T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

Central to adaptive immunity is the interaction between the αß T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and ß-chain are overlaid with the α-chain and ß-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

How MHC molecules grab citrullinated peptides to foster rheumatoid arthritis.

The molecular immunologist's dream is to elucidate a fundamental biochemical process that explains the basis of an affliction that affects millions of people, and that, precisely understood, might yield a rational approach to diagnosis, prevention, or therapy. In this issue of JBC, Ting et al. report proteomic, biochemical, and structural analyses that better explain how the antigen-presenting HLA-DR4 molecules bind citrullinated peptides to provoke rheumatoid arthritis (RA), a chronic autoimmune disease that affects 0.5-1% of the population.

Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4(+) monozygotic twins discordant for rheumatoid arthritis.

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell- and B cell-epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4-related (antigen DR4(-) HLA-DR4)(+) woman with early RA, her healthy monozygotic twin and an unrelated HLA-DR3(+) woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7-aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)-17/IL-4/IL-10/IL-6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261-273), the K264 carbamylated T cell epitope (carT261-273), the native B cell epitope (B359-369) or the R360 citrullinated B cell epitope (citB359-369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4(+) twins, but not in the DR3(+) RA. The collagen-specific activation of CD4(+) T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL-17 production in the RA twin, but a greater IL-4 and IL-10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL-6/IL-10 ratio in the RA twin compared to a greater IL-10 production in the healthy twin. Our data suggest that circulating collagen-specific T and B cells are found in HLA-DR4(+) subjects, but only RA activated cells express co-stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

Identification of small-molecule inhibitors against human leukocyte antigen-death receptor 4 (HLA-DR4) through a comprehensive strategy.

Rheumatoid arthritis (RA) is an autoimmune disease mediated by T-lymphocytes and associated with the human leukocyte antigen-death receptor 4 (HLA-DR4). The HLA-DR4 protein selectively interacts with the antigenic peptides on the cell surface and presents them to the T cell receptor (TCR) on CD4+ T cells. The HLA-DR4-antigen-TCR complex initiates the autoimmune response and eventually causes the chronic inflammation within patients bodies. To inhibit HLA-DR4-restricted T cell activation, an ideal approach is to discover non-T cell stimulating substrates that specifically bind to HLA-DR4. In this paper, a comprehensive structure-based design strategy involved de novo design approach, pharmacophore search, and dock method was presented and applied to "simplify" the known binding peptide ligand of HLA-DR4 and identified specific small-molecule inhibitors for HLA-DR4. The designed three-step strategy successfully identified five nonpeptide ligands with novel scaffolds from a chemical library containing 4 × 10(6) commercially available compounds within a tolerable computing time. The identified five chemicals, BAS-0219606, T0506-2494, 6436645, 3S-71981, and KM 11073, are all non-T cell stimulators and are able to significantly inhibit HLA-DR4-restricted T cell activation induced by type II collagen (CII) 263-272 peptide. IC(50) for the best two potentials, BAS-0219606 and T0506-2494, was 31 and 17 µM, respectively, which is equivalent or better than the known peptide ligands. It is hopeful that they can be used as effective therapeutic means for further treatment of RA patients. In addition, the comprehensive strategy presented in this paper exhibited itself to be an effective flow line from peptide ligands to small-molecule inhibitors and will have applications to other targets.

Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association.

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

Structure of a complex of the human alpha/beta T cell receptor (TCR) HA1.7, influenza hemagglutinin peptide, and major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRB1*0401): insight into TCR cross-restriction and alloreactivity.

The alpha/beta T cell receptor (TCR) HA1.7 specific for the hemagglutinin (HA) antigen peptide from influenza A virus is HLA-DR1 restricted but cross-reactive for the HA peptide presented by the allo-major histocompatibility complex (MHC) class II molecule HLA-DR4. We report here the structure of the HA1.7/DR4/HA complex, determined by X-ray crystallography at a resolution of 2.4 A. The overall structure of this complex is very similar to the previously reported structure of the HA1.7/DR1/HA complex. Amino acid sequence differences between DR1 and DR4, which are located deep in the peptide binding groove and out of reach for direct contact by the TCR, are able to indirectly influence the antigenicity of the pMHC surface by changing the conformation of HA peptide residues at position P5 and P6. Although TCR HA1.7 is cross-reactive for HA presented by DR1 and DR4 and tolerates these conformational differences, other HA-specific TCRs are sensitive to these changes. We also find a dependence of the width of the MHC class II peptide-binding groove on the sequence of the bound peptide by comparing the HA1.7/DR4/HA complex with the structure of DR4 presenting a collagen peptide. This structural study of TCR cross-reactivity emphasizes how MHC sequence differences can affect TCR binding indirectly by moving peptide atoms.

In Silico Characterization of B Cell and T Cell Epitopes for Subunit Vaccine Design of Salmonella typhi PgtE: A Molecular Dynamics Simulation Approach.

Typhoid fever is an acute illness in humans, caused by Salmonella typhi, a gram-negative bacterium. Outer membrane proteins of S. typhi have strong potential for its use in the development of subunit vaccine against typhoid. In the current study, peptide-based subunit vaccine was constructed from outer membrane protease E (PgtE) against S. typhi. B cell and T cell epitopes were identified at fold level with a validated three-dimensional modeled structure. T cell epitopes from PgtE (IHPDTSANY) have 99.5% binding to a maximum number of major histocompatibility complex class I and class II alleles. They also bind to the typhoid-resistant human leukocyte antigen (HLA) alleles DRB1*0401. PgtE epitopes were docked with HLA-DR4 (PDB ID: 1D5M) and a contact map was constructed. A simulation search for the binding site for full flexibility of the peptide from CABS- (Cα, Cß, side-chain)-dock shows stable interactions. Molecular dynamics simulation studies revealed that the PgtE-epitope complex structure was more stable throughout the simulation (20 ns) and interaction did not change the radius of gyration. In conclusion, computational analysis, molecular docking, and molecular dynamics (MD) simulation of PgtE-epitope complex were used to elucidate the binding mode, and the dynamical changes of epitopes were more suitable for vaccine development against typhoid.

Dynamics Determine Signaling in a Multicomponent System Associated with Rheumatoid Arthritis.

Strategies that target multiple components are usually required for treatment of diseases originating from complex biological systems. The multicomponent system consisting of the DR4 major histocompatibility complex type II molecule, the glycopeptide CII259-273 from type II collagen, and a T-cell receptor is associated with development of rheumatoid arthritis (RA). We introduced non-native amino acids and amide bond isosteres into CII259-273 and investigated the effect on binding to DR4 and the subsequent T-cell response. Molecular dynamics simulations revealed that complexes between DR4 and derivatives of CII259-273 were highly dynamic. Signaling in the overall multicomponent system was found to depend on formation of an appropriate number of dynamic intramolecular hydrogen bonds between DR4 and CII259-273, together with the positioning of the galactose moiety of CII259-273 in the DR4 binding groove. Interestingly, the system tolerated modifications at several positions in CII259-273, indicating opportunities to use analogues to increase our understanding of how rheumatoid arthritis develops and for evaluation as vaccines to treat RA.

Natural ligand motifs of closely related HLA-DR4 molecules predict features of rheumatoid arthritis associated peptides.

Rheumatoid arthritis (RA), one of the most common autoimmune disorders, is believed to be mediated via. T lymphocytes and genetic studies have shown that it is strongly associated with HLA-DR4. The DR4 subtypes DR4Dw4, DR4Dw14 and DR4Dw15 represent increased risk factors for RA, whereas DR4Dw10 is not associated with the disorder. Our study determines and compares the natural ligand motifs of these MHC class II molecules and identifies 60 natural ligands. At relative position 4 (P4), only the RA-associated DR4 molecules allow, or even prefer, negatively charged amino acids, but do not allow those which are positively charged (Arg, Lys). In the case of DR4Dw10 the preference for these amino acids is reversed. The results predict features of the putative RA-inducing peptide(s). A remarkable specificity, almost exclusively for negative charges (Asp, Glu), is found at P9 of the DR4Dw15 motif. This specificity can be ascribed to amino acid beta57 of the DR beta chain, and gives an important insight into the beta57-association of another autoimmune disease, insulin-dependent diabetes mellitus type I.

Disease-stage variance in functional CD4(+) T-cell responses against novel pan-human leukocyte antigen-D region presented human papillomavirus-16 E7 epitopes.

Given the anticipated clinical importance of helper and regulatory CD4(+) T cells reactive against human papillomavirus-16 E7 in the cervical carcinoma setting, we performed this study to identify novel E7-derived T helper (Th) epitopes and to characterize functional anti-E7 Th responses in normal donors and patients with cervical intraepithelial neoplasia I-III or cervical cancer. Candidate pan-HLA-DR (D region) binding peptides were identified and synthesized based on results obtained using a predictive computer algorithm, then applied in short-term in vitro T-cell sensitization assays. Using IFN-gamma/IL-5 (interleukin 5) enzyme-linked immunospot assays as readouts for Th1-type and Th2-type CD4(+) T-cell responses, respectively, we identified three E7-derived T helper epitopes (E7(1-12), E7(48-62), and E7(62-75)), two of which are novel. Normal donor CD4(+) T cells failed to react against these E7 peptides, whereas patients with premalignant cervical intraepithelial neoplasia I-III lesions displayed preferential Th1-type responses against all three E7 epitopes. Th1-type responses were still observed to the E7(48-62) but not to the E7(1-12) and E7(62-75) peptides in cancer patients, where these latter two epitopes evoked Th2-type responses. Notably all responders to the E7(1-12) and E7(62-75) peptides expressed the HLA-DR4 or -DR15 alleles, whereas all responders to the E7(48-62) peptide failed to express the HLA-DR4 allele. Our results are consistent with a model in which cervical cancer progression is linked to an undesirable Th1- to Th2-type shift in functional CD4(+) T cell responses to two novel E7-derived epitopes. These peptides may prove important in vaccines to promote and maintain protective Th1-type antihuman papillomavirus immunity and in the immune monitoring of treated patients harboring HPV-16(+) malignancies.

A chaperone-assisted high yield system for the production of HLA-DR4 tetramers in insect cells.

MHC tetramers have become essential tools for the analysis of antigen specific responses of CD8+ and CD4+ T cells. However, the use of MHC class II tetramers is hampered by the relatively low yields of most current expression systems. We have devised an insect cell/baculovirus expression system in which yields of 50-70 mg of recombinant HLA-DR4 molecules, with or without covalently linked peptide, per liter of insect cell supernatant, are routinely obtained. These yields are rendered possible by an optimized design and use of DRalpha and DRbeta expression cassettes and by co-expression of a housekeeping chaperone of the endoplasmic reticulum, calreticulin, which, due to its co-secretion, increases secretion of HLA-DR molecules two- to threefold. A tetramer produced in the system specifically was shown to stain an HLA-DR4 restricted T cell line obtained from a healthy donor by in vitro priming, but which recognizes a type I diabetes autoantigen. Co-expression of chaperones may represent a general strategy for enhancing yields of recombinant proteins expressed in insect cells and facilitate production of MHC class II tetramers in the future.

Subgrouping of DR4 alleles by DNA heteroduplex analysis.

Amplified DNA molecules from six DR4 alleles at the DRB1 locus were denatured and cross-hybridized pairwise. Several of the DNA heteroduplexes thus generated were found to possess distinct mobilities in polyacrylamide gel electrophoresis. The degree of retardation as compared to homoduplexes depends strongly on the position of mismatched nucleotide pairs. In critical positions, the type of mispairing also influences the number of heteroduplex bands since a transversion-type substitution yields two reciprocal pairings (purine-purine or pyrimidine-pyrimidine) whereas a transition-type substitution generates symmetrical (purine-pyrimidine) pairings. Based on their heteroduplex pattern the DR4 alleles can be subdivided in four groups: group I = DRB*0401, group II = 02 and 06, group III = 03 and 04, and group IV = 05. Each group can be recognized by the heteroduplex bands generated by cross-hybridizing with group II reference DNA (either the 02 or 06). This subgrouping is obtained with a single electrophoretic run and without the use of probes. However, the alleles within groups II and III, and notably the alleles 03 and 04, which are both present in the Caucasoid population, can be distinguished only by oligonucleotide hybridization (dot blot) analysis. With this limitation, the method can be recommended either in conjunction with dot blot typing or independently, thus avoiding completely the use of probes in the cases where it is not essential to discriminate between 03 and 04. The data also show that distinguishable heteroduplexes may be generated by a single mismatch. This opens the possibility of applying the same technique to genetic systems of lower degree of polymorphism.

Sequence of a new HLA-DR4 allele with an unusual residue at position 88 that does not seem to affect T-cell allo recognition.

New HLA alleles can be identified by unorthodox patterns observed during low-resolution typing performed with sequence specific oligonucleotide probes (SSOP). One of the best examples is locus DRB1, where allelic subtypes are characterized by a combination of a limited number of residues located in three hypervariable regions of exon 2. HLA-DR oligotyping analysis of a female caucasoid bone marrow donor led to the identification of an individual that typed as DRB1*11, DRB3*02, DRB4*01, DQB1*0301-0302. This donor was, however, typed by serology as DR11 DR4, DR52, DR53, DQ7 DQ8. PCR-SSP typing for DR4 subtype revealed an amplification pattern typical for DRB1*0404. After sequencing the entire exon 2, a new DRB1 allele was identified: DRB1*04var that is identical to DRB1*0404, except for one nucleotide at codon 88 resulting in a Ser-->Arg exchange. This mutation had prevented amplification with the DR generic primers. Cellular typing by three HTCs-DRB1*0404/DW14 from the 9th Workshop showed that this DRB1*04var typed exactly like a DW14 cell. This suggests that residue 88 does not affect T cell recognition.

Analysis of monoclonal antibodies specific for unique and shared determinants on HLA-DR4 molecules.

The specificities for seven mAbs to HLA-DR4 were determined initially using homozygous BCLs and L-cell transfectants expressing wild-type DR molecules. Three antibodies (NFLD.D1, NFLD.M1, and NFLD.D7) bound all DR4 molecules, but only one was specific for DR4. Four antibodies (NFLD.D2, NFLD.D3, NFLD.D8, and NFLD.D10) reacted with some but not all DR4 subtypes and had extra reactions, particularly with DR gene products associated with susceptibility to RA. To localize the antibody-binding epitopes on DR4 molecules, the antibodies were then analyzed on transfectants expressing hybrid genes, which were generated by exon shuffling of DRB1*0403 and DRB1*0701. Two of the pan-DR4 antibodies bound epitopes that require the beta 2 domain while the third mapped primarily to the HVR-I region. One antibody NFLD.D10 to subtypes of DR4 mapped to residues 40-97 on DR beta 1*0403 chains. Comparison of reaction patterns with amino acid sequences suggest that the antibodies against subtypes of DR4 are specific primarily for a region containing sequences postulated to determine susceptibility to RA.

Reassembly and reconstitution of separate alpha and beta chains of human leukocyte antigen DR4 molecule isolated from Escherichia coli.

The class II major histocompatibility complex molecules play a major role in presentation of exogenous antigenic peptides to the CD4 positive helper T cell. These are heterodimeric cell surface glycoproteins consisting of alpha- and beta-chains. In the present study, we cloned and expressed the alpha- and beta-chain of HLA-DR4 lacking the transmembrane and cytoplasmic domain separately in Escherichia coli using the pET-5a expression vector system. The expressed alpha- and beta-chains were purified in a denaturing condition by an ion exchange chromatography on Q-Sepharose and a gel filtration chromatography on Sephacryl S-200, respectively. The recombinant proteins were refolded and reassembled by removing the denaturing agent and concomitant reoxidation of the disulfide bond. The refolded heterodimeric rDR4 molecule was resolved by 12.5% SDS-PAGE in a nonreducing condition and confirmed by Western blot using polyclonal antibody against DR-alpha and the monoclonal antibody (L243) for the conformationally correct DR molecule. The rDR4 molecules were reconstituted with a 50-fold molar excess biot-HA (307-319), and the bound peptides to the heterodimer complex were determined by a microplate assay coated with L243 antibody using Extravidin-HRP conjugate.

Interactions of the human class II major histocompatibility complex protein HLA-DR4 with a peptide ligand demonstrated by affinity capillary electrophoresis.

The interactions of empty recombinant major histocompatibility complex (MHC) class II molecules (DRA1*0101/DRB1*0401) with a known peptide ligand [the HA(307-319) fragment of influenza virus hemagglutinin] were studied by capillary electrophoresis. Using an alkaline buffer system with the addition of non-ionic or zwitterionic detergent and high sensitivity laser-induced fluorescence detection, both slowly and rapidly equilibrating binding could be demonstrated. This was accomplished using a pre-equilibration approach as well as migration shift experiments where receptor molecules were added to the electrophoresis buffer. This system may be useful for the study of both peptide binding to MHC molecules and screening for inhibition or amplification of binding by other ligands as well as for the study of the interactions of T-cell receptors with MHC-peptide complexes.

[The inhibitory effect of hyporesponsive peptide on human leukocyte antigen-DRbeta1 specific T cell activation].

OBJECTIVE: To evaluate the inhibitory effect of human leukocyte antigen (HLA)-DRbeta1 specific collagen II (CII) peptides with substitutions of TCR binding residues on T cell activation, and explore a new therapeutic strategy for T cell mediated autoimmune diseases by interfering with antigen recognition of T Cell receptor (TCR). METHODS: Non-TCR binding peptides were designed by computer modeling based on interaction of HLA DR1. The modified CII263-272. Intracellular transfer of the modified CII peptide and its binding to HLA DR1 were studied using confocal microscopy and fluorescence-activated cell sorter (FACS). The effects of altered peptides on T cell activation were evaluated using an antigen presenting system consisting of HLA-DR1 transgenic APC and CII specific T cells. RESULTS: Computer modeling showed the side chains of 263 (F), 266 (E) fit in the peptide binding groove, and form hydrogen bond with alpha1, beta1 chain of HLA-DR1. The side chains of TCR specific 267 (Q) and 270 (K) protruded out of the groove, which might be TCR recognizing residues. The modified CII peptides with intact HLA-DR1 binding residues were bound to intracellular HLA-DR1 and expressed on cell surface. The modified peptides with single residue substitution of 267-270 and consecutive substitution of 268-270 showed a hyporesponsive T cell activation. Altered peptides 270A, sub268-270 could significantly suppress the T cell activation induced by CII263-272. CONCLUSION: The altered peptides with substitution of TCR binding residues are hyporesponsive in T cell activation, and may competitively inhibit the T cell activation in T cell mediated autoimmune diseases.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy.

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE(-/-) mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane ß-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.

Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers.

Successful immunity requires that a limited pool of αß T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by 'self' major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA(306-318)) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA(306-318) presented by multiple MHC-II alleles.