T-cell clones identify three distinct epitopes associated with HLA-Dw14.

Alloreactive T-cell clones were used to study allodeterminants associated with the HLA-DR1, -DR4, and -DRw14 allelic families. Three clones derived by priming against the DR4,Dw14 alloantigen were tested against a panel of HLA-D homozygous B-cell lines and homozygous and heterozygous peripheral blood lymphocytes. Each clone was blocked by monoclonal antibodies specific for HLA-DR, but not HLA-DQ or -DP, molecules, and each showed a unique pattern of allorecognition when tested against the cell panel. Clone 14B appeared to recognize a specific sequence, termed L67-A74, comprised of amino acids in the third hypervariable region of the alpha-helix of the DR beta 1 molecule, and expressed on certain DR1-, DR4-, and DRw14-positive cells. Clone EMO25 recognized the same L67-A74 sequence, but only when expressed on DR4-positive cells, suggesting a role for residues in the first and second hypervariable regions of DR4-positive DR beta 1 molecules in T-cell recognition. Clone EM036 also recognized the L67-A74 sequence, but only when expressed on DR4,Dw14.1-positive cells, implicating residues at positions 57 and 86 of the alpha-helix in T-cell recognition. These results demonstrate the range of specific T-cell responses that are possible against alloepitopes expressed by a single class II allele (Dw14), and are an indication of the diverse regions of the class II molecule that can contribute to allorecognition sites.     

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.     

HLA-Dw14 and HLA-DR3 haplotypes share a functional determinant recognized by a human alloreactive T-cell clone

In the process of studying the fine specificity of HLA class II molecules, we identified an alloreactive T-cell clone raised to a HLA-Dw14 homozygous cell line that was specifically stimulated by Dw14+ homozygous typing cells but negatively with cells expressing the HLA-Dw4,-Dw10, -Dw13, and -Dw15 subspecificities of DR4. Of interest, this clone was also equivalently activated by stimulation with all DR3 cells and cell lines tested. Negative responses were obtained using a panel of 87 non-DR3 and non-Dw14 cells, including cell lines of the Tenth Histocompatibility Workshop. A monoclonal antibody inhibition study revealed the relevant stimulating determinant to be on HLA-DR molecules in both Dw14- and DR3-positive cells. A comparison of the DR beta 1-chain-inferred amino acid sequences suggests that formation of a topologically equivalent stimulating determinant would involve the participation of two noncontiguous regions of the third diversity region of DR beta 1. The putative recognition conformation detected by the clone is most probably specified by the presence of a valine at position 86 and a nonnegatively charged residue at positions 70, 71, and 74, since these are the only residues where DR3 and Dw14 are distinguishable from all other HLA-DR types. These findings illustrate that the functional ability of class II molecules is not necessarily either illustrated or predicted by serologic typing or by simple considerations of amino acid sequence.     

Shared molecular markers of genetic predisposition to seropositive rheumatoid arthritis

Rheumatoid arthritis is associated with the human class II major histocompatibility complex antigens known as HLA-DR4. HLA-DR4 can be subdivided by cellular typing into five subtypes: Dw4, Dw10, Dw13, Dw14, and Dw15. By traditional serologic methods, 60-80% of rheumatoid arthritis patients type HLA-DR4 compared to approximately 20% of the general population. It has been demonstrated, using a panel of four alloreactive T-cell clones, each of which recognized HLA-DR4, Dw14 homozygous typing cells, that cells from all of a group of 23 rheumatoid arthritis patients could be recognized by one or more of these clones regardless of the patients' serologic typing. As the expressed polymorphism of the DR molecule is accounted for by the beta 1 gene, this gene was amplified, using the polymerase chain reaction, and sequenced. Seven patients whose cells were recognized by one of the DR4, DW14-specific T-cell clones, T431, were analyzed. All of these patients shared a common sequence in the third hypervariable region of the DR beta 1 chain gene. The sequence identified is the one normally associated with DR4, Dw14 and DR1. Patients and DR4-positive controls whose cells did not stimulate this clone did not share this sequence. These results suggest that this hypervariable region might be an important contribution to a restriction site for the putative causative agent(s) in rheumatoid arthritis.     

Functional analysis of MHC class II-restricted T cells derived from a Caucasian with a DR4, Dw15, DQw8 haplotype

Rabies virus-specific CD4+ T lymphocyte clones were isolated from a Caucasian male vaccine recipient (DR4/7, DQw2/w8; DPw4) and studied for their major histocompatibility complex restricting elements. None of the rabies-specific T-cell clones could be induced to proliferate to antigen by either lymphoblastoid cells or DR-transfected L cells expressing DR4 molecules of the Dw subtypes commonly found on Caucasian individuals (Dw4, Dw10, Dw13, Dw14). The HLA-Dw subtype of the rabies vaccine recipient was determined by conventional mixed lymphocyte culture, and the results revealed that this individual had a DR4 (Dw15), DR7 (Dw7) phenotype. The presence of the DR4, Dw15 antigen was confirmed by nucleotide sequencing of the DR4B1 gene corresponding to the DRB1*0405 allele. Significant antigen-induced T-cell proliferative responses were obtained with two DR4, Dw15, DQw4 homozygous lymphoblastoid cell lines of Japanese origin (HAS-15 and KT-3) and with a L-cell transfectant expressing the DR4, Dw15 molecule. The existence of the DR4, Dw15 antigen in the Japanese has been reported to be associated with the DQw4 specificity. However, the presence of DQw8 (previously designated DQw3.2) and the absence of DQw4 in the lymphoblastoid cells of the Caucasian rabies vaccine was confirmed with monoclonal antibodies IVD12 (anti-DQw7 + DQw8 + DQw9) and HU46 (anti-DQw4) and by the reactivity of a DQw8-restricted antigen-specific T-cell clone. These studies indicate, contrary to previous findings, that the DR4, Dw15 molecule may be present in Caucasian (non-Japanese) individuals in association with DQw8.     

T-cell receptor V beta selectivity in T-cell clones alloreactive to HLA-Dw14.

The HLA-DR4 subtypes Dw14 and Dw4 are T-cell-defined allospecificities encoded by the DRB1*0404 and DRB1*0401 genes, respectively. Although these allelic subtypes differ in only two amino acids, allorecognition between Dw14 and Dw4-positive individuals is brisk. This provides an opportunity to analyze T-cell receptor (TCR) usage in a very limited and specifically targeted case, namely the Dw4 anti-Dw14 allogeneic T-cell response. The variable (V), diversity (D), and joining (J) region sequences of the TCR beta chain from two different Dw14-specific alloreactive T-cell clones derived from a Dw4 donor were examined. Clone EMO25 recognized the Dw14.1, Dw14.2, and Dw15 subtypes, which share a DRB1 polymorphism at codon 71 on a DR4 background, while clone EMO36 reacted with only the Dw14.1 subtype associated with polymorphisms at codons 71 and 86. TCR beta cDNA from each clone was amplified using an anchored polymerase chain reaction (PCR) and subsequently expanded with V beta- and C beta-specific primers for asymmetric PCR and direct DNA sequencing. Both clones were found to express the same TCR V beta 8.2 gene segment; however, they have several different residues within the V beta-D beta-J beta junctional regions. V beta 8 usage was also enriched in polyclonal cells obtained from mixed lymphocyte cultures performed between the Dw4 and Dw14 responder-stimulator combination from which EMO25 and EMO36 were derived.     

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.     

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)     

Effects of localized HLA class II beta chain polymorphism on binding of antigenic peptide and stimulation of T cells.

The relationship between HLA-DR1 polymorphism and recognition of antigen by T cells was investigated. Two allelic variants of HLA-DR1, which differ by amino acid substitution at positions 85 and 86 of the beta chain, were characterized for the effect of substitution on recognition of foreign antigen by DR1-restricted T cells. Substitution of alanine and valine for valine and glycine residues at positions 85 and 86 of the DR1 beta chain resulted in deficient T-cell stimulation as demonstrated by the requirement for higher concentrations of antigen to induce maximal levels of T-cell proliferation, induction of lower levels of proliferation at optimal antigen concentrations, and slower kinetics of formation of stimulatory peptide-DR1 complexes. Direct binding studies employing both biotinylated and radioiodinated forms of antigenic peptide demonstrated quantitatively lower levels of peptide bound to substituted DR1 molecules and low levels of site-specific binding as assessed by competitive inhibition analyses. The effect of MHC class II polymorphism on peptide-binding affinity as opposed to induction of appropriate peptide conformation and the impact of polymorphism at DR1 beta chain positions 85 and 86 on allorecognition of HLA-DR1 are discussed.     

A supertypic HLA class II determinant shared by DR1 and DRw9, and crossreactive with DR2, defined by human monoclonal antibody

A human monoclonal antibody Pez.2F5, produced by a lymphoblastoid cell line, has been established in vitro by Epstein-Barr virus (EBV) transformation of B lymphocytes isolated from the blood of a volunteer immunized with allogeneic peripheral blood leukocytes (PBLs). The antibody reacted with a new supertypic determinant expressed on all lymphoblastoid cell lines homozygous for HLA-DR1, -2, and -w9. The genetic linkage of the Pez.2F5 determinant to the HLA region was demonstrated by family segregation studies. Quantitative absorption studies indicated that DR2-positive cells required more Pez.2F5 antibody for lysis, and since their absorption capacity was significantly lower than that of DR1- or DRw9-positive cells, it is likely that the Pez.2F5 determinant of the DR2 haplotype is crossreactive but not identical with the determinant found on the latter haplotypes. In addition, on a test panel of HLA-typed B lymphocytes, Pez.2F5 showed perfect correlation with DR1 and DRw9, but reacted with only a fraction of DR2-positive cells. The Pez.2F5 determinant was found to be absent from resting T lymphocytes, but its expression could be identified on IL-2-dependent T-cell lines by cytotoxicity and flow cytofluorometric analysis. By sequential immunoprecipitation and SDS gel analysis of antigens of DR1 cells it was determined that the Pez.2F5 determinant is carried by HLA class II DR molecules. Thus, the Pez.2F5 is the first described human monoclonal antibody able to immunoprecipitate HLA class II-related molecules.     

Further DNA sequence microheterogeneity of the HLA-DR4/Dw13 haplotype group: Importance of amino acid position 86 of the DRβ1 chain for T-cell recognition

Using the polymerase chain reaction we have isolated and sequenced cDNA clones corresponding to the polymorphic first domain of the DRβ1 chain from the DR4, “Dw13” cell line, JHa. We have found that the JHa DRβ1 allele differs from previously reported Dw13 alleles by a single amino acid substitution at position 86. The functional relevance of this polymorphism is supported by the reactivity pattern of a T-cell clone, E38. E38 is an alloreactive T-cell clone which reacts with all Dw14 stimulator cells and all Dw13-positive cells tested except the “Dw13”- positive homozygous typing cell line JHa. Inhibition studies with monoclonal antibodies revealed the stimulating determinant to be on DR and not on DQ or DP molecules. These data indicate that position 86 of the DRβ1 chain can play an important role in the formation of determinants recognized by T cells.     

Clonal analysis of HLA-DR and -DQ associated determinants: Their contribution to Dw specificities

In order to investigate the distribution of epitopes recognized by T-cell clones directed against HLA class II products, bulk primed cell populations were generated using cells matched for class I determinants but disparate for class II determinants. Cells were cloned by single cell deposition (FACS IV) or limiting dilution (1 cell/3 wells), and assayed for proliferative and cytolytic function with panels of well-characterized cells. All cytolytic clones generated from an anti-DR4/Dw4/DQw3 priming combination or an anti-DR2/Dw2/DQw1 priming combination lysed essentially all targets sharing the same Dw type as the sensitizing cell. In some cases, other targets were also lysed. For instance, some clones were lytic to targets bearing the same DR antigen but another Dw subtype including a few clones lytic to virtually all cells carrying that DR specificity. An occasional target cell expressing a different DR antigen from the sensitizing cell was also lysed by these clones, in some cases to the same extent of lysis seen on the specific target. Monoclonal antibody inhibition studies identified three groups of clones: the DQ directed clones and clones apparently directed at more than one DR product. However, the number of molecules detected for each haplotype remains to be investigated. Our data indicate that determinants detected on both DR and DQ products are associated with the Dw type of the sensitizing cell showing that there is polymorphism recognized by T cells on both DR and DQ that is subtypic to the serologically defined specificities. Thus, it appears that the bulk T-cell response is a composite of individual clones recognizing distinct determinants on these class II molecules. The implications of these findings for studies of HLA restricted recognition are discussed.     

The impact of naturally occurring DR3 microvariants, DRw17 and DRw18, on T-cell allorecognition.

The limited amino acid sequence differences between the DR3 microvariants, DRw17 and DRw18, are found in the second variable region of the DR beta chain (residues 26 and 28) as well as in framework residues 47 and 86. Using selected responder/stimulator combinations, alloproliferative T-lymphocyte clones (TLC) were generated which recognize either a supertypic DR3-related determinant(s) or only those T-cell recognition determinants created by the four amino acids which differ between DRw17 and DRw18. Results indicate that the microvariation creates potent T-cell recognition determinants while leaving the DR3-related determinant(s) unaffected. Several TLC were generated which recognize the DRw18 molecule strongly and the DRw52c molecule weakly reflecting the sequence similarity between these molecules. In addition, one TLC was generated which recognizes DRw18 and DRw14,Dw9 but not DRw14,Dw16 molecules, a result not predicted by linear amino acid sequence comparisons. The intricate and sometimes unpredictable allorecognition patterns observed demonstrate that the molecular context of a specific amino acid sequence is as important as the actual sequence in forming a T-cell recognition site and, thus, in shaping the immune response profile of a given allele.     

Allo-cross-reactivity of a human neuraminidase-specific T cell clone dependent on presentation of an endogenous B cell-specific antigen.

T cells specific for foreign antigen recognize a complex of peptides and self-major histocompatibility complex (MHC) molecules and can also cross-react with allo-MHC molecules. It remains controversial, however, what alloreactive T cells exactly recognize. It has been proposed that alloreactive T cells recognize endogenous peptides presented by allo-MHC molecules. To test this hypothesis, we examined an influenza virus-specific T cell clone (6H5), specific for neuraminidase N2 and restricted by HLA-DR1. In the absence of influenza virus, this clone cross-reacted with HLA-DR1Dw1+ but not with HLA-DR1Dw20+ Epstein-Barr virus-transformed lymphoblastoid cells (B-LCL). Cold target inhibition experiments and the rearrangement pattern of the T cell receptor beta chain indicated that 6H5 was a monoclonal T cell population most likely using the same T cell receptor for both responses. To determine whether determinants other than HLA-DR1Dw1+ B-LCL or activated B cells, but, surprisingly, not to other cell types expressed HLA-DR1Dw1, including monocytes and transfected L cells. These experiments further support the concept that recognition of allogeneic MHC (in this case HLA-DR1Dw1) may result from a cross-reactivity of T cells specific for a complex of foreign antigen and self-MHC (neuraminidase N2 and HLA-DR1Dw20). Furthermore, allorecognition of T cell clone 6H5 appears to depend upon the recognition of a complex of allogeneic MHC and a cell-type specific endogenous peptide presented by activated B cells.     

Identification of cross-reactive T cell restriction epitopes located on the DR7 beta 1 and DR beta 4 molecules.

L cell fibroblasts transfected with HLA class II cDNA clones isolated from a cDNA library produced from a DR7 homozygous cell line were used as antigen-presenting cells (APC) for three HLA DR-restricted, diphtheria toxoid-specific T-cell clones in order to assess the antigen-presenting ability of the transfectants and to define the class II restriction of each clone. Class II-expressing transfectants are capable of presenting antigen to antigen-specific T-cell clones, although the transfectants are less efficient at antigen presentation than conventional APC. Paraformaldehyde fixation of transfectants prior to antigen pulsing abrogated antigen presentation, demonstrating that the transfectants require antigen processing. Antigen presentation by transfectants is completely inhibited by CD4-specific monoclonal antibodies (mAb) and one of four DR-specific mAb, whereas antigen presentation by conventional APC is only partially inhibited. Both the DR alpha:DR7 beta 1 and DR alpha:DR beta 4 (DR omega 53) molecules of the DR7 allotype serve as restriction elements for the diphtheria toxoid-specific T-cell clones. One clone is restricted by the DR7 beta 1 molecule, another clone by the DR beta 4(DR omega 53) molecule, and a third clone by a cross-reactive T cell epitope on DR7 beta 1 and DR beta 4(DR omega 53) molecules. The two DR beta 4(DR omega 53)-restricted clones react, however, differently with a panel of HLA-DR DR omega 53-positive human peripheral blood lymphocytes used as APC. Therefore the data presented here clearly document that the DR beta 4 (DR omega 53) chain may serve as restriction elements for DT-specific T-cell clones. They also provide the first evidence for functional cross-reactivity of the products of two different DR beta loci and in addition emphasize the high complexity of the supertypic HLA-DR omega 53 specificity.     

HLA-DR peptide-induced alloreactive T cell lines reveal an HLA-DR sequence that can be both "dominant" and "cryptic": evidence for allele-specific processing.

Previously, we reported on a T cell line, ThoU6, which we obtained through stimulation of DPw3+ cells with a synthetic "DR3 peptide" with a sequence identical to the third hypervariable region of the DRB1*0301 chain. This T cell line recognizes both the synthetic peptide presented by DPw3 as well as DR3+ DPw3+ stimulator cells. This implies that the synthetic DR3 peptide has a natural counterpart in DR3-positive cells. Here we describe the recognition pattern of another T cell line that was sensitized with the same synthetic DR3 peptide. This T cell line, BieU6, shows both HLA-DRw13/Dw18 (self)-restricted recognition of the synthetic DR3 peptide and allorecognition towards DR13/Dw19, a molecule which is highly homologous to Dw18, in the absence of synthetic peptide. These results suggest that the epitope formed by the Dw18 molecule plus the synthetic DR3 peptide and recognized by T cell line BieU6 mimics the Dw19 molecule. The potential role for a Dw19-specific peptide is discussed. The inability of T cell line BieU6 to recognize Dw18+ DR3+ cells indicates that, in this case, the synthetic DR3 peptide is "cryptic", i.e. does not have a natural counterpart that is effectively presented to T cells. Mapping of the shortest peptides recognized by T cell lines ThoU6 and BieU6 indicate that these sequences are fully overlapping. We, therefore, suggest that the antigen-presenting molecules, HLA-DPw3 and HLA-Dw18, differ in their accessibility for self peptides derived from the third hypervariable region of DR molecules. These observations may be explained by allele-specific processing.     

CD4+T cells restricted by DQ not by DR support CD8+T cells to grow

Much attention has been paid whether there are any differences in regulating the human immune response between HLA-DR and -DQ molecules encoded by the genes within the HLA class II multigene family. Previous studies have suggested that HLA DQ molecules control low responsiveness through activating CD4 T cells which generate CD8 positive T cells, whereas HLA -DR molecules control high responsiveness through activating CD4 helper T cells. To examine this model we investigated the streptococcal cell wall antigen (SCW) specific T cell lines restricted by either DR or DQ molecule. To identify the restricting molecules, L cell transfectants expressing DQw1, DR2AB1 or DR2AB5 from Dw12 haplotype or DQw4, DR4 or DRw53 from DW15 haplotype were used. 1. From individuals with Dw12 which is a low responder haplotype to SCW, T cell clones specific to SCW and restricted by HLA-DQw1 or DR2 were identified, whereas from individuals with Dw15 which is a high responder haplotype, only DR4 or DRw53 restricted T cell clones were identified and DQw4 restricted T cells were never observed. 2. SCW specific CD4 T cells restricted by DQw1 were able to support the growth of CD8 positive cells, whereas those restricted by DR4 could not do so. 3. The CD8 T cells also required autologous antigen presenting cells and SCW to grow, and they completely blocked the immune response to SCW in vitro. These observations clearly demonstrated the distinct function of HLA-DQ and -DR molecules in regulating the human immune response to SCW.     

On the relative immunogenicity of DR alloantigens: T cell recognition of HLA-DR2a and HLA-DR2b

The HLA-DR2 haplotype encodes two highly polymorphic DR molecules, DR2a and DR2b. Because little is known regarding the relative immunogenicity of different HLA-DR molecules, we have studied the T-cell recognition of DR2a and DR2b molecules from the DRw15, Dw2 haplotype. A series of DR2-specific alloreactive T-cell clones were analyzed with murine L-cell transfectants expressing either the DR2a or the DR2b molecules as stimulator cells in proliferation assays. Somewhat surprisingly, both DR2a and DR2b were capable of stimulating DR2-specific T-cell clones with equal magnitude and similar frequency. In addition, DR2a and DR2b are functionally distinct, that is, no clone was identified which was stimulated by both DR2a and DR2b molecules.     

Molecular mimicry by major histocompatibility complex molecules and peptides accounts for some alloresponses.

One explanation offered for the uniquely high precursor frequencies of T cells which recognize allogeneic major histocompatibility complex (MHC) molecules, and their lack of self-MHC restriction, is that the alloreactive cells are polyclonal populations the primary specificity of which is self-MHC plus peptide X1, X2, ... Xn. These are postulated to cross-react with allo-MHC plus peptides Y1, Y2, ... Yn. It has been further suggested that the structural basis for the crossreactivity between different MHC alleles is the similarity in amino acid sequence of that part of the molecule predicted to make contact with the T cell receptor (TcR). In order to test this concept, T cells were obtained with dual specificity for influenza haemagglutinin (HA), restricted by HLA-DR1Dw1, and for DR4Dw4/Dw14 expressed on allogeneic human B cell lines, and the specificity of one clone was studied in detail. The exposed, TcR-contacting surfaces of these two DR molecules are predicted to be identical. Although the HA-specific response was stimulated by DR1-expressing mouse DAP.3 transfectants, DAP.3 cells expressing the alloantigen DR4Dw4 were unable to stimulate, possibly because of a failure to present the necessary human peptide for anti-DR4 allorecognition. Therefore, the effects of pulsing the DR4Dw4-expressing DAP.3 cells with the HA peptide were examined. This peptide is known to bind to both DR1 and DR4. Addition of the HA peptide restored the anti-DR4Dw4 response. These data support the concept that allorecognition in some responder/stimulator combinations can be explained by cross-reactivity at the level of the MHC molecule and the peptide.     

Mapping of allospecific T-cell recognition sites encoded by the HLA-DR4 beta 1-chain

Genes of the major histocompatibility complex (MHC) influence the immune system by their central role in the activation of T lymphocytes, which have to corecognize antigen in the association with MHC-encoded cell surface molecules. The location and number of sites on HLA class II molecules that interact with T-cell receptors remains unknown. Using a set of ten alloreactive human T-cell clones we have defined the molecular basis of T-cell interaction sites on the HLA-DR4 molecule. At least seven unique determinants are recognized that are confined to an immunodominant region encoded by the third hypervariable region (hvr) of the HLA-DR beta 1-chain. Substitutions at amino acid positions 71, 74, and 86 contribute similarly to determinants recognized by alloreactive T cells. A cluster of tightly overlapping sites stimulatory for distinct T cells is contained within the segment of amino acid residues 71 to 86, which is expressed within the HLA-DR4 as well as HLA-DR1 haplotype. Five of the ten T-cell clones are stimulated by HLA-DR1+ cells, suggesting that allospecific T-cell receptors directly interact with protein structures determined by the third hvr. These data provide evidence that the third hvr of the HLA-DR beta 1-chain encodes for a functional domain on the surface of the molecule that is recognized by a polyclonal T-cell response.