleles predispose individuals to RA, DRB1 molecule can modulate disease by shaping T-cell repertoire in the thymus by providing self-peptides and presented by DQ molecules. Using A beta o.DQ8 transgenic mice, we tested ability of peptides derived from HV3 of DR molecules, implicated in RA positively or negatively, to activate T cells. While the peptides derived from RA susceptible DR molecule were poor binders and poor in activating T cells, the peptides derived from RA resistant DR molecules were high affinity binders and efficient T-cell activators. Our experiments suggest that high affinity DR peptides could induce tolerance to autoimmunity while the low affinity peptides could be permissive to autoimmunity. Using peptide from DRB1*0402 molecule, known to be associated with resistance to RA, prior to induction of collagen induced arthritis prevents the onset of disease. Thus, self-peptides derived from HLA molecules could potentially generate tolerance or autoimmunity depending on their binding affinity with HLA molecules.     

Binding of peptides to HLA-DQ molecules: peptide binding properties of the disease-associated HLA-DQ({alpha}1*0501, {beta}1*0201) molecule

Peptide binding to DQ molecules has not previously been described.Here we report a biochemical peptlde-blndlng assay specificfor the BQ2 [I.e. DQ(1^*0501, ß1^*0201)] molecule. Thismolecule was chosen since It shows a strong association to diseasessuch as celiac disease and insulin-dependent diabetes mellitus.Initially we radlolabelled some selected peptides and testedthem for binding to affinity-purified DQ2 molecules. One ofthe peptides, a Mycobacterium bovis (MB) 65 kDa 243–255Ypeptide, displayed a good slgnal-to-noise ratio and was thuschosen as an indicator peptide in the DQ2 binding assay. TheMB 65 kDa 243–255Y peptide bound to DQ2 In a strictlypH-dependent fashion, with optimal binding around pH 5 and onlyweak binding at pH 7.4. The association of the MB 65 kDa 243–255Ypeptide to DQ2 was slow, but once formed, the peptide-HLA complexeswere very stable. The binding of peptides to DQ2 was specific,as shown in Inhibition experiments with a panel of 47 peptides,differing in length, sequence, and origin. The binding of peptidesto DR3 was tested in a similar assay with a Mycobacterium tuberculosis65 kDa 3–13 peptide as the binding indicator. DQ2 andDR3 molecules bound to different sets of peptides. However,the peptide binding to DQ2 and DR3 showed, In general, similarcharacteristics with respect to pH dependence and kinetic parameters,Indicating that the overall rules for peptide binding to DQmolecules are the same as those previously shown for human DRand murlne I-A and I-E molecules.     

Differences in self-peptide binding between T1D-related susceptible and protective DR4 subtypes

HLA-DR0401, 0403 and 0405 are associated with variable T1D susceptibilities when linked with a common HLA-DQ8 (DQA110301/DQB110302). It is unknown how the modest differences within the peptide binding regions of DR4 subtypes lead to distinct autoimmune risks. Since all Class II HLA molecules share the same intracellular compartments during biosynthesis, it is possible that DQ and DR compete with one another to bind and present antigenic peptides. As such, it is reasonable to hypothesize that a strong DR4 self-peptide binder down-modulates DQ8 epitope presentation more than a weak one. In this study, we first examined the binding of the peptides derived from two putative beta-cell autoantigens – GAD65 and insulin. Protective DR0403 bound similar number of self-peptides as susceptible DR0401 while highly susceptible DR0405 bound substantially less self-peptides than rest two molecules. Kinetic assays were used to further compare the stability of peptide:DR complexes formed between DR0401, 0403 and selected GAD65 peptides, which also bound DQ8. Two peptides with naturally processed DQ8 epitopes bound protective DR0403 with longer half-life and lower dissociation rate than susceptible DR0401, confirming DR0403 as a better peptide competitor than DR0401. The distinguishing peptide binding features of DR0401, DR0403, and DR0405 highlighted in this study help to explain the hierarchy of genetic associations between T1D and these DR4 subtypes. The enhanced peptide competition of DR0403 leads to a down-modulation of DQ8 epitope presentation, as compared to weak competitors such as DR0401 and DR0405, and therefore contributes to disease protection.     

T-cell responses against products of oncogenes: Generation and characterization of human T-cell clones specific for p21 ras-derived synthetic peptides

Peptides derived from mutated human proto-oncogenes bound to HLA may represent a novel type of tumor-specific antigen. Mutated ras genes are the oncogenes most frequently identified in human cancer. The transforming genes carry a mutation in codons 12, 13, or 61. We have investigated whether the T-cell repertoire of healthy individuals contains T cells capable of recognizing and responding to oncogene-derived peptides. Synthetic peptides derived from mutated p21 ras proto-oncogenes, covering mutations at codons 12 or 13 were selected. It was feasible to elicit T-cell responses and isolate several new T-cell clones (TCC) with specificity for a number of different mutated ras peptides after repeated in vitro immunization. Fout TCC wer characterized with respect to the fine specificity and HLA restriction. TCC B and I were restricted by HLA-DR molecules, and recognized the mutated p21 ras-derived peptide carrying Arg and Lys at residue 12, respectively. TCC E and F were restricted by HLA-DQ molecules, the former being specific for a mutated p21 ras-derived peptide with Val in position 13 and the latter more broadly reactive. Peptide competition experiments with a panel of ten peptides derived from p21 ras indicated that all could bind to HLA-DQ molecules of the T-cell donor, while several were also able to bind his HLA-DR molecules. These results show that several p21 ras mutations resulting in aa substitutions at residues 12 or 13 could be recognized by T cells derived from precursor T cells of relatively low frequency present in the normal repertoire of a single donor.     

Preferential presentation of herpes simplex virus T-cell antigen by HLA DQA1*0501/DQB1*0201 in comparison to HLA DQA1*0201/DQB1*0201

The HLA DQA1 locus is polymorphic. Haplotypes containing HLA DQA1*0501, but not HLA DQA1*0201, together with HLA DQB1*0201 are associated with Grave's disease and celiac sprue. In this report, we demonstrate a functional correlate of DQA1 polymorphism. T cells infiltrating a herpes simplex virus (HSV) lesion from a HLA DQ 2,7 individual yielded a virusspecific CD4+ clone restricted by DQ2. Presentation of viral peptide and protein segregated with DQA1 allele, because cell lines bearing DQA1*0501/DQB1*0201 heterodimers presented antigen in proliferation and cytotoxicity assays much more efficiently than cell lines bearing DQA1*0201/DQB1*0201. Binding of viral peptide to cell lines bearing DQA1*0201, in comparison to DQA1*0501, was only moderately reduced and may not explain this effect. Truncation and substitution analyses of peptide binding and T-cell activation were performed to determine which viral peptide residues contacting TCR might therefore be presented in an altered conformation by DQA1*0201/DQB1*0201. Residues 432, 435, 437, 438, and 440 (position P1, P4, P6, P7, and P9) contributed to DQ2 binding, whereas residues 431, 433, 434, and 436 (positions P-1, P2, P3, and P5) contributed to TCR contact. Differential presentation of peptide by HLA DQ2 heterodimers varying at the DQA1 locus may have relevance to host defense and the pathogenesis of HLA DQ2-associated autoimmune diseases. Human Immunology 53, 195-205 (1997).     

T Cells from the Peripheral Blood of Coeliac Disease Patients Recognize Gluten Antigens when Presented by HLA-DR, -DQ, or -DP Molecules

Coeliac disease (CD) is a T-cell mediated immunological disease of the small intestine which is precipitated in susceptible individuals by ingestion of gluten. We recently reported that gliadin-specific T cells can be found in the small intestinal mucosa of CD patients, and that a preponderance of these T cells was restricted by the CD-associated DQ(%aL*0501, βl*0201)heterodimer. Here we report studies on whether the same is found for gliadin specific T cells in the peripheral blood of CD patients. T-cell responses towards gluten antigens in vitro were found for both most CD patients and healthy controls. Gluten-specific T-cell clones (TCC) were established from four CD patients. Although a large proportion of these TCC were restricted by DQ molecules, including the CD-associated DQ(α1*0501, β1*0201) heterodimer, several were restricted instead by DR or DP molecules. Thus, gluten-derived peptides can be presented to T cells by several different HLA elass-II molecules, and the preferential DQ(#aL1*0501, β1*0201) restriction of gluten-specific T cells in the small intestinal mucosa of CD patients is less pronounced than for similar T eells in the peripheral blood.     

Structural model for T-cell recognition of HLA class II-associated alloepitopes.

In an effort to investigate the structure-function relationship of HLA class II molecules vis-à-vis alloepitope expression, cloned T-cell reagents were used to define polymorphic epitopes associated with DR and DQ molecules. DNA sequences of genes encoding allelic or isotypic DR or DQ molecules that appear to express the same T-cell-defined epitopes were compared in an attempt to identify association of shared sequences with shared epitopes. When sequence sharing is associated with shared epitope expression, we suggest that it is the shared sequence that encodes the epitope in question. Based on the hypothetical three-dimensional structure of the class II molecule, an approximation is made as to which parts of the HLA class II molecule are involved in alloepitope expression. T-cell clones were generated from cells primed against HLA-DR2 haplotypes representing the cellularly defined subgroups Dw2 or Dw21 (previously designated MN2, FJ0, or Tb24). Those clones determined to be DR- or DQ-directed based on monoclonal antibody inhibition assays were tested by panel cell analysis utilizing DR2-positive and DR2-negative target cells. The data support the concept that amino acids 67, 70, 71, and 74 for DR molecules and amino acids 57, 70, and 71 for DQ molecules, which appear to comprise one face of the alpha helix, are of primary importance in T-cell recognition. In other cases, sharing of both the second hypervariable region (amino acids 25-33) and the third hypervariable region (amino acids 67-74) appears necessary to explain epitope sharing for DR molecules. We emphasize that the involvement of these two hypervariable regions may indicate that alloepitope expression involves the complex of class II molecule plus peptide, with the second HVR primarily involved in determining which peptides are bound and the third in T-cell receptor (TcR) recognition and/or peptide binding; we do not rule out that conformational changes of the second HVR can induce conformational changes in the third HVR. Finally, shared alloepitopes detected by some clones could not be explained based on shared primary sequences.     

Memory T cells of a patient with follicular thyroid carcinoma recognize peptides derived from mutated p21 ras (Gln-->Leu61).

Point mutations in ras genes resulting in substitutions of amino acid Gly in positions 12 and 13, and Gln in position 61 of the ras gene product p21, are commonly found in human tumors. Peptides derived from aberrant p21 may elicit a tumor specific T cell response, provided that these peptides can bind to HLA molecules of the tumor and the patient has T cells able to recognize the corresponding peptide-HLA complex. Here we report that CD4+ T cells of memory type (CD45RO+) from a patient with a follicular thyroid carcinoma respond against a synthetic peptide derived from aberrant p21 ras having a Gln-->Leu substitution at position 61. Such responses were not observed when T cells from healthy volunteers or cancer patients where this mutation does not usually occur were stimulated with this peptide. The responding T cells did not cross-react with the corresponding peptide derived from native p21 ras nor did they recognize peptides carrying other substitutions in position 61. T cells clones were generated which recognized this Leu61 peptide when presented by HLA-DQ8 molecules. These T cell clones also recognized the corresponding intact p21 ras protein. By using several different synthetic peptides, a peptide with optimal stimulatory capacity was defined. Performing polymerase chain reaction and oligonucleotide probing we were, however, not able to detect the p21 ras gene encoding the Gln-->Leu substitution in DNA from tumor biopsies from the patient. This may indicate that tumor cells harboring the mutation leading to the Gln-->Leu substitution had been eliminated and that tumor progression was due to cells that had deleted the mutated ras gene. The finding that ras derived peptides and recombinant mutated p21 ras are immunogenic in man may form the basis for the development of cancer immunotherapy based on synthetic oncogene derived peptides.     

Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α₅ and α₆ integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α₅ and α₆-positive cells than in the peripheral blood. In addition, there were significantly fewer α₆ integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of α₅ and α₆ integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (α₅β₁) and VLA-6 (α₆β₁) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.     

Binding of gluten-derived peptides to the HLA-DQ2 (α1*0501, β1*0201) molecule,assessed in a cellular assay

The nature of the immunopathogenic relationship underlying the very strong association of coeliac disease (CD) to the HLA-DQ (A1*0501, B1*0201) genotype is not known, but probably relates to binding of gluten-derived epitopes to the HLA-DQ (α1*0501, β1*0201) heterodimer (DQ2). These epitopes have not yet been defined. In this study we have tested the binding of various gluten-derived peptides to DQ2 in a cellular assay using Epstein–Barr virus (EBV)-transformed B lymphocytes and murine fibroblast transfectants. One of these peptides (peptide A), which has previously been shown to exacerbate the CD lesion in vitro and in vivo, was found to bind to DQ2, albeit only moderately, lending further credence to its possible role in the pathogenesis of CD. The nature of peptide A's binding to DQ2 was explored with truncated and conservative point substituted analogues and compared with the published DQ2 binding motif, the results of which explain the observed level of binding.     

Interactions of HLA-B*4801 with peptide and CD8

Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8α homodimers weakly due to the presence of a threonine residue at position 245 in the α₃ domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8α homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8α, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.     

Residue 67 in the DR β1*0101 and DR β1*0103 chains strongly influences antigen presentation and DR-peptide molecular complex conformation

Abstract: Two closely-related molecules, DR(α,β*0101) and DR(α,β*0103), whose β chains only differ by three amino acids at positions 67, 70, and 71, and six intermediate molecules obtained by site-directed mutagenesis were used to ascertain the respective roles of the three polymorphic residues. Substitutions at positions 70 (D→Q), 71 (E→R) and 67 (I or L→F) strongly affected HA 306–318-specific T-cell recognition. The consequences of the substitution of residue 67 by a phenylalanine depended on the modified HLA-DR molecule. Although this substitution completely inhibited peptide-specific DR1-restricted T-cell recognition, its manifestations on the DR103-restricted T-cell response were variable (abolishing proliferation of some cell lines and not others), no matter what the peptide presented was (HA 306–319 or HIV P25 peptides). We also observed that inhibition of the proliferation of an alloreactive anti-DR103 T-cell clone, caused by a substitution at position 70, was completely canceled by substitution of residue 67 by a phenylalanine. The observations based on functional experiments, thus, suggest that residue 67 plays an important role in determining conformation of the peptide presented to the T cells. Molecular modeling was used to predict changes induced by amino acid substitutions and highly supports functional data. Substitution of residue 67 by a phenylalanine could have repercussions on the structure of HLA-DR molecule/peptide complexes and affect T-cell recognition.