Splenic but not thymic autoreactive T cells from New Zealand Black mice respond to a dominant erythrocyte Band 3 peptide

Previous work from our laboratory suggested that erythrocyte Band 3 peptide 861-874 is the dominant epitope recognized by splenic T cells from adult New Zealand Black (NZB) mice that are developing autoimmune haemolytic anaemia (AIHA). Here, it is shown that splenic T cells from 6-week-old NZB mice mount a vigorous in vitro proliferative response to peptide 861-874 and some other selected Band 3 peptides. As the donors grow older, splenic T cells respond to an increasing number of Band 3 peptides and the magnitude of their response also becomes greater. Splenic T cells from 3-week-old NZB mice still responded vigorously to peptide 861-874 and Band 3. By contrast, neither thymocytes nor single-positive CD4-enriched thymus cells from NZB mice responded to peptide 861-874 or Band 3, although they responded to concanavalin A (Con A). However, thymocytes from mice expressing a transgenic T-cell receptor (TCR)-specific for myelin basic protein (MBP) peptide Ac 1-9 responded vigorously to Ac 1-9. It is considered that the T-cell response of NZB mice to Band 3 is initially focused on peptide 861-874 and later spreads to other Band 3 peptides as the disease progresses and that peptide 861-874-reactive T cells are primed in the periphery rather than the thymus.     

Hb(64-76) epitope binds in different registers and lengths to I-Ek and I-Ak

The nature of peptide binding to MHC molecules is intrinsically degenerate, in what, one given MHC molecule can accommodate numerous peptides which are structurally diverse, and one given peptide can bind to different alleles. The structure of the MHC class II molecules allows peptides to extend out of the binding groove at both ends and these residues can potentially influence the stability and persistence of peptide/class II complexes. We have previously shown that both I-E(k) and I-A(k)-restricted T cell hybridomas could be generated against the Hb(64-76) epitope. In this study, we characterized the binding register of the Hb(64-76) epitope to I-A(k), and showed that it was shifted by one residue in comparison to its binding to I-E(k), and did not use a dominant anchor residue at P1. This conclusion was further supported by the modeling of the Hb(64-76) epitope bound to I-A(k), which revealed that all of its putative anchor residues fit into their corresponding pockets. We identified the naturally processed Hb epitopes presented by both I-E(k) and I-A(k), and found that they consisted of different species. Those associated with I-A(k) being 20-22 residues long, whereas, those found to I-E(k) contained 14-16 residues. These findings suggested that the lack of a dominant P1 anchor could be compensated by the selection of longer peptides. Overall, these studies revealed the Hb(64-76) epitope bound to I-E(k) and I-A(k) in distinct registers and lengths, demonstrating the plasticity MHC molecules have in generating distinct TCR ligands from the same amino acid sequence.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.     

Modulation of TCR recognition of MHC class II/peptide by processed remote N- and C-terminal epitope extensions

N- and C-terminal extensions of naturally processed MHC class II-bound peptides may affect TCR recognition. In fact, residues immediately flanking the minimal epitope on either side can contact the MHC groove and modify the interaction with a TCR. We report now that residues much farther away from the peptide core can also modulate TCR recognition in a functional antigen presentation system. To show this, we isolated from the same donor DR5-restricted T cell clones, specific for the HIV-1 RT(248-262) sequence and differing in their ability to respond to recombinant antigens obtained by insertion of the epitope in different positions of schistosomal, human, or murine glutathione-S-transferase (GST). We found that the reactivity profile of individual clones was related to their TCR fine specificity, suggesting that processing can generate determinants focused onto the same epitope, but antigenically distinct. In addition, we analyzed the response of this panel of T-helper cell clones against GST-derived recombinant antigens in which the epitope was flanked by stretches of polyalanine or polyserine on either side. These spacers had different effects on TCR recognition suggesting that secondary structures outside the core peptide may influence MHC/epitope complex recognition over a distance of 15-30 residues from the determinant.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

Rational optimization of tumor epitopes using in silico analysis‐assisted substitution of TCR contact residues

Altered peptide ligands with increased affinity of the peptide–MHC complex for the TCR provide an alternative strategy to natural T‐cell epitopes for cancer immunotherapy, as they can recruit and stimulate stronger T‐cell repertoires. However, it remains unclear how alterations of the TCR contact residues improve the interaction between the peptide–MHC complex and the TCR molecule. In this study, we introduced a molecular simulation strategy to optimize a tumor immunodominant epitope NY–ESO‐1_157–165 by the substitution of the potential TCR contact residues. We correlated molecule simulation with T‐cell activation capacity assay and detected the effect of modifications of TCR contact residues on T‐cell recognition. An agonist peptide W5F with substitution at Trp5 with Phe was identified and it exhibits a stronger ability to induce a cross‐reactive CTL response with the WT peptide. Additionally, the W5F‐induced CTL could be maintained with the WT peptide and possess higher capacity in lysing native NY–ESO‐1‐expressing tumor cells. These results provide important insights into the enhanced immunogenicity of epitopes through substitution at the TCR contact sites and revealed a novel molecular simulation approach for rational therapeutic peptide vaccine design.     

Limited plasticity in T cell recognition of modified T cell receptor contact residues in MHC class II bound peptides

The balance between specific and degenerate T cell recognition of MHC class II bound peptides is crucial for T cell repertoire selection, and holds important implications for protective immunity versus autoimmunity. To investigate the degree of degeneracy in T cell recognition, we applied selected modifications to T cell receptor (TCR) contact residue amino acids in the MHC class II bound epitope gpMBP72-85. By using glycosylated amino acids, as an example of a posttranslational modification, large alterations were applied. Small modifications were accomplished by exchanging an arginine residue for a citrulline or an ornithine residue. Finally, the unmodified TCR contact residue side chains were shifted one atom position to the left, using peptoid residues. Both these large and subtle changes in the wild type (WT) peptide caused lack of recognition by WT peptide specific monoclonal and polyclonal T cells. Furthermore, T cells specific for the modified peptides did not cross recognize the WT peptide. Using a set of additional compounds, we investigated the specificity of these T cell populations into detail. Our data reveal a strongly limited plasticity in T cell recognition, and a high specificity for TCR contact residue side chains.     

Murine models of systemic lupus erythematosus: B and T cell responses to spliceosomal ribonucleoproteins in MRL/Fas(lpr) and (NZB x NZW)F(1) lupus mice

(NZB x NZW)F(1) and MRL/Fas(lpr) lupus mice present a similar phenotype with a spectrum of autoantibodies associated with very severe nephritis. It is thought, however, that in contrast to other lupus-prone mice such as MRL/Fas(lpr) mice, (NZB x NZW)F(1) mice do not generate autoantibodies to ribonucleoproteins (RNP) Sm/RNP. In this study, we demonstrate that contrary to previous reports, the autoimmune response directed against Sm/RNP antigens also occurs in NZB x NZW mice. CD4(+) T cells from unprimed 10-week-old NZB x NZW mice proliferate and secrete IL-2 in response to peptide 131-151 of the U1-70K protein, which is known to contain a T(h) epitope recognized by CD4(+) T cells from MRL/Fas(lpr) mice. Peptide 131-151, which was found to bind I-A(k) and I-E(k) class II MHC molecules, also bound both I-A(d) and I-E(d) molecules. This result led us to also re-evaluate longitudinally the anti-Sm/RNP antibody response in NZB x NZW mice. We found that 25-week-old mice do produce antibodies reacting with several small nuclear and heterogeneous nuclear (hn) RNP proteins, such as SmD1, U1-70K and hnRNP A2/B1 proteins. The fine specificity of these antibodies was studied with overlapping synthetic peptides. The same antigenically positive and negative peptides were characterized in MRL/Fas(lpr) and NZB x NZW mice in the three proteins. This new finding can help to understand the mechanisms involved in the development of the anti-Sm/RNP antibody response and, particularly, the role played by non-MHC genes in this autoimmune response.     

Characterization of in vivo expanded OspA-specific human T-cell clones

A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.     

Recognition of allo-peptide is governed by novel anchor imposition and limited variations in TCR contact residues

Immune specificity of a T cell is determined by the TCR contact residues exposed on the antigenic peptide/MHC complex. Naturally processed, biallelic epitopes from H7 minor histocompatibility (mH) antigen vary in position 7 (p7) from aspartic acid (D) to a glutamic acid (E), which differ by an additional methylene (-CH(2)) in the side chain. Here, we show that this variation generates a strong anti-H7a or anti-H7b cytotoxic T cell responses. Further, the H7 allelic peptides use p6 asparagine as their central anchor residue and amino acid variations in either the canonical p5 or the predicted p6 anchor positions in the antigenic epitope were detrimental for TCR recognition. In addition, introduction of any other amino acids, except asparagine, in the polymorphic p7 significantly abolished the ability of anti-H7b TCR recognition. This demonstrates that only an asparagine with an amine group as a side chain instead of a charged oxygen radical could effectively stimulate the anti-H7b specific T cells. Our findings provide evidence that mH antigen-specific TCRs are highly stringent in recognizing their cognate epitopes.     

Autoreactive T cell specificity in autoimmune hemolytic anemia of the NZB mouse

Splenic T cells from Coombs'-positive New Zealand Black (NZB) mice proliferated consistently in vitro in response to the integral red blood cell (RBC) membrane protein Band 3, the antigen previously shown to be the target for the pathogenic RBC autoantibodies. The responding cells predominantly express CD4 and the proliferative response is blocked by antibodies to the NZB major histocompatibility complex class II but not by antibodies to an irrelevant H-2 haplotype. NZB splenic T cells also proliferated in response to the internal membrane skeleton protein spectrin. By contrast, T cells from BALB/c and DBA2 mice, which bear the same H-2 haplotype as NZB mice, but which do not develop autoimmune hemolytic anemia (AIHA), fail to respond to Band 3. It is considered that these results support the hypothesis that Band 3-reactive T cells provide help for the production of pathogenic anti-Band 3 autoantibodies in NZB mice. T cells from Coombs'-negative NZB mice as young as 3 weeks old proliferated in response to Band 3; moreover, the RBC from Coombs'-negative mice bore elevated levels of autoantibody as judged by a sensitive direct enzyme-linked anti-globulin test. Thus, the pathology of AIHA develops at a much earlier age than was thought previously.     

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H-2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG(38-51) peptide important for peptide binding to I-Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I-Ab. In addition, G38 and W39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I-Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC-binding residue of the MOG(38-51) peptide and most likely occupies the p1 pocket of I-Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG(38-51) peptide to study their therapeutic potential in the EAE model.     

Characterization of HLA-DR- and TCR-binding residues of an immunodominant and genetically permissive peptide of the 16-kDa protein of Mycobacterium tuberculosis

The 16-kDa protein of Mycobacterium tuberculosis represents an important antigenic target during bacillary latency and, consequently, should be considered as candidate subunit vaccine component. In this study, we have used CD4 T cell clones that recognize the peptide p91-110, an immunodominant and genetically permissive epitope, in the context of five different HLA-DR molecules and truncated and substituted variants of this peptide, to identify the minimal binding sequence (HLA-DR-binding core) and the minimal stimulatory sequence (TCR-binding core), as well as the residues that contact HLA-DR molecules and the TCR. We have found a common 9-mer sequence, spanning amino acids 93-101, as the binding core for HLA-DR1, -DR11, -DR13 and -DR7, but a longer (13-mer) sequence spanning amino acids 92-104 was required for binding to the HLA-DR15 molecules. F(93) was required for binding to all the tested HLA-DR molecules, hence allowing us to identify it as the N-terminal primary anchor residue (P1). Additionally, the binding requirements for other residues varied considerably between the tested alleles: A(94) for HLA-DR15, V(99) for HLA-DR1, -DR15, -DR11 and -DR7, R(100) for HLA-DR11 and -DR13, and L(104) for HLA-DR15. Concerning the residues of p91-110 peptide required for binding to the TCR, the pepscan analysis results would support the contention that P(-1) E(92), P6 F(98) would be important TCR contact sites because their substitutions led to full loss of T cell activation. Moreover, P8 R(100) is found to be critical residue in binding to HLA-DR11- and -DR13-restricted T cell clones, without influencing binding to the relevant HLA-DR molecule. Our results could be useful to design peptides with altered HLA anchor residues or TCR interaction sites to achieve remarkable increase in activity and to study their vaccine potential.     

Modulation of T cell response to hGAD65 peptide epitopes

Human CD4 T cell responses to an epitope of hGAD65 (GAD = glutamic acid decarboxylase), residues 555-567, are modulated by interaction with an altered peptide ligand containing modifications at TCR contact residues. Using different HLA-DR4 molecules with polymorphisms at sites corresponding to peptide binding pockets p1 and p9, we tested the effect of additional modifications in the altered peptide ligand (APL) designed to increase the avidity of the MHC-peptide interaction and therefore the efficiency of TCR signaling. Modification of the peptide or the MHC molecule which enhanced the p1 interaction also enhanced the antagonist activity of the modified APL. In contrast, modifications at p9 led to a reversal in APL function, resulting in agonist activity. Molecular homology modeling of these MHC-peptide interactions suggests a structural basis for this functional dichotomy in which topographically remote variations lead to unique interaction effects.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

Analysis of functional sites on a peptide antigen, p43-58, in I-A or I-E-restricted T cell responses.

It has been shown that two different sites (an agretope and an epitope) on a peptide antigen function independently in T cell responses to the antigen. By virtue of these sites, antigens, MHC molecules, and TCRs constitute trimolecule complexes which eventually result in T cell activation. In our previous reports, we have defined that residues 46 and 54 on synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58, AEGFSYTDANKNKGIT) and its analogs function as an agretope and residue 50 as an epitope in both I-Ab and I-Ak-carrying mice. In the present study, to extend our method to the other MHC class II molecules (I-E), we used two peptide antigens, 46D50V54R and 50V54R, which had been prepared by substitution of amino acids at positions, 46, 50 and 54 or 50 and 54 of p43-58 D, V, R or V, R, respectively, and compared the immunogenicity with those of other peptide analogs. The 46D50V54R was shown to be non-immunogenic in I-Ab-carrying mice and the 50V54R was non-immunogenic in I-Ak-carrying mice. In contrast, the 46D50V54R or 50V54R could induce I-E-restricted proliferative responses of T lymphocytes in I-Eb/k- or I-Ek/k-carrying mice, respectively. Furthermore, residues 46 and 54 were shown to function as agretopes and residue 50 as an epitope in the I-E-restricted responses as they did in the I-A-restricted responses, even though some differences were seen between peptide-I-E interaction and peptide-I-A interaction. These agretopes and epitope functioned independently.     

Microheterogeneity in the recognition of a HLA-DR2-restricted T cell epitope from a meningococcal outer membrane protein

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA-DR2-restricted T cell clones recognizing the 49-61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3⁺CD4⁺CD8^?TcRct/β⁺, were selected by restimulation with the synthetic peptide OMP(49-61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine-specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single-residue substitution analogues of the minimal epitope OMP(50-58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49-61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, He 51 and Phe 54 act as MHC-anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since He 51 and Phe 54 can be substituted by conservative residues [as OMP(50-58) and OMP(49-61) analogues] and with Ala [as OMP(49-61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different V_α and V_β genes (which encompass the putative MHC-binding regions first and second complementarity-determining regions, CDR1 and CDR2) different modes of interaction with the HLA-DR2 molecule indeed are likely to occur.