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.     

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.     

Restricted TCR Valpha gene rearrangements in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis

T cell responses to myelin basic protein (MBP) are thought to play an important role in the pathogenesis of multiple sclerosis (MS). The response to the 83-99 region of MBP represents a dominant response to MBP in patients with MS and is associated with HLA-DR2 that is linked with susceptibility to MS. Although T cell clones reactive to various regions of MBP have been found to exhibit heterogeneous TCR Vbeta gene usage in patients with MS, it is unclear whether T cell clones uniformly recognizing the 83-99 peptide of MBP in the context of the same DR molecule would have restricted TCR V gene rearrangements and recognition motifs. In this study, a panel of DR2- or DR4-restricted T cell clones specific for the MBP83-99 peptide were derived from 11 patients with MS and examined for TCR V gene usage by PCR and the recognition motifs using analog peptides. Our study revealed that despite a few T cell clone pairs having similar recognition motifs and shared sequence homology in the CDR3, the overall recognition motifs of MBP83-99-specific T cells were considerably diverse. Interestingly, the DR2-restricted T cell clones displayed a biased V gene usage for Valpha3 and Valpha8, while Vbeta gene rearrangements were highly heterogeneous. This study provided experimental evidence suggesting a limited heterogeneity in TCR Valpha gene rearrangements of MBP-reactive T cells in DR2 patients with MS.      

The Diversity of Antigen-Specific TCR Repertoires Reflects the Relative Complexity of Epitopes Recognized

Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 K^d. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252–260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170–179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant H-2 K^d molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/K^d complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.     

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.     

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.     

Analyses of TCR clustering at the T cell-antigen-presenting cell interface and its impact on the activation of naive CD4+ T cells

The role of micrometer-scale clustering of TCRs at the T cell-antigen-presenting cell (APC) interface in T cell activation is an area of active investigation. Here we have investigated the impact of variations in the extent of TCR clustering on the activation of naive CD4+ T cells. These T cells are derived from transgenic (tg) mice expressing TCRs (172.10 and 1934.4) specific for the N-terminal nonapeptide of MBP bound to I-A(u), and are associated with murine experimental autoimmune encephalomyelitis (EAE). The 172.10 TCR has a approximately 4-fold higher affinity for antigen relative to the 1934.4 TCR, allowing us to compare the properties of two tg T cells of different avidities. We observe that variations in large-scale TCR clustering at the T cell-APC interface do not correlate well with the extent of activation (CD25 or CD69 up-regulation and IL-2 or IFN-gamma production). Efficient activation can also be achieved in the absence of micrometer-scale TCR clustering, indicating that this is not a prerequisite for the effective stimulation of naive T cells.     

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.     

MHC Class II-Dependent Peptide Antigen Versus Superantigen Presentation to T Cells

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.     

The central residues of a T cell receptor sequence motif are key determinants of autoantigen recognition in murine experimental autoimmune encephalomyelitis

The autoreactive response in murine experimental autoimmune encephalomyelitis (EAE) is dominated by an oligoclonal expansion of V beta 8(+) CD4(+) T cells. These T cells recognize the immunodominant N-terminal nonapeptide of myelin basic protein (MBP1-9) associated with the MHC class II molecule, I-A(u). Amongst the autoreactive cells, T cells bearing TCR containing the CDR3 beta motif Asp-Ala-Gly-Gly-Gly-Tyr (DAGGGY) play a dominant role in the disease process. Here we have investigated the molecular basis for antigen recognition by a representative TCR (172.10) that contains the DAGGGY motif. The roles of the three glycines in this motif in the corresponding TCR-peptide-MHC interactions have been analyzed using a combination of site-directed mutagenesis and surface plasmon resonance. Our data show that mutation of either of the first two glycines (G97, G98) to alanine results in soluble, recombinant TCR that do not bind to recombinant antigen at detectable levels. Mutation of the third glycine (G99) of the 172.10 TCR results in a substantial decrease in affinity. The importance of the triple glycines for antigen recognition provides an explanation at the molecular level for the recruitment of T cells bearing the DAGGGY motif into the responding repertoire during EAE induction.     

Prevention and treatment of experimental autoimmune encephalomyelitis with clonotypic CDR3 peptides: CD4+ FoxP3+ T‐regulatory cells suppress interleukin‐2‐dependent expansion of myelin basic protein‐specific T cells

T‐cell receptor (TCR)‐derived peptides are recognized by the immune system and are capable of modulating autoimmune responses. Using the myelin basic protein (MBP) TCR 1501 transgenic mouse model, we demonstrated that TCR CDR3 peptides from the transgenic TCR can provide a protective effect when therapy is initiated before the induction of experimental autoimmune encephalomyelitis (EAE). More importantly, TCR CDR3 peptide therapy can ameliorate the disease when administered after EAE onset. Concurrent with the therapeutic effects, we observed reduced T‐cell proliferation and reduced interleukin‐2 (IL‐2) levels in response to stimulation with MBP‐85‐99 peptide in splenocyte cultures from mice receiving TCR CDR3 peptides compared with that of control mice. Moreover, we found that Foxp3⁺ CD4 T cells from mice protected with TCR CDR3 peptide are preferentially expanded in the presence of IL‐2. This is supportive of a proposed mechanism where Foxp3⁺ T‐regulatory cells induced by therapy with MBP‐85‐99 TCR CDR3 peptides limit expansion and the encephalitogenic activity of MBP‐85‐99‐specific T cells by regulating the levels of secreted IL‐2.     

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.     

Identification of core structure and critical T cell receptor contact residues in an antigenic peptide by measuring acidification rates

A silicon-based biosensor microphysiometer measures real time cell response by monitoring an increase in extracellular acidification rate in response to ligands for specific membrane receptors. We used the microphysiometer to identify the minimal structure and critical residues of an antigenic peptide for its interaction with T cell receptor (TCR) using a synthetic peptide analog of human myelin basic protein (MBP) corresponding to residues 84–102 [MBP(83–102)Y⁸³]. MBP(83–102)Y⁸³ peptide analogs were allowed to interact with TCRs on a DRB5 1 0101-restricted Herpes virus saimiri (HVS) transformed human T cell clone (SS8T) which also contains major histocompatibility complexes (MHC) class II (DR2) molecules. Cultured SS8T cells were exposed to 11 N-terminus and 11 C-terminus truncated peptides separately in the microphysiometer chambers to determine the minimal amino acid residues required for the T cell response. In parallel, 13 analogs of the MBP(83–102)Y⁸³ peptide with single alanine substitutions were tested in this assay to identify critical amino acid residues involved in TCR interactions. A minimal core length of MBP(91–100) peptide and residues F-91, K-93, N-94, I-95 and V-96 were essential for TCR interaction. Acidification rate measurements correlated well with enhanced levels of γ-IFN (interferon gamma) and TNF-β cytokine production and suggested that the increase in the extracellular acidification rate is a direct result of early T cell signaling events.     

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide/MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide/MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II/antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP/MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide/MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen/MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.     

Functional degeneracy of residues in a T cell peptide epitope contributes to its recognition by different T cell hybridomas.

Synthetic antigen Poly EYK(EYA)5 induces T cells of narrowly defined fine specificity as represented by the two I-Ad-restricted T cell hybridomas, A.1.1 and B.1.1. Both these hybridomas recognize the minimum 15-amino-acid peptide sequence EYK(EYA)4. We have characterized the residues involved in the recognition of EYK(EYA)4 peptide by these hybridomas with synthetic peptides and discovered a distinct functional hierarchy for the residues in the sequence. Even with the repeating tripeptide (EYA)5, which is recognized by B.1.1 cells, the residues that are essential cluster near the middle of the sequence but not near the N- or C-terminal region. Different MHC binding and TCR contacting residues were found for each of the hybridomas. The results suggest that different T cells either recognize different parts of the peptide MHC complex or that the peptide binds to MHC in multiple conformations. This was supported by the fact that Poly EYK(EYA)5 is alpha-helical but the peptides used here showed only a slight propensity to adopt this structure and it did not correlate with their functional activity. We also found that (EYA)5 does not compete with EYK(EYA)4 in the stimulation of A.1.1 cells despite its obvious capacity to interact with I-Ad when it stimulates B.1.1 cells. This may be because these peptides have a low affinity for Ia and therefore only appropriate TCR interactions would stabilize the antigen-Ia complex. In conclusion, antigen-MHC-TCR interaction appears to be a dynamic process which allows recognition of different residues of a T cell determinant by different T cells.     

T cell recognition motifs of an immunodominant peptide of myelin basic protein in patients with multiple sclerosis: structural requirements and clinical implications

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83 – 99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83 – 99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83 – 99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81 – 95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83 – 99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.     

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.     

Conserved T-cell receptor class II major histocompatibility complex contact detected in a T-lymphocyte population.

T-cell receptor (TCR) interacts with an antigenic peptide deeply buried in the major histocompatibility complex (MHC) molecule. How class II MHC is contacted by TCR during antigen recognition remains largely elusive. Here we used a panel of I-Ek mutants to identify two I-Ek residues that were frequently contacted by TCR among a large pool of T cells specific for the same antigen. The restricted TCR interaction with I-Ek was independent of the antigen peptides. We also identified a dominant heteroclitic residue on I-Ek, beta81H, in which mutation led to increased recognition of antigens in individual T-cell clones. Moreover, both the conserved TCR-I-Ek interaction and the heteroclitic TCR-I-Ek recognition were detected in T lymphocytes freshly isolated from mice primed with the specific antigens. The identical TCR-I-Ek interaction in a heterogeneous T-cell population suggested the dominance of invariant TCR-class II MHC interaction.     

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.     

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.