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.     

The specificity of alloreactive T cells is determined by MHC polymorphisms which contact the T cell receptor and which influence peptide binding

The separate contributions to allorecognition of peptide-binding and T cell receptor-contacting residues of an allogeneic HLA-DR molecule were investigated by site-directed mutagenesis. Alloreactive T cell clones were generated from a combination of responder (DR1Dw1,DR4Dw14) and stimulator (DR1Dw1, DR4Dw10) whose DR products differed at only three amino acid positions, two of which are predicted to interact with the T cell receptor (67 and 70), and one with bound peptide (71). Transfected murine DAP.3 cells expressing the wild type and mutated forms of DR4Dw10 in which the codons for residues 70 and/or 71 had been altered towards DR4Dw14 were used to stimulate a panel of anti-DR4Dw10 T cell clones. Substitutions at either position 70 or 71, or the combination of the two, led to loss of recognition by the alloreactive T cell clones. This implies that residues involved in peptide binding and residues involved in interaction with the T cell receptor are important for this panel of alloreactive T cell clones. The specificity of these alloreactive T cells for exposed polymorphic residues on the allogeneic MHC molecule was further demonstrated by the inhibitory effects of synthetic peptides, derived from the alpha-helix of the beta 1 domain of the DR4Dw10 molecule.     

Comparison of TCR-alpha beta sequences of cross-reactive anti-DR alloreactive T-cell clones: identification of possible contact residues based on charge complementarity between TCR chains and DR determinants.

We analysed alloreactive T-cell clones selected for their differential recognition of DR variants differing in the third hypervariable region (hvr) of the DRB1 gene (amino acid positions 67-70-71). This polymorphism leads to two main hvr3 types: a basic form (Leu67-Gln70-Arg/Lys71) and an acidic form (Ile67-Asp70-Glu71) where residue 70 is probably directly accessible to the TCR on DR beta chains. The TCRs have been sequenced. Three DRw13-reactive clones use similar V alpha 2 and V beta 13 gene family members but differ mainly by their cross-reactivity towards acidic or basic DR4 variants and by the sequence of CDR3 on their TCR alpha and/or beta chains. One anti-DRw13 clone cross-reacts with most specificities sharing the DRw13 type of hvr3 and reciprocally one anti-DRBon (DRB1*0103) clone cross-reacts with DRw13. These two clones use similar V beta genes and share negative charges in CDR2 alpha at position 56. They also share these negative charges in CDR2 alpha with two other clones reacting specifically with DRBon, the acidic variant of DR1. We hypothesized that the selective recognition of positively or negatively charged residues on the DR beta chain would necessitate reciprocal charges on the TCR complementarity determining regions (CDRs) responsible for this interaction. This facilitated identification of those residues of the TCR that possibly interact with the hvr3 determinant of HLA-DR. From these observations the mechanisms allowing the recognition of alloantigens by these T-cell clones are discussed.     

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.     

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes

The contributions of individual amino acids in the polymorphic β chain and the conserved chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the β₁ domain and 11 positions in the ₁ domain. The β₁-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR₁ domain were based on the sequences of other human and mouse class II chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the β₁-domain -helix caused substantial reduction in the responses of all the clones. Substitutions at multiple positions in the β₁-domain floor and in the ₁ domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the β₁ domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules. Human Immunology 40, 311–322 (1994)     

Binding of a major T cell epitope of mycobacteria to a specific pocket within HLA-DRw17(DR3) molecules.

CD4+ T cells recognize antigenic peptides bound to the polymorphic peptide-binding site of major histocompatibility complex (MHC) class II molecules. The polymorphism of this site is thought to dictate which peptides can be bound and thus presented to the T cell receptor. The mycobacterial 65-kDa heat-shock protein (hsp65) peptide 3-13 is an important T cell epitope: it is immunodominant in the mycobacterium-specific T cell response of HLA-DR3+ individuals but, interestingly cannot be recognized in the context of any other HLA-DR molecules. We, therefore, have tested whether the hsp65 epitope p3-13 is selected for T cell recognition in the context of only HLA-DR3 molecules by an unique binding specificity for HLA-DR3. Using biotinylated peptides and EBV-transformed BLCL comprising all known HLA class II specificities, we find that p3-13 binds to HLA-DRw17(DR3) but not to any other HLA-DR molecule. Conversely, a control peptide p307-319 influenza hemagglutinin binds to all known HLA-DR molecules but only weakly to HLA-DRw17 and HLA-DR9. Peptide binding could be inhibited by excess unbiotinylated competitor analogue as well as by anti-DR monoclonal antibodies but not by anti-class I-, anti-DP- or anti-DQ monoclonal antibodies. The amino acid sequence of DRw17 molecules differs uniquely at five positions from the other DR beta 1 sequences. Three of these five residues (positions 26, 71 and 74) are potential peptide contacting residues. These residues map closely together in the hypothetical three-dimensional model of the DR molecule and, thus, most probably form a positively charged pocket, critical for the binding of p3-13. Interestingly, p3-13 does not bind to a DR3 variant, the DRw18 molecule. The DRw18 beta 1 chain differs from DRw17 at two major positions, close to or within the DRw17-specific pocket. These substitutions drastically change the structure and charge of the pocket and thus presumably abrogate its ability to bind p3-13.     

T cell receptor and peptide-contacting residues in the HLA-DR17(3) β1 chain

Previously, we have proposed that the β1 residues 9–13, 26, 28 and 86 in HLA-DR17, the most common subtype of DR3, might be critical for the binding of an immunodominant, mycobacterial epitope (peptide 3–13 of the 65-kDa heat shock protein). In order to examine directly (i) which DR17 residues are involved in peptide binding, (ii) whether the same or other DR17 residues are involved in the binding of different peptides, and (iii) whether subtle differences in the mode of peptide binding can influence T cell stimulation, we have now systematically mutated 15 highly polymorphic DR17β1 residues, located in the proposed peptide binding groove of DR17, and examined the effect thereof on binding and presentation of two peptides, hsp65 p3–13 and p56–65 of the 30/31-kDa secreted mycobacterial protein. Mutations in residues 28 (D → H) and 86 (V → G) completely eliminated binding of p3–13 and significantly reduced binding of p56–65. A mutation in residue 26 (Y → F) decreased binding of p3–13 but did not affect binding of p56–65. Substitutions of amino acid residues 28, 67, 71 and 86 in the DR17β1 chain abrogated peptide-specific stimulation of both the p3–13- and the p56–65-specific T cell clones, while specific stimulation by only one peptide was eliminated by substitution at positions 26 and 74 (p3–13) and by substitution of residues 11 and 37 (p56–65). The observation that substitution of several other peptide-contacting DR17β1 chain residues does not significantly affect peptide binding but does affect T cell stimulation, suggests that these substitutions alter the conformation of the bound peptide.     

Contribution of T-cell receptor-contacting and peptide-binding residues of the class II molecule HLA-DR4 Dw10 to serologic and antigen-specific T-cell recognition.

The relative contributions of putative T-cell receptor (TCR)-contacting and peptide-binding residues of a major histocompatibility complex (MHC) class II restriction element to serologic and antigen-specific T-cell recognition were investigated by site-specific mutagenesis. Amino acids 70 and 71 in the DR beta 1 domain of DR4 Dw10 are uniquely differnet from the other Dw subtypes of DR4. Residue 70 is predicted to be located at the membrane-distal surface of the class II molecule, where it may influence T-cell recognition by a direct interaction with a TCR. Residue 71 is predicted to form part of the antigen-binding groove where its influence on T-cell recognition may be mediated indirectly via an effect on peptide binding. Transfected murine L cells were produced expressing the products of DR4 Dw10B genes in which the codons for residues 70 and 71 had been mutated towards DR4 Dw14. Support for the predicted orientations of beta-chain residues 70 and 71 was lent by the observation that only residue 70 plays an important role in the formation of a serologic determinant. Mutation of this residue was sufficient to produce recovery of recognition by a human monoclonal antibody, NI, which has specificity for all the DR4 subtypes with the exception of DR4 Dw10. The human T-cell clone HA1.7, specific for influenza virus hemagglutinin (HA) peptide 307-319 and restricted by DR1 Dw1, exhibits degeneracy of MHC restriction on the DR4 Dw subtypes with the exception of DR4 Dw10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of Ras Oncogene Peptides to Purified HLA-DQ(αl*0102, ß1*0602) and -DR(α, ß1*0101) Molecules

Mutated oncogene peptides may be presented to T cells by HLA molecules. To be able to design the optimal peptides for stimulation of T cells in individuals with different HLA molecules, it is important to analyse the binding characteristics of oncogene peptides to HLA. HLA-DQ6 (DQ(α1*0102, ß1*0602)) and HLA-DRI (DR(α, ßl*0101)) molecules were purified from lysates of homozygous EBV-transformed cell lines. Purified HLA molecules were then tested for their ability to bind synthetic peptides in gel filtration assays. A _p21 ras oncogene peptide (previously found to stimulate DQ6-restricted T-cell clones) and an influenza matrix peptide were labelled with ¹²⁵I and served as indicator peptides for binding to DQ6 and DR1 respectively. Binding of homologous truncated and mutated _p21 ras peptides and unrelated peptides was then evaluated by their capacity to inhibit binding of the indicator peptides. _p21 ras-derived peptides were found to bind to both DQ6 and DR1 molecules indicating the existence of a promiscuous binding motif in these peptides. The binding affinities seemed to vary between the different peptides, but the amino acid substitutions resulting from natural mutations were not critical for binding. Notably, the results obtained for DQ6 in the biochemical peptide binding assay correlated well with results obtained in a functional assay using T-cell clones as probes.     

Position 71 in the α helix of the DRβ domain is predicted to influence peptide binding and plays a central role in allorecognition

Despite all the structural and functional data that have been accumulated regarding major histocompatibility complex (MHC) class II molecules during recent years, the relative contribution of putative T cell receptor (TcR)-contacting residues and peptide-binding MHC polymorphisms to MHC-restricted and allospecific T cell responses remains a point of contention. Some authors emphasize the importance of direct interaction between the allospecific TcR and polymorphic MHC residues whereas other emphasize the role of naturally processed MHC-bound peptides. We have previously described a new HLA-DRB1 allele: DR BON (DRB1*0103). This gene differs from DRB1*0101 by six base pairs clustered in the third variable region of the second exon leading to three amino acid changes at positions 67, 70 and 71 of the β chain of the HLA-DR molecule. To define the respective role of these residues in allorecognition, we have performed site-directed mutagenesis on the DRB1*0103 allele to create six mutants which are intermediary between the DR BON and the DR1 alleles. These mutant cDNA were expressed in mouse fibroblasts and the transfectants with the highest expression of class II molecules were used as stimulators for a panel of ten anti-DR BON and five anti-DR1 alloreactive T cell clones. We demonstrate that the residue at the peptide-binding position 71 is of paramount importance in the alloresponse of these clones. In addition some clones were sensitive to amino acid substitution at the TcR-contacting position 70, while substitution at position 67 affects very few clones. The dominance of residue 71 was also observed with an influenza hemagglutinin-specific HLA-DR BON-restricted T cell line.     

Inhibitory effects on HLA-DR1-specific T-cell activation by influenza virus haemagglutinin-derived peptides

Collagen (CII) 263-272 peptide, an autoantigen in rheumatoid arthritis, is a specific human leukocyte antigen (HLA)-DR1/4-binding peptide recognized by T-cell receptors (TCR). The affinity of influenza virus haemagglutinin (HA) 306-318 peptide for the antigen-binding groove of HLA-DR1/4 molecules is higher than that of CII263-272. The HLA-DR1/4-binding residues of HA306-318 are located in the region 308-317. Altered HA308-317 peptides with substitutions of TCR-contact residues may inhibit HLA-DR1/4-specific T-cell activation by blocking the antigen-binding site of HLA-DR1/4 molecules. To evaluate the role of altered HA308-317 peptides in HLA-DR1-restricted T-cell activation, we synthesized three altered HA308-317 peptides. The specific binding of altered HA308-317 peptides to HLA-DR1 molecules was examined using flow cytometry. Effects of altered HA308-317 peptides on HLA-DR1-specific T-cell hybridoma were studied by measuring T-cell proliferation and surface expression of CD69 or CD25. The results showed that altered HA308-317 peptides were able to bind to HLA-DR1 molecules and competed with CII263-272 or wildtype HA308-317 peptide. Compared with wildtype CII263-272 or HA308-317, altered HA308-317 peptides did not stimulate significant T-cell proliferation and CD69 or CD25 expression. Furthermore, the altered HA308-317 peptides inhibited HLA-DR1-specific T-cell activation induced by CII263-272 or wildtype HA308-317 peptide, which may suggest an effective therapeutic strategy in inhibition of HLA-DR1-specific T-cell responses in autoimmunity.     

Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells

To understand the effect of human MHC class II polymorphism on antigen recognition, we analyzed the memory T cell response to three tetanus toxin epitopes defined by three short synthetic peptides (p2, p4 and p30). We found that p2 and p30 are universally immunogenic, since they are recognized by all primed donors, irrespective of their MHC haplotypes. The analysis of specific clones indicates that both peptides are very promiscuous in their capacity to bind to class II. p30 can be recognized in association with DRw11(5), 7, 9 and with DPw2 and DPw4, while p2 can be recognized in association with DR1, DRw15(2), DRw18 (3), DR4Dw4, DRw11(5), DRw13(w6), DR7, DRw8, DR9, DRw52a and DRw52b. On the contrary, the third peptide, p4, can be recognized by only half of the donors in association with only DRw52a and DRw52c. Analysis of truncated peptides shows that p30 contains three distinct epitopes, each recognized in association with different class II molecules. Therefore, the restriction specificity is already set at the level of the peptide-MHC complex and, in all cases, T cells discriminate p30 bound to different class II molecules. On the contrary, p2 contains only one epitope, which is recognized in association with all DR molecules. In this case we found two different restriction patterns. Some clones are monogamous, since they recognize the peptide in association with one DR allele, while others are promiscuous, since they recognize by peptide in association with several different DR molecules. Thus, in this case, the restriction specificity is also set at the level of the T cell receptor. We suggest that both the promiscuous binding of peptides and the promiscuous recognition by T cells are dependent on the particular structure of the DR molecules, having a monomorphic alpha chain associated with a polymorphic beta chain.     

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.     

An allo-specific peptide derived from HLA-A2 is presented by HLA-DQ7

The peptide motifs of two HLA class II molecules, DR11 and DQ7, were determined from natural peptides. The EBV transformed B cells JVM (HLA-A2-DRB1^*1102-DQA1^*0501-DQB1^*0301) were cultured to a final yield of 2 10¹⁰ cells. DR11 and DQ7 molecules were immunopurified and peptides were extracted after acid elution and separated by reverse-phase HPLC. Five peptides from DR11 and five from DQ7 were sequenced using Edman degradation and other peptides were analysed by pool sequencing. Peptides were from 11 to 15 amino acids in length and P often occurred at the second residue for both DR5 and DQ7. The peptide motif for DR11 was I at position i and K or R at position i + 4. For DQ7 the most significant signal was A in the middle of the peptide as also described by Falk et al. The source of one peptide eluted from DQ7 was a polymorphic part of the HLA-A2 heavy chain (from 56th to 69th amino-acid). It was also exactly the same peptide than the synthetic peptide used by Krensky et al in the 10th international workshop to modulate lysis by HLA-A2-specific cytotoxic T lymphocytes. The biochemical characterisation of this peptide from HLA-DQ7 strongly supports functional tests showing an indirect presentation of alloantigens by MHC molecules.     

MULTIPRED2: a computational system for large-scale identification of peptides predicted to bind to HLA supertypes and alleles

MULTIPRED2 is a computational system for facile prediction of peptide binding to multiple alleles belonging to human leukocyte antigen (HLA) class I and class II DR molecules. It enables prediction of peptide binding to products of individual HLA alleles, combination of alleles, or HLA supertypes. NetMHCpan and NetMHCIIpan are used as prediction engines. The 13 HLA Class I supertypes are A1, A2, A3, A24, B7, B8, B27, B44, B58, B62, C1, and C4. The 13 HLA Class II DR supertypes are DR1, DR3, DR4, DR6, DR7, DR8, DR9, DR11, DR12, DR13, DR14, DR15, and DR16. In total, MULTIPRED2 enables prediction of peptide binding to 1077 variants representing 26 HLA supertypes. MULTIPRED2 has visualization modules for mapping promiscuous T-cell epitopes as well as those regions of high target concentration - referred to as T-cell epitope hotspots. Novel graphic representations are employed to display the predicted binding peptides and immunological hotspots in an intuitive manner and also to provide a global view of results as heat maps. Another function of MULTIPRED2, which has direct relevance to vaccine design, is the calculation of population coverage. Currently it calculates population coverage in five major groups in North America. MULTIPRED2 is an important tool to complement wet-lab experimental methods for identification of T-cell epitopes. It is available at http://cvc.dfci.harvard.edu/multipred2/.     

Substitutions in the HLA-DRα chain differentially affect DR7-restricted T-cell recognition of rabies virus antigen

To investigate the functional roles of DRα residues in T-cell recognition, 20 mutants of the DRα chain were constructed by site-directed mutagenesis. These DRα mutants were expressed with WT DR(β1∗0701) on mouse L cells and used as APC for four DR7-restricted T-cell clones specific for rabies virus antigens. The results indicate that the DRα residues are differentially involved in recognition of rabies virus antigen by different T-cell clones. Mutations in the floor of the antigen-binding groove (positions 9, 11, 22, and 24), on the α-helix (47, 55, 65, 66, and 72), and surprisingly on the outer loop (15, 18, and 19), abrogated recognition by at least one T-cell clone. Most of these residues appear to be involved in either peptide or TCR contact, based on the DR1 crystal structure. The involvement in T-cell recognition of DRα residues located in the outer loop outside the binding groove suggests that these residues may directly contact TCR, or indirectly contribute to the conformation of peptide sitting in the groove.     

Binding of collagen II peptides to several subtypes of DR4 and DR1 molecules.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a chronic inflammation of the synovial membrane. Several studies have shown that the susceptibility towards RA is confered by some HLA-DR alleles such as DR401, 404, and 101. We studied the binding of overlapping peptides of the CB11 fragment of the human collagen II on DR molecules associated or not associated with disease susceptibility. The experiments were performed by binding inhibition of the biotinylated HA (306-318) peptide on human homozygous lymphoblastoid B cell lines expressing the molecules implicated (DR401 and DR101) or not (DR402 and DR103) in RA. Among 23 peptides of collagen II tested, we highlighted 5 peptides capable of binding on the molecules associated with RA. Three of these peptides contain the specific anchor residues to bind DR401 or DR101 molecules. One of them strongly inhibited the binding of HA on DR401 and DR101, but not on DR103. This peptide was directly biotinylated and will be used in direct binding experiments on other DR molecules. The immunogenicity of these peptides will be also assessed on T cells obtained from blood or synovial membrane of several patients. Altogether these results will allow to define immunogenic collagen II epitopes potentially implicated in RA.     

Diverse usage of human T-cell receptor gene segments in HLA-DR1 allospecific T-cell clones

T-cell recognition of alloantigen involves both the MHC molecule and its associated peptide ligand. To understand the relationship between the specificity of alloantigen recognition and the structure of TCR molecules, we have investigated TCR gene utilization by sequencing TCR genes from well-defined allospecific Tlymphocyte clones. Alloreactive TLC consisted of a panel of clones primed to recognize DR l-related alloantigens. Our sequencing results revealed extensively diverse, but nonrandom, usage of TCR AV and BV gene segments and essentially no conservation in CDR3 or junctional sequences. Such observations are consistent with allospecific TCR that interact with MHC molecules on a generic level while recognizing specific peptides. They also reduce potential enthusiasm for anti-TCR therapy in allograft rejection.     

Flexibility in T-cell receptor ligand repertoires depends on MHC and T-cell receptor clonotype.

T-cell receptors (TCR) recognize peptides complexed to self-major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence-related and -unrelated ('mimicry') epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen-DR1 (HLA-DR1)-restricted T-cell clones specific for HA p307-319, we identified several diverse combinations of peptide-MHC complexes that are functionally equivalent in their ability to trigger T-cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self- as well as non-self-MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA-DR molecule and varies among different TCRs that both recognize the HA p307-319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA-restriction pattern of the TCR: T-cell recognition of the wild-type peptide that was absent in the context of a non-self HLA-DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.     

Mutations in the substrate binding site of human heat‐shock protein 70 indicate specific interaction with HLA‐DR outside the peptide binding groove

Heat‐shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I‐ and II‐restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA‐DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA‐DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA‐DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild‐type Hsp70 whereas HLA‐DR binding remains unaffected. The removal of the C‐terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA‐DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA‐DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA‐DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA‐DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70‐chaperoned peptides might thereby be directly transferred into the binding groove of HLA‐DR, so enabling enhanced presentation of the peptide on antigen‐presenting cells and leading to an improved proliferation of responding T cells as shown previously.