Extraordinary cross-reactivity of an autoimmune T-cell receptor recognizing specific peptides both on autologous and on allogeneic HLA class II molecules

A T-cell receptor's (TCR) recognition of a human leukocyte antigen (HLA)-peptide complex (pHLA) is normally described as being restricted by the HLA molecule and specific for the peptide. This is, however, not always true. Several TCRs have been described, which cross-react with other peptides bound to the restricting HLA molecule. This phenomenon has been considered a variant of molecular mimicry and is suggested to be one of the mechanisms behind autoimmunity. The positive selection of T cells in the thymus imposes low-affinity recognition of the TCRs toward self-pHLA, which increases the probability of the TCR to be promiscuous by nature, and further implies that the T-cell repertoire contains TCRs prone to be autoreactive and thus able to induce autoimmunity. We present an autoimmune TCR showing extreme cross-reactivity to several pHLA comprising both own HLA class II restriction element and allogeneic HLA class II restriction elements in complex with both self-derived and microbially derived peptides. The existence of such a significant cross-reactivity in the context of distinct HLA-DR molecules might be more common among autoimmune TCRs than previously anticipated and potentially reveals a new way of designing altered peptide ligands for therapeutic use.     

Coevolution of T-cell receptors with MHC and non-MHC ligands

The structure and amino acid diversity of the T-cell receptor (TCR), similar in nature to that of Fab portions of antibodies, would suggest that these proteins have a nearly infinite capacity to recognize antigen. Yet all currently defined native T cells expressing an α and β chain in their TCR can only sense antigen when presented in the context of a major histocompatibility complex (MHC) molecule. This MHC molecule can be one of many that exist in vertebrates, presenting small peptide fragments, lipid molecules, or small molecule metabolites. Here we review the pattern of TCR recognition of MHC molecules throughout a broad sampling of species and T-cell lineages and also touch upon T cells that do not appear to require MHC presentation for their surveillance function. We review the diversity of MHC molecules and information on the corresponding T-cell lineages identified in divergent species. We also discuss TCRs with structural domains unlike that of conventional TCRs of mouse and human. By presenting this broad view of TCR sequence, structure, domain organization, and function, we seek to explore how this receptor has evolved across time and been selected for alternative antigen-recognition capabilities in divergent lineages.     

Evidences of conformational changes in class II Major Histocompatibility Complex molecules that affect the immunogenicity

The N-terminal part of class II-associated invariant chain peptide (CLIP) is assumed to interact with an accessory peptide-binding site on the class II Major Histocompatibility Complex (MHC) molecule, and promote a conformational modification. We have linked this immunoregulatory segment (residues 81-88) to the N-terminus of the influenza hemagglutinin (HA) 307-319 epitope in order to evaluate relationships between the MHC conformational changes and their implication in immune responses. Our chimeric peptide, named CLIP-HA, bind with the same affinity to class II HLA-DR1 molecules as the HA peptide, and is normally recognized by HA-specific T cells. Interestingly, the presence of the N-terminal CLIP region enhances the rate of association to soluble DR1 molecules but prevents the formation of SDS-resistant complexes. These features suggest the existence of HLA-DR1 conformational changes induced by the chimeric peptide. Furthermore, while in vitro HA and CLIP-HA peptides associated to DR1 could not be differentiated based on T-cell recognition, in vivo the CLIP residues strongly impaired the immunogenicity of HA epitope as assessed in HLA-DR1 transgenic mice. Our study demonstrates for the first time that MHC conformational changes, revealed at molecular level, may influence the immunogenicity.     

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.     

Extensive T cell receptor cross-reactivity on structurally diverse haptenated peptides presented by HLA-A2

Previous studies have shown that individual TCRs are able to effectively recognize multiple peptide/MHC complexes that have varying degrees of structural diversity. These TCR cross-reactivities have usually been demonstrated by using peptides that have different amino acid sequences. To further examine the extent to which TCRs can accommodate structurally diverse ligands, we analyzed human TCR cross-reactivity to eight structurally distinct haptens that are coupled to the HLA-A2-binding Tax peptide with a lysine substitution at position 5 (Tax-5K, LLFG[K-hapten]PVYV). The results demonstrate that 71% percent of the haptenated-peptide-induced CTL lines could cross-react on at least one other hapten. We compared the effects of HLA-A2 mutants with substitutions at known TCR contact sites for recognition by hapten-cross-reactive CTL. Recognition of the A2 mutants was remarkably similar whether they were presenting the immunizing or the cross-reactive peptide, indicating that similar amino acid contacts are made by the TCR during recognition of both complexes. Thus, hapten cross-reactivity is apparently accomplished without major adjustments to the interaction between the TCR and the surface of the HLA-A2 molecule. Collectively, these results suggest that TCRs possess the molecular flexibility to accommodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule.     

The structural basis of T-cell allorecognition

Foreign allogeneic major histocompatibility complex (MHC) class I and class II molecules elicit an exceptionally vigorous T-cell response. A small component of the alloresponse comprises CD4+ T cells that recognize allogeneic MHC indirectly after processing into peptide fragments that are bound and presented by self-MHC class II. The majority of alloreactive T cells directly recognize intact allogeneic MHC molecules expressed on foreign cells. Some alloreactive T-cell interactions with allogeneic MHC molecules are indifferent to the bound peptide, but evidence suggests that most show specificity to peptide. The vigor and diversity of the direct alloreactive T-cell response can therefore be explained by summation of numerous responses to each of the peptides in the novel set bound by allogeneic MHC molecules. Structural studies definitively show that the overall mechanism of T-cell receptor (TCR) recognition of self-MHC and allogeneic MHC molecules is similar. Many alloreactive T cells recognize several different combinations of MHC and bound peptide that do not necessarily possess structural homology. Flexibility within the TCR structure allows adaptation to the different contact surfaces. Crossreactivity seems to be an intrinsic property of the TCR required, because a single TCR must possess the ability to interact with both self-MHC during positive selection and at least one combination of foreign antigenic peptide presented by self-MHC. Recognition of allogeneic MHC molecules is an inadvertent consequence of the need for TCR crossreactivity.     

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.     

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.     

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)     

Implications of a Fab-like structure for the T-cell receptor

The antigen-specific receptor of T lymphocytes (TCR) and the Fab moiety of immunoglobulins are expected to fold into similar three-dimensional structures because of their identical protein domain organization, the conservation of key residues and their overall sequence homology. However, T cells mostly appear to recognize short peptide antigens bound to MHC class I or class II presenting molecules. A complete model of the human leucocyte antigen molecule (HLA-A2) reconstructed from the alpha-carbon coordinates was used to investigate the putative organization of a TCR/peptide/HLA-A2 complex. In this article, Jean-Michel Claverie and co-workers show that the respective geometries of a Fab-like TCR structure and of the HLA-A2 antigen binding site suggest a model where the third variable regions of both chains of the TCR mainly interact with the peptide antigen, while the first and/or second less variable regions are in position for making contact with residues pointing up from the alpha 1 and alpha 2 helical regions of the HLA-A2 molecule.     

MHC restriction is imposed on a diverse T cell receptor repertoire by CD4 and CD8 co-receptors during thymic selection

Mature αβ T cells recognize foreign antigenic peptides presented by MHC molecules but do not recognize native antigenic proteins; features known as MHC restriction. How MHC restriction is imposed on αβ T cells has intrigued immunologists for several decades. One model proposes that germline-encoded elements in the T cell receptor (TCR) variable regions are evolutionarily conserved to only recognize MHC. However, we propose an alternative model that posits that MHC restriction is imposed by CD4 and CD8 co-receptors during thymic selection. Thus, we think that TCRs are structurally able to recognize a huge diversity of ligands but only TCRs with MHC specificity survive thymic selection.     

The inhibitory effect of altered collagen II peptide on HLA-DRB1-restricted T-cell activation

It has been known that rheumatoid arthritis (RA)-associated antigenic peptides CII263-272 are coupled with human leucocyte antigen (HLA)-DRB1 and recognized by T-cell receptor (TCR), which in turn induced T-cell proliferation and pathogenesis of RA. Non-T-cell-stimulating type II collagen (CII) peptides might be generated by removing the amino acids responsible for TCR contact and keeping the HLA-DR-binding residues intact. In this study, a panel of altered CII peptides (APs) with consecutive or single substitutions of the TCR-contacting residues were synthesized. Through peptide binding and T-cell activation assays, we demonstrated that altered CII263-272 peptides with substitution of the TCR-contacting residues did not or barely induced T-cell activation; one of the best non-T-cell-stimulating peptide AP268-270 inhibited the binding of wild-type CII263-272 to HLA-DR1 and T-cell activation triggered by wild-type CII263-272 and HA306-318 in a dose-response manner. These data suggest that removal of the TCR-contacting residues of CII263-272 leads to HLA-DRB1 binding and low T-cell-stimulating peptides, which could potentially inhibit the T-cell response induced by HLA-DRB1-binding antigenic peptides.     

Binding energetics of T-cell receptors: correlation with immunological consequences.

Recent crystal structures of the T-cell receptor (TCR) engaged with peptide–major histocompatibility complex (MHC) ligands suggest the TCR binds to such ligands in a conserved orientation, with key Vα residues contacting the α helices. TCR mutagenesis has permitted energy measurements of these interactions. Here, Thomas Manning and David Kranz review binding energetics data that might explain the inherent reactivity of TCRs with MHC products.     

Recognition of disparate HA and NS1 peptides by an H-2Kd-restricted, influenza specific CTL clone.

CTLs (CD8+) are known to recognize exogenous peptide in the context of class I MHC molecules. We observed that an influenza subtype H1 and H2 cross-reactive CTL clone B7, which was stimulated by a fusion protein containing a portion of HA2 subunit of A/PR/8 virus HA, recognized a synthetic peptide (residues 515-526) of the HA2 subunit of A/PR/8 virus strain. This CTL clone also recognized a structurally disparate NS1 peptide 50-68 of the same A/PR/8 virus. We examined the recognition of the NS1 peptide 50-68 and the HA peptide 515-526 by the subcloned CTL clone, B7-B7. Cold target inhibition experiments showed that the recognition of the HA peptide by the CTL clone B7-B7 could be competed by NS1 peptide-treated target cells and vice versa. The recognition of both NS1 peptide and HA peptide by the CTL clone B7-B7 was restricted by the same allele, H2Kd. In addition, this NS1 peptide requires approximately a 600-fold higher concn for optimal CTL recognition than did the HA peptide. We conclude that the TCR on clone B7-B7 recognizes the HA peptide or the NS1 peptide as comparable complexes with the same class I MHC molecules, although there is no obvious homology in the primary sequences of HA 515-526 and NS1 50-68 peptides. CTLs elicited with certain antigens appear to recognize distinctively different antigens complexed to the same presenting MHC molecule.     

Specific stimulation of MHC-transgenic mouse T-cell hybridomas with xenogeneic APC

From the recombinant human leukocyte antigen (HLA)-DR1/H2-E(k) major histocompatibility complex (MHC) class II-transgenic mice, we have generated two CD4(+) T-cell hybridomas specific for peptides which were derived from human prostatic acid phosphatase (PAP) complexed to the human class II molecule HLA-DR1. Both hybridomas strongly react to PAP-pulsed antigen-presenting cells (APC) from transgenic mice. Interestingly, these hybridomas also responded to PAP antigen presented by HLA-DR1-positive human APC. The species-mismatched T-cell stimulation occurs despite the biologic discordance in participating accessory molecules, which are required for the optimal T-cell-APC interaction. Our results demonstrate various degrees of functional interaction between coreceptors, costimulatory molecules, and integrins, which are expressed on the surface of T-cell hybridomas and heterologous APC.     

B-Cell and T-Cell Epitopes in Anti-factor VIII Immune Responses

Adequate hemostasis is achieved for many hemophilia A patients by infusion of plasma-derived or recombinant factor VIII (FVIII), but unfortunately, a significant subset of patients develop an immune response in which anti-FVIII antibodies, referred to clinically as “inhibitors,” interfere with its procoagulant activity. Inhibitors are the subset of anti-FVIII antibodies that bind to surfaces on FVIII (B-cell epitopes) that are important for its proper functioning in coagulation. Less antigenic FVIII molecules may be designed by identifying and then modifying the amino acid sequences of inhibitor B-cell epitopes. Conversely, characterization of these epitopes can yield important information regarding functionally important surfaces on FVIII. The production of inhibitor antibodies is driven by T cells. T cells recognize FVIII as foreign when FVIII-derived peptides bind to major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells. The class II–peptide complexes must then be recognized by T-cell receptors (TCRs). T-cell stimulation requires sustained association of antigen-presenting cells and T cells through formation of a class II–peptide–TCR complex, and peptide sequences that mediate this association are termed “T-cell epitopes.” MHC class II tetramers that bind FVIII-derived peptides and recognize antigen-specific TCRs are proving useful in the characterization of human leukocyte antigen-restricted T-cell responses to FVIII.     

Degenerate recognition of T cell epitopes: impact of T cell receptor reserve and stability of peptide:MHC complexes

The concept of molecular mimicry suggests that microbial pathogens might activate antigen-specific T cells that then cross-react with endogenous antigens and result in the generation of autoimmunity. Here we discuss several under-represented factors impacting the ability of TCRs to recognize a wide spectrum of related peptide:MHC (pMHC) ligands. Two of these factors include the affinity of the peptide for MHC and the level of TCR expression. Thymocytes that recognize peptides with low affinity for MHC avoid negative selection, but mature T cells, by virtue of increased TCR expression, will proliferate in response to these same unstable pMHC complexes. While the expression of a reserve of antigen receptors expands the potential number of epitopes for which a T cell can cross-react, phosphatase activity provides a tuning mechanism to increase the threshold level of activation. Thus, degenerate recognition of T cell epitopes involves the stability of the peptide:MHC complex, the number of TCR expressed, and the level of phosphatase activity.     

Conformational changes in MHC class I molecules

In this article we review the role of MHC conformation, including peptide-induced MHC conformation, in forming antibody (Ab), T-cell receptor (TCR), and natural killer (NK) cell receptor epitopes. Abs recognize conformational major histocompatibility (MHC) epitopes that often are influenced by the identity of MHC-bound peptide. Diverse TCRs recognize a common docking site on peptide/MHC complexes and directly contact peptide. Human NK cell inhibitory receptors (KIR) appear to recognize limited regions of the HLA α₁ helix. DX9+ KIR specifically focus on HLA-B residues 82 and 83. However, NK cells recognize much broader regions of HLA class I molecules and are sensitive to bound peptides. Thus, several classes of lymphocyte receptors are peptidespecific. Peptide specificity could be the result of direct contact with the receptor, or to conformational shifts in MHC residues that interact with both receptor and bound peptide.