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The Journal of Immunology, 1999, 162: 6451-6457.
Copyright © 1999 by The American Association of Immunologists

Highly Cross-Reactive T Cell Responses to Myelin Basic Protein Epitopes Reveal a Nonpredictable Form of TCR Degeneracy1

Christine Loftus*, Eric Huseby{dagger}, Priya Gopaul{ddagger}, Craig Beeson* and Joan Goverman2,{dagger},{ddagger}

Departments of * Chemistry, {dagger} Immunology, and {ddagger} Molecular Biotechnology, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We identified two nonoverlapping epitopes in myelin basic protein presented by I-Au that are responsible for mediating tolerance induction to this self-Ag. A large number of T cells expressing diverse TCRs are strongly cross-reactive to both epitopes. Surprisingly, the TCR contact residues in each peptide are highly dissimilar. Furthermore, functional TCR contacts cannot be interchanged between the two epitopes, indicating that the TCR contacts in each peptide can only be recognized within the context of the other amino acids present in that peptide’s sequence. This observation indicates that both buried and exposed residues of each peptide contribute to the sculpting of completely distinct antigenic surfaces. We propose that the cross-reactive TCRs adopt mutually exclusive conformations to recognize these dissimilar epitopes, adding a new dimension to TCR degeneracy. This unpredictable TCR plasticity indicates that using just the TCR contacts on a single epitope to define other cross-reactive peptides will identify only a subset of the complete repertoire of cross-reactive epitopes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ Th cells recognize peptides that are associated with MHC class II molecules on the surface of APC. Despite a high degree of Ag specificity associated with each TCR, numerous studies have demonstrated flexibility in Ag recognition (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Degeneracy in TCR recognition was first observed in the form of allo-recognition in which T cells selected by interaction with self-MHC/peptide complexes also respond at a high frequency to Ag associated with allo-MHC molecules (13, 14, 15, 16, 17). In addition, many TCRs can recognize variants of wild-type peptides. These "altered peptides" induce T cell responses that range from full activation to strong antagonism (8, 18, 19, 20). This ability to transmit different signals depending on the peptide ligand presumably allows a TCR to trigger positive selection when engaging a ligand in the thymus but trigger full activation of effector functions when a different ligand is engaged in the periphery (8, 12, 21, 22).

T cell recognition of peptides with more limited sequence homology to the wild-type peptide has also been described (1, 2, 5). Synthetic peptides with minimal sequence similarity to the natural ligand are often able to trigger T cell activation if one or two wild-type amino acid residues that interact with the TCR are retained (1, 2, 3, 5). These studies indicated the importance of particular amino acid residues within the bound peptide as TCR contact residues. A molecular basis for these observations was provided by crystal structures of peptide/MHC complexes showing residues in particular positions in the bound peptide available for interaction with the TCR because they were pointing directly up from the MHC groove (23). The involvement of these residues in TCR binding has been further confirmed by crytallographic studies of peptide/MHC/TCR complexes (24, 25, 26, 27, 28).

TCR degeneracy has also been demonstrated using combinatorial peptide libraries to determine the optimal peptide residues for TCR recognition at each position (10, 11). These experiments have suggested that multiple residues could be accommodated at many positions within a peptide without abolishing TCR recognition. Furthermore, optimal residues for stimulation were defined for every peptide position, and synthetic peptides with all of these optimal residues resulted in the strongest T cell stimulation. In these systems, a more stimulatory residue at one contact position could compensate for a less optimal residue in another contact position. These studies led to a model in which each TCR contact residue in the bound peptide contributes independently to facilitate interaction with the TCR, and the overall strength of interaction is the sum of the individual contributions (10, 11).

Our interest in TCR degeneracy arose from studies of T cells specific for myelin basic protein (MBP).3 We previously identified T cells present in MBP-deficient mice that are tolerized by the endogenous expression of MBP in wild-type mice (29). A large portion of these T cells exhibited a dual specificity for two adjacent but nonoverlapping epitopes within MBP. Sequence analysis of TCR V genes demonstrated that these cross-reactive T cells exhibited a very diverse set of Ag-specific receptors. To understand how two distinct epitopes bound to the same MHC class II molecule could stimulate a large set of cross-reactive TCRs, we characterized the core peptides that formed these epitopes and identified the residues in each peptide that are accessible to the TCR. We report here that the TCR contacts in the two cross-reactive epitopes exhibit no structural similarity to each other. Dissimilarity between the antigenic surfaces is emphasized by the observation that exchanging an important TCR contact residue from one epitope with the peptide residue in the analogous position in the other epitope abolishes T cell recognition. These data indicate that the TCR contact residues in these epitopes are not recognized independently. Instead, recognition of the TCR contact residues is context dependent and, for these two epitopes, is mediated by mutually exclusive conformations of individual TCRs. The ability of TCRs to exhibit this type of degeneracy greatly limits the ability to identify all possible cross-reactive epitopes based on the sequence of a single epitope.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of T cell hybridomas

Hybridomas were generated as previously described (29). Briefly, T cells were isolated from the draining lymph nodes of H-2u MBP-/- (shiverer) mice (29) previously immunized with 75 µg murine MBP and stimulated for 72 h with 30 µM MBP131–150 before fusion. MBP was purified from mouse brains (Pel-Freeze Biologicals, Rogers, AR) (30). The hybridomas were screened for positive responses to both MBP and MBP131–150 using HT-2 cells to detect IL-2 production before cloning by limiting dilution.

Peptide synthesis

Peptides were synthesized with standard Fast F-moc chemistry on an Applied Biosystems 431A peptide synthesizer (Foster City, CA) and labeled on the N terminus with the N-hydroxysuccinimidyl ester of 5(6)-carboxyfluorescein before cleavage. Cleavage from the resin was achieved with 85% trifluoracetic acid, 10% water, and 5% thioanisole, and crude peptides were then purified by reverse-phase HPLC (acetonitrile/water gradient with 0.1% trifluoracetic acid). Identity of the purified peptides was confirmed by electrospray mass spectrometry, and concentrations of peptide solutions were obtained through absorbance measurements of the fluorescein label in pH 8.9 buffer at 495 nm.

Dissociation rates of peptide/MHC complexes

I-Au protein was isolated as described previously (31). In brief, cells expressing I-Au were lysed and passed over a lentil lectin column that was subsequently eluted with methyl mannoside onto an affinity column (10.3.6 Ab). The protein was eluted from the Ab column with Na2CO3, pH 11.5, purity was assessed with silver-stained SDS-PAGE, and concentrations were determined with a micro bicinchoninic acid assay (Pierce, Rockford, IL). To measure the rate of dissociation of fluorescein-labeled peptides, a solution of I-Au protein and an excess of peptide were incubated at pH 5.3 at 37°C for 24 or 48 h. Unbound peptide was then removed by size exclusion (Sephadex G50-SF) at 4°C. The reaction mixture was separated by high performance size exclusion chromatography using a 60- or 30-cm by 7.5-mm TSK3000SW column (Toso Haas, Montgomeryville, PA) and a fluorescence detector. At the beginning of the dissociation, the initial amount of labeled peptide bound to the MHC was measured as the peak height of the peptide/MHC fraction. After subsequent incubations at 37°C, the relative peak height at each time was used as a measure of peptide still bound to the protein. Half-times of dissociation were obtained from single exponential fits to the dissociation data.

T cell stimulation

T cell responses to Ag were assessed by measuring the amount of IL-2 present in culture supernatants after incubating 1 x 105 T cell hybridomas with 5 x 105 irradiated spleen cells and either 20 µM peptide, 3 µM MBP, or no Ag. Incubations were conducted in duplicate or triplicate for 48 h in 96-well round-bottom plates in a total volume of 200 µl growth media containing DMEM supplemented with 10% FCS. Supernatant (50 µl/well) was transferred to another 96-well plate and frozen at -80°C. Relative amounts of IL-2 in the supernatants were determined by adding IL-2-dependent HT-2 indicator cells (1 x 104/well) and measuring the proliferation of the cells after 24 h by adding [3H]thymidine for the last 8 h of incubation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specificity of T cell hybridomas

Previous studies of T cell hybridomas specific for MBP121–140 divided the hybridomas into three groups based on their fine specificity for Ag (29). The first group recognized only the MBP121–140 peptide, the second group recognized MBP121–140 and the nested peptide MBP126–140, and the third group recognized MBP121–140, MPB126–140, and the partly overlapping peptide MBP131–150. However, the group III hybridomas did not respond to MBP131–140, the only region of overlap between MBP121–140 and MBP131–150. Thus, group III hybridomas exhibit an unusual specificity in that these T cells could be stimulated by two distinct epitopes, one within MBP121–140 and the other within MBP131–150. To confirm that the dual specificity for the two epitopes was encoded by individual TCRs, the TCR {alpha}- and ß-chain genes from two different group III hybridomas were cloned and independently transfected into a recipient hybridoma lacking TCR expression. In both cases, expression of the transfected genes from a single TCR was sufficient to confer strong recognition of both MBP121–140 and MBP131–150 (data not shown).

Prediction of MHC-binding epitopes

To predict how sequences within this region of MBP might bind to I-Au, we evaluated existing structural information on the I-Au molecule. Crystal structures of other murine MHC class II molecules complexed with antigenic peptides have revealed a highly conserved backbone conformation of bound peptides (23, 32, 33). Thus, for each residue in a peptide bound to a MHC class II molecule, it is possible to predict whether the side chain is pointing directly down into the binding groove, up toward the TCR, or somewhere in between (Fig. 1GoA).



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  		FIGURE 1. Predicted alignment of the two MBP epitopes. A, The backbone conformation of a generic polyalanine peptide bound to a MHC class II protein with the P1–P9 positions labeled. Coordinates were derived from the crystal structure of OVA323–339 peptide bound to I-Ad (32). B, Predicted alignments for the two epitopes in which P1, P4, P6, and P9 positions are represented with downward arrows and P2, P5, and P8 positions are represented with upwards arrows, respectively.

 
Although a crystal structure has not been determined for I-Au, the properties of the binding groove have been well described by peptide binding studies and molecular modeling (31, 34, 35, 36, 37). The side chain of the residue at the P1 position (i.e., the P1 side chain) points down into a binding pocket of I-Au that is relatively large and hydrophobic. The P2 side chain points up and out of the binding groove and is the first potential T cell contact. The P3 side chain occupies a very shallow, solvent-exposed pocket, and the P4 side chain points down into a pocket that is smaller than the P9 pocket. The major T cell contact residue of the I-Au motif is generally found at the P5 position, where the side chain is pointed directly up and out of the groove. The P6 side chain points down into the groove, filling an unusually large, hydrophobic pocket bounded by I-Au residues Tyr ß30 and Phe ß11. The binding preferences of the P6 pocket are best illustrated by studies of MBP1–11/I-Au complexes in which a strong preference for tyrosine at the P6 position is observed (37). The P7 side chain occupies a shallow, solvent-exposed pocket situated near the MHC ß72 Glu residue, a potential partner for salt bridge formation. The P8 side chain points upward and is the final potential T cell contact. The P9 side chain is positioned in an MHC pocket of moderate size and polarity.

Hypothetical alignments for the two MBP epitopes were proposed based on the structural characteristics of the I-Au binding groove, the primary sequence of MBP121–150, and our preliminary binding studies (29). To test our predictions, core epitopes comprised of 11 residues were synthesized because the flanking P(-1) and P10 residues contribute to peptide/MHC stability by forming hydrogen bonds between MHC side chains and the peptide backbone (23, 38, 39). The MBP peptides 125–135 and 136–146 were predicted to be the core epitopes as shown in Fig. 1GoB. In both alignments, a large, aromatic tyrosine side chain fills the P6 pocket, forming a primary binding anchor for the peptide.

Verification of predicted binding epitopes

Peptides of varying lengths within MBP121–150 and sequence variations of the two core peptides were synthesized and tested for their ability to bind to I-Au and stimulate T cells. The combination of the T cell stimulation data with measurements of peptide/MHC stability (Table IGo) was used to determine the extent to which a specific peptide side chain interacts with either the MHC binding groove, the TCR, or both. The half-time (t1/2) of dissociation of each peptide/I-Au complex was measured by incubating fluorescein-labeled peptide with detergent soluble I-Au and measuring the dissociation rate by chromatography. T cell stimulation was assessed by measuring IL-2 production in response to Ag stimulation. Response to each peptide was tested using panels of six or nine different T cell hybridomas. The number of hybridomas that responded with a weak (0–10%), moderate (10–50%), or complete (50–100%) response relative to the core peptide is reported.


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  		Table I. Peptide variants of MBP121–150 used in study

 
Length variation peptides define core epitopes

A properly aligned 11-mer peptide should exhibit the most stable binding because it encompasses most of the MHC binding interactions. The peptide within MBP121–140 that formed the most stable complex with I-Au is the MBP125–135 peptide (Fig. 2Go), strongly supporting the hypothesis that the core epitope consists of these 11 residues. Extension of the peptide to include residues outside of the core region did not increase binding and, as the core was truncated, binding decreased (Table IGo and Fig. 2Go). In addition to exhibiting the most stable binding to I-Au, MBP125–135 stimulates all T cell hybridomas in the panel as well as MBP121–140 (Table IGo). The truncated MBP127–140 peptide stimulated all T cells despite much weaker binding (t1/2 = 5 h), while further truncation of the P2 residue, a putative TCR contact, eliminated much of the T cell stimulation (Table IGo).



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  		FIGURE 2. Definition of the core epitopes using peptides with extensions at the N and C termini. A, Within MBP121–140, the 125–135 peptide was most stable. B, Within MBP131–150, the 136–146 peptide was most stable. Dissociation half-times (t1/2) were measured as described in Table IGo.

 
The MBP136–146 peptide also exhibited a very long half-life, and extensions at either end of MBP136–146 did not increase the binding stability. Thus, every residue required for the maximal stability is contained in MBP136–146 (Fig. 2Go). Binding of the MBP132–144 peptide is greatly diminished (t1/2 = 2 h) due to the loss of the P9 residue. However, this level of binding is sufficient for the peptide to fully stimulate many of the T cells (Table IGo). The MBP132–144 peptide does retain all of the residues predicted to be accessible to the TCR. We had also considered MBP138–148 as a possible second core epitope because it would align D143 with the D130 at the P5 position of the first epitope. However, this alignment would position the Y142 side chain into the very shallow P4 pocket and the K139 side chain into the nonpolar P1 pocket. It was also found that extending MBP136–146 to MBP136–148, a peptide that includes the P8 and P9 residues of this alternate alignment, did not improve MHC binding.

Responses to core peptides reveal extensive TCR cross-reactivity

Analyses of T cell responses to the core peptides redefined the Ag specificity of the hybridomas that respond to this region of MBP. All of the hybridomas that were tested from group II (10/10) and group III (12/12) exhibited cross-reactivity for MBP125–135 and MBP136–146. The cross-reactive response of the group II hybridomas to MBP136–146 was unexpected because these T cells were previously distinguished from group III by their inability to respond to the longer MBP131–150 peptide that contains this core epitope. Our previous studies showed that the small number of MBP121–150-specific T cells that escape tolerance in wild-type mice consist of both group I and group II T cells. Therefore, the ability of group II T cells to recognize the two core epitopes in this region indicates that T cells exhibiting cross-reactivity are found in the periphery of wild-type mice.

Five group I hybridomas that only recognized MBP121–140 peptide were also tested for cross-reactivity to the two core peptides. In contrast to group II and group III hybridomas, none of these hybridomas responded significantly to MBP136–146. Three group I hybridomas did respond to MBP125–135, and two did not respond to any peptide tested except MBP121–140. Apparently recognition by some of the group I T cells requires additional residues at either the amino- or carboxy-terminal ends of MBP125–135. Representative responses of group I, II, and III hybridomas are shown in Fig. 3Go. The T cell hybridomas used for the data in Table IGo were comprised of both groups II and III.



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  		FIGURE 3. Cross-reactivity of MBP-specific T cells for the MBP125–135 and 136–146 core epitopes. Individual T cell hybridomas previously defined as group I, II, and III (29) were tested for their ability to recognize the core peptides as well as the longer MBP peptides previously used to define Ag fine specificity. Top panels show the responses of two different group I hybridomas representative of those that do (left) and those that do not (right) recognize MBP125–135. Bottom panels show representative responses of a group II (left) and group III (right) hybridoma. IL-2 production by the hybridomas in response to stimulation with different peptides was measured as proliferation of HT-2 indicator cells.

 
Hybridomas from all specificity groups respond to MBP121–140, consistent with the fact that these hybridomas were obtained by stimulating MBP-primed T cells with MBP121–140 in vitro before fusion. To determine whether T cells specific for MBP131–150 exist that do not cross-react with MBP121–140, additional hybridomas were generated by immunizing MBP-deficient animals with MBP and stimulating cells from the draining lymph nodes with MBP131–150 before fusion. Twelve independent hybridomas were obtained with this protocol, and all responded to both MBP121–140 and MBP131–150. Thus, there do not appear to be T cells that can recognize MBP131–150 without also recognizing MBP121–140.

A diverse set of TCRs exhibit cross-reactivity to MBP125–135 and MBP136–146

The degenerate recognition of MBP125–135 and MBP136–146 was observed for more than 30 different T cell hybridomas (data not shown). Remarkably, the repertoire of V{alpha} and Vß genes expressed on these hybridomas is quite diverse. Our previous studies demonstrated that nine different V{alpha} subfamilies were represented among 29 V{alpha} genes expressed on group II and group III T cells, and seven Vß subfamilies were represented among 34 Vß genes. Furthermore, a diverse set of J{alpha} and Jß gene segments were associated with these V genes (29). A comparison of the CDR3 sequences for a set of V{alpha} and Vß genes expressed on group II and group III hybridomas (Table IIGo) illustrates that there are no obvious highly conserved structural features in these TCRs that could account for the shared specificity of the T cells.


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  		Table II. Sequences of V gene regions for selected T cell hybridomas

 
Single amino acid substitutions that affect MHC binding

The function of specific residues in the two epitopes was studied using peptides containing single amino acid substitutions. Substitutions in each epitope are divided into those that essentially affect only MHC binding and those that retained sufficient MHC binding but primarily lost T cell recognition relative to the wild-type sequences. Our predicted alignments for the MBP125–135 and MBP136–146 core peptides were predicated on placing a tyrosine side chain in the P6 pocket (Y131 and Y142, respectively). In support of this assignment, substitution of these tyrosine residues with the polar glutamic acid completely eliminated binding for both peptides (Table IGo). Although the P1 pocket of I-Au appears to be fairly large and hydrophobic, both predicted epitopes have a glycine at the P1 position that would not place a side chain into the P1 pocket. Replacement of the P1 glycine with a larger nonpolar residue, such as isoleucine, was expected to be well tolerated and potentially increase binding. An increase in stability was observed for G126I in the first core epitope, and wild-type stability was maintained for G137I in the second core epitope (Table IGo). In contrast, the P3 and P7 pockets are very shallow and therefore are expected to be much less tolerant of changes in the side chains. Substitutions at either the P3 or P7 positions in both core peptides diminished MHC binding (Table IGo).

Single amino acid substitutions that affect T cell recognition

Substitution of a peptide residue that strongly affects T cell stimulation while retaining sufficient MHC binding suggests that the peptide side chain at that position is directed outside of the groove and available for interaction with a TCR. However, the accessibility of a residue does not guarantee that it is essential for recognition by all T cells that recognize this epitope. The diversity in group II and III TCRs suggests that there may be a corresponding diversity in the TCR residues that interact with these peptides. To assure that mutations of a TCR contact not detected by one hybridoma would likely be detected by another, a panel of nine hybridomas differing in V gene usage was used to assess the effects of substitutions on TCR recognition.

All of the hybridomas were extremely sensitive to substitution of the P5 residue on both core peptides, while mutations of the P2 and P8 residues had differential effects (Fig. 4Go). Of particular importance, substituted peptides in which the wild-type P5 residues were interchanged between the first and second epitope (i.e., D130A for the first and A141D for the second) were not recognized by any T cell hybridomas. Therefore, even though each cross-reactive TCR can recognize both an aspartic acid and an alanine at the P5 position, the aspartic acid can only be recognized in the context of MBP125–135 and the alanine only in the context of MBP136–146.



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  		FIGURE 4. Effects of substitution of residues predicted to be accessible to the TCR. Most of the substitutions abolished T cell recognition even though MHC binding was sufficient for complete activation. Dissociation half-times (t1/2) and the number of hybridomas that responded in each of the three stimulation categories are listed as presented in Table IGo.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the studies described here, we investigated the structural basis for the degenerate TCR recognition of two MBP epitopes. Our analyses using length-variation peptides defined two core epitopes within the MBP121–150 region, MBP125–135 and MBP136–146. Alignment of these peptides in the MHC groove predicted the "topology" of each peptide when bound to its ligand, i.e., which residues should be primarily involved in MHC binding and which should be accessible to the TCR. The effects of numerous amino acid substitutions on MHC binding and T cell recognition were then used to confirm the predicted alignments for each core peptide. Finally, peptide residues that appeared to be accessible to the TCR were further analyzed to determine which of these residues contributed to the specificity of T cell recognition ("functional" TCR contacts) and which were at the TCR/MHC interface but were not strictly required for recognition. Similarities between the functional TCR contact residues in the two epitopes would suggest that cross-reactivity occurs by a molecular mimicry mechanism (40, 41), while a lack of similarity would point to an alternate mechanism.

In MBP125–135, the aspartic acid at the P5 position contributed strongly to the specific recognition of this epitope. Substitution of the aspartic acid with an alanine (D130A) had only a minor effect on MHC binding but eliminated recognition by nearly all T cells. The arginine residue at the P2 position also appears to be an important functional TCR contact in that only a few T cells tolerated its mutation to an alanine residue (R127A). The serine at the P8 position behaved differently: the side chain is accessible to the TCR but does not appear to contribute significantly to recognition. The loss of stimulation following the replacement of serine with glutamic acid (S133E) confirms that this residue is at the TCR/MHC interface. However, substitution of the serine with an alanine (S133A) had little effect on recognition, indicating that the serine side chain itself is not strictly required.

Defining the functional TCR contacts in the second epitope was less straightforward. Mutation of the phenylalanine at the P2 position produced similar effects to those observed for the serine at the P8 position in MBP125–135. Substitution of this phenylalanine with a tyrosine (F138Y) had no effect on MHC binding but abolished recognition by most T cells. This disruption of recognition due to insertion of an oxygen atom at this position strongly suggests that this residue lies at the TCR/MHC interface. However, substitution of the phenylalanine with the much smaller alanine residue (F138A) did not affect either MHC binding or T cell stimulation. Therefore, the affinity between the TCR and MHC ligand does not strictly require interaction with the phenylalanine. At the P5 position, mutation of the alanine to an aspartic acid (A141D) did not diminish MHC binding but eliminated recognition by most hybridomas. Although this result demonstrates that the specificity of peptide recognition is dependent on the alanine, binding of the methyl side chain of alanine to a TCR pocket is unlikely to contribute substantially to binding affinity. Similarly, substitution of the alanine residue at the P8 position to an aspartic acid abolished all T cell recognition. The same reservation in attributing significant TCR binding affinity to the methyl side chain of an alanine applies to residues at both the P5 and P8 position.

Because none of the residues that we had so far evaluated in MBP136–146 seemed well suited as functional TCR contacts, the residues at the P3 and P7 positions of this epitope were also studied. Residues at these positions are potential TCR contacts in that they reside in shallow, solvent-exposed pockets. Substitution of the aspartic acid at the P7 position with an alanine (D143A) retained sufficient MHC binding but abolished recognition by all T cells. Thus, this aspartic acid appears to be a functional TCR contact. In contrast, substitution of the lysine with an alanine at the P3 position (K139A) had little effect on T cell recognition. Together, these results indicate that the functional TCR contacts in the second epitope are distinct from those in the first epitope.

The observation that the peptide residues most important for specificity of T cell recognition differ between the two peptides rules out the mechanism of molecular mimicry. Instead, our results suggest a mechanism for recognition by which the contribution of each TCR contact is dependent upon the context of the entire peptide sequence. This is best illustrated by the fact that an aspartic acid residue at the P5 position is necessary for TCR recognition of the first epitope, while an aspartic acid at the equivalent position on the second epitope prohibits T cell recognition. The inability to tolerate an exchange of a functional TCR contact suggests that TCR residues have substantially shifted when bound to the second epitope. In this alternate TCR conformation, binding to an aspartic acid at the P5 position is no longer permitted. An aspartic acid at the P7 position is required for binding to the second epitope. This raised the possibility that the TCR is directed into position by an aspartic acid residue whose position is shifted in the second epitope. However, specific recognition by the cross-reactive TCRs is not simply dependent on the presence of an aspartic acid at either the P5 or P7 position. If this were the case, then T cells would have recognized the second epitope substituted with an aspartic acid at the P5 position (A141D).

Our experiments suggest that the TCR conformation adopted for recognition of the first epitope structurally excludes the recognition of the second epitope, and vice versa. Thus, not only are distinct conformations of the TCR required for binding each of the two epitopes, but these TCR conformations are mutually exclusive. Furthermore, the observation that over 30 diverse T cells are cross-reactive for the two epitopes suggests that this ability of a TCR to adopt at least two alternate, mutually exclusive conformations is not a rare event.

Recent studies have proposed several mechanisms by which degeneracy of TCR recognition may occur. It has been well established that peptide residues buried within the binding groove of the MHC can have strong influences on T cell recognition (18, 42). Other experiments have shown that mutation of one TCR contact residue on a peptide can alter the recognition of a second residue in that peptide (43). While both of these observations could be explained by only a change of the peptide conformation, experiments conducted by Ono et al. (44) suggest that substitution of a peptide residue buried in the MHC can have consequences that are more global in nature. Conservative mutations of TCR contact residues in a viral peptide were found to alter the pattern of CTL recognition of MHC residues. It was concluded that alterations in the TCR conformation allow it to bind related ligands. The striking feature of these results is that many of the TCR contacts on the MHC protein were remote from the substituted peptide residue, indicating that changes in the peptide structure can induce conformational changes throughout the MHC surface.

Our results demonstrate that when MBP125–135 and MBP136–146 separately bind to I-Au, two distinct antigenic surfaces are generated that are uniquely defined by the peptide sequences. Our conclusion that TCR recognition of a peptide residue might occur only in the context of one peptide sequence and be prohibited in a different sequence is in direct contrast with the suggestion that TCR contacts function independently of each other (10, 11). The optimization of TCR contacts independently of each other has been used to identify cross-reactive epitopes; however, this is only successful when the overall conformation of the TCR bound to each epitope is similar. If, as observed here, binding of two cross-reactive peptides to the same MHC molecule produces unrelated antigenic surfaces, then a TCR bound to one surface may be required to adopt a conformation that excludes the ability to recognize the other surface. Thus, MBP125–135 and MBP136–146 would never have been predicted to be cross-reactive by simply comparing the functionally important TCR contacts in the two epitopes. The mutually exclusive contexts in which the two MBP epitopes are recognized indicate that TCR degeneracy is likely to be much broader and less predictable than previously believed. Therefore, an attempt to predict potential cross-reactive epitopes based only on the analysis of T cell contact residues in a particular peptide sequence will be intrinsically limited in scope.

Our studies do not imply that the induction of tolerance in T cells specific for MBP121–150 depends upon their cross-reactivity for two distinct epitopes within this region. It is possible that only one of these epitopes is actually presented in vivo and is responsible for mediating tolerance to the MBP protein. In this regard, it is interesting to note that all hybridomas that were obtained by stimulating T cells in vitro with MBP131–150 before fusion responded to both MBP121–140 and MBP131–150. We also have no evidence to suggest that this form of degeneracy is limited to tolerogenic epitopes. In fact, the degeneracy exhibited by the T cells described here was only discovered during our analyses of the immune response to a self-Ag because the two cross-reactive MBP epitopes are adjacent to each other in the primary sequence of the protein. The cross-reactivity of these TCRs was revealed because the set of synthetic peptides used to define their fine specificity happen to include nonoverlapping peptides that contained each epitope. While this close proximity allowed us to detect the cross-reactive epitopes, it is not required or even expected that such cross-reactive epitopes be derived from the same protein. Therefore, we envision the ability of TCRs to assume different conformations to recognize distinct antigenic surfaces to be a fundamental property of TCR recognition that increases the potential diversity of the TCR repertoire.


   Acknowledgments
 
We are grateful to Dr. T. Brabb and A. Perchelett for critical reading of the manuscript.


   Footnotes
 
1 These studies were supported by National Science Foundation Grant MCB-9722374 (to C.B.) and National Institutes of Health Grant R01 NS35126 (to J.G.). J.G. is supported in part by a Junior Faculty Award (2080-A2) from the National Multiple Sclerosis Society, and E.H. is supported by a National Institutes of Health Training Grant CA09537-13. Back

2 Address correspondence and reprint requests to Dr. Joan Goverman, Department of Molecular Biotechnology, Box 357650, University of Washington, Seattle, WA 98195. E-mail address: Back

3 Abbreviation used in this paper: MBP, myelin basic protein. Back

Received for publication January 27, 1999. Accepted for publication March 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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