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.     

Molecular basis of antigen recognition by insulin specific T cell receptor

The TCR alpha/beta chains recognize antigen peptides bound to the groove of the MHC class II molecule. The crystal structure analyses of the TCR/peptide/MHC class II complexes have revealed that the Valpha chains play a significant role in antigen recognition. However, molecular details which amino acid residues of the Valpha chain are able to contribute to fine antigen specificity are not clearly understood. Previously, we have classified a panel of T hybrids specific for insulin isotypes from different species of animals into four groups based on response profiles to these antigens. In particular, the group III (pork insulin > or = beef insulin hierarchy of responsiveness) and IV (pork insulin > beef insulin hierarchy of responsiveness) T hybrids are interesting, since these TCR alpha/beta chains with marked different antigen specificities demonstrate identical gene usages and very similar sequences. To specifically address the molecular requirements for insulin recognition by TCR, the TCR alpha and beta chain genes from these group III and IV T hybrids were transfected into 58 alpha-beta- T hybrid. The experiments suggested that CDR3alpha dictates the fine antigen specificity. Then, we have introduced a series of mutations into position 95 of CDR3alpha. The mutation experiments clearly indicated that position 95alpha determines the antigen specificity of the group III and IV T hybrids.     

Interface-disrupting amino acids establish specificity between T cell receptors and complexes of major histocompatibility complex and peptide

T cell receptors (TCRs) bind complexes of cognate major histocompatibility complex (MHC) and peptide at relatively low affinities (1-200 microM). Nevertheless, TCR-MHC-peptide interactions are usually specific for the peptide and the allele encoding the MHC. Here we show that to escape thymocyte negative selection, TCRs must interact with many of the side chains of MHC-peptide complexes as 'hot spots' for TCR binding. Moreover, even when the 'parental' side chain did not contribute binding affinity, some MHC-peptide residues contributed to TCR specificity, as amino acid substitutions substantially reduced binding affinity. The presence of such 'interface-disruptive' side chains helps to explain how TCRs generate specificity at low-affinity interfaces and why TCRs often 'accommodate' a subset of amino acids at a given MHC-peptide position.     

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.     

Modulation of T cell responses with MHC-derived peptides

T cells are activated by an interaction of their TCRs with a complex made up of antigenic peptide bound to the interhelical groove of MHC molecules. The helices lining the antigen binding groove of MHC molecules are felt to contribute several contact residues for TCR binding. Peptides derived from the amino acid sequences of these helices may be capable of modulating immune responses and aiding in the dissection of immune recognition. These studies address the effects of a peptide derived from the sequence of amino acids 68-83 of the IAk beta 1 domain (IAk 68-83) predicted to represent a portion of an antigen-binding helix on the IAk molecule. The IAk 68-83 peptide is bound by a monoclonal anti-IAk antibody and inhibits its binding to IAk-bearing cells. The IAk 68-83 peptide inhibits antigen-dependent activation of the IAk+con-albumin restricted T cell clone D10.G4, and this effect is more pronounced at lower doses of antigen-presenting cells. The free peptide has a small effect in limiting binding of anticlonotypic antibodies to D10.G4, and a multivalent form bound to BSA has a more pronounced effect in this regard. The BSA-peptide conjugate, when fluoresceinated, specifically stained D10.G4 cells, and this was specifically competed by unfluoresceinated IAk 68-83 peptide-BSA conjugate, as well as by anticlonotype. These results suggest that peptides derived from the predicted helical region of MHC class II molecules may have a direct interaction with T cell receptors. Such peptides may be capable of modulating immune responses in a physiologically significant manner.     

T cell receptor crossreactivity as a general property of T cell recognition

TCR recognition of MHC/peptide complexes directs many aspects of T cell biology, including thymic selection, survival of na?ve T cells and differentiation into effector and memory T cells. It was widely thought that TCR recognition is highly specific, with an individual T cell being capable of only recognizing a particular peptide and closely related sequence variants. By considering the structural requirements for peptide binding to MHC molecules and TCR recognition of MHC/peptide complexes, we demonstrated that T cell clones could recognize a number of peptides from different organisms that are remarkably distinct in their primary sequence. These peptides are particularly diverse at those sequence positions buried in pockets of the MHC binding site, while a higher degree of similarity is present at a limited number of peptide residues that create the interface with the TCR. Many examples have now been documented for human and murine T cells, indicating that TCR crossreactivity represents a general feature of TCR recognition.     

Unique role of thyroxine in T cell recognition of a pathogenic peptide in experimental autoimmune thyroiditis

We have previously demonstrated the importance of iodination and the requirement of the thyroxine residues in thyroglobulin (Tg) for the stimulation of two clonotypically distinct murine T cell hybridomas reactive against human and mouse Tg. We are now able to show that these T cell hybridomas only recognize an 11-residue peptide containing a thyroxine structure that has iodine at two positions on each ring. This iodination state is critical for recognition by these hybridomas as a peptide containing de-iodinated thyroxine is nonstimulatory. Furthermore we have demonstrated that a peptide lacking the thyroxine residue or containing de-iodinated thyroxine cannot block the recognition of the thyroxine-containing peptide. We suggest that in our system the thyroxine residue is involved in binding to major histocompatibility complex (MHC) class II molecules. We have also been able to show that the thyroxine residue is available for contact by the T cell receptor (TCR) as recognition of the peptide/H-2A^k complex is blockable by an antibody directed against thyroxine. Using substituted peptides, we have been able partially to define the residues within the peptide that are critical for recognition of the 11-residue peptide by our hybridomas. From our data, we suggest that the thyroxine residue may bind the MHC and TCR, while the residues identified in the peptide backbone as important for the stimulation of the hybridomas may bind only the TCR.     

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.     

T cell inducing determinants contain a hierarchy of residues contacting the T cell receptor.

T cells through their antigen specific T cell receptor recognize a bi-molecular ligand composed of an antigenic peptide bound to a MHC molecule. Several T cell inducing determinants have been extensively characterized by single amino acid substitutions. In this review, we have summarized our characterization of four immunodominant determinants. Each of these determinants possessed a single amino acid residue which was absolutely critical for the recognition by T cells. From these data we propose a hypothesis that there is a hierarchy in the T cell contact residues of a determinant, composed of a single primary residue, and a few secondary residues.     

T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms

Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.     

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.     

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.     

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.     

T cells and their eons-old obsession with MHC

T cells bearing receptors made up of α and β chains (TCRs) usually react with peptides bound to major histocompatibility complex proteins (MHC). This bias could be imposed by positive selection, the phenomenon that selects thymocytes to mature into T cells only if the TCRs they bear react with low but appreciable affinity with MHC + peptide combinations in the thymus cortex. However, it is also possible that the polypeptides of TCRs themselves do not have random specificities but rather are biased toward reaction with MHC. Evolution would therefore have selected for a collection of TCR variable elements that are prone to react with MHC. If this were to be so, positive selection would act on thymocytes bearing a pre biased collection of TCRs to pick out those that react to some extent, but not too well, with self MHC + self-peptides. A problem with studies of this evolutionary idea is the fact that there are many TCR variable elements and that these differ considerably in the amino acids with which they contact MHC. However, recent experiments by our group and others suggest that one group of TCR variable elements, those related to the mouse Vβ8 family, has amino acids in their CDR2 regions that consistently bind a particular site on an MHC α-helix. Other groups of variable elements may use different patterns of amino acids to achieve the same goal. Mutation of these amino acids reduces the ability of T cells and thymocytes to react with MHC. These amino acids are present in the variable regions of distantly related species such as sharks and human. Overall the data indicate that TCR elements have indeed been selected by evolution to react with MHC proteins. Many mysteries about TCRs remain to be solved, including the nature of auto-recognition, the basis of MHC allele specificity, and the very nature and complexity of TCRs on mature T cells.     

Unusual features of self-peptide/MHC binding by autoimmune T cell receptors

Structural studies on T cell receptors (TCRs) specific for foreign antigens demonstrated a remarkably similar topology characterized by a central, diagonal TCR binding mode that maximizes interactions with the MHC bound peptide. However, three recent structures involving autoimmune TCRs demonstrated unusual interactions with self-peptide/MHC complexes. Two TCRs from multiple sclerosis patients bind with unconventional topologies, and both TCRs are shifted toward the peptide N terminus and the MHC class II beta chain helix. A TCR from the experimental autoimmune encephalomyelitis (EAE) model binds in a conventional orientation, but the structure is unusual because the self-peptide only partially fills the binding site. For all three TCRs, interaction with the MHC bound self-peptide is suboptimal, and only two or three TCR loops contact the peptide. Optimal TCR binding modes confer a competitive advantage for antimicrobial T cells during an infection, whereas altered binding properties may permit survival of a subset of autoreactive T cells during thymic selection.     

Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude

Cell-mediated immunity stems from the proliferation of naive T lymphocytes expressing T cell antigen receptors (TCRs) specific for foreign peptides bound to host major histocompatibility complex (MHC) molecules. Because of the tremendous diversity of the T cell repertoire, naive T cells specific for any one peptide:MHC complex (pMHC) are extremely rare. Thus, it is not known how many naive T cells of any given pMHC specificity exist in the body or how that number influences the immune response. By using soluble pMHC class II (pMHCII) tetramers and magnetic bead enrichment, we found that three different pMHCII-specific naive CD4(+) T cell populations vary in frequency from 20 to 200 cells per mouse. Moreover, naive population size predicted the size and TCR diversity of the primary CD4(+) T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides are stronger immunogens than others.     

T cell receptor specificity for major histocompatibility complex proteins

The ligands for alpha beta T cell receptors (alphabetaTCRs) are usually major histocompatibility complex (MHC) proteins bound to peptides. Although there is evidence that T cell receptor variable regions have been selected evolutionarily to bind MHC, the rules governing this interaction have not previously been apparent. However, recent solved structures of T cell receptors with related variable regions bound to MHC plus peptides suggest that some amino acids in variable region CDR1 and CDR2s almost always react in a consistent way with MHC. These amino acids may therefore have been selected evolutionarily to predispose T cell receptors toward recognition of MHC ligands.     

Structural analysis of a peptide--HLA class II complex: identification of critical interactions for its formation and recognition by T cell receptor

An assay for the binding of peptides to major histocompatibility complex (MHC) class II proteins on the surface of cells has been used to determine the relative importance of the amino acids composing an influenza haemagglutinin T cell determinant in binding. The important contact residues were identified by the effect substitution of each residue with biotinylated lysine had on the ability of the peptide to bind. The spacing of the critical residues within the peptide sequence was consistent with the central core, of approximately eight amino acids, adopting a helical conformation. The terminal residues were less constrained and might not be part of a regular conformation. Increasing the helical propensity of the determinant, by simply acetylating and amidating the peptide, resulted in an analogue that was able to stimulate a specific T cell clone at significantly lower concentrations than the natural sequence. A potential location for the peptide in the binding site was postulated based on the presence of complementary amino acids in the class II molecule and supported by screening a large number of peptide analogues for their ability either to bind the restriction element or to stimulate T cell proliferation.     

Evidence for preferred MHC class II-TCR recognition independent of the source of bound peptide

The interaction between TCR and peptide-MHC is well described in terms of the recognition of the peptide, but the recognition of the MHC is less well understood. At issue is whether particular V gene products may have higher affinity for some MHC over others and to what extent the bound peptide influences V gene selection. We examined this issue by developing T cell lines in which the presenting MHC class II molecule has a constant TCR contact region, while the presented peptides vary. If there is an affinity between particular V genes and the specific MHC used, only a subset of the V genes will be associated with the response. Indeed, in all the cell lines analyzed, there was a reproducible usage of a limited number of Vbeta genes, regardless of the bound peptides. This Vbeta-gene constraint was independent of the CDR3 sequence, compatible with the lack of involvement of specific peptides. Our results support the hypothesis that certain V gene products may have a preference for interacting with a particular MHC molecule, and this could have an impact in selectively controlling immune responses.