To define the relative contributions of HLA and peptide contacts with TCR complementarity-determining region (CDR) 3 residues in T cell recognition, systematic mutagenesis and domain swapping was conducted on two highly similar TCRs that both respond to the influenza hemagglutinin (HA) peptide, HA307–319, but with different HLA restrictions. Despite the primary sequence similarity of these TCRs, exchange of as little as two CDR3 residues between them completely abrogated responsiveness. At position 95 within CDR3α, various substitutions still allowed for some degree of recognition. One modest substitution, alanine for glycine (essentially the addition of a methyl group), significantly broadened the specificity of the TCR. Transfectants expressing this mutant TCR responded strongly in the context of multiple HLA-DR alleles and to HA peptide variants with substitutions at each TCR contact residue. These results suggest that the conformations of CDR3 loops are crucial to TCR specificity and that it may not be reliable to extrapolate from primary sequence similarities in TCRs to similarities in specificity. The ease with which a broad specificity is induced in this mutant TCR has implications for the mechanisms and frequency of alloreactivity and promiscuity in T cell responses.

The human TCR recognizes a diverse array of potential ligands that can be formed by combinations of antigenic peptides and polymorphic HLA-restricting elements. The structural diversity in the TCR necessary to support this recognition is generated through rearrangement of multiple germline gene segments and the action of somatic diversification mechanisms. The functional similarity of Ig and TCR, together with their primary sequence similarity, first suggested that TCR and Ig might adopt comparable three-dimensional structures.

The complex structure of the ligand recognized by TCR, consisting of both an HLA-presenting molecule and a bound peptide, might be expected to impose restrictions on the orientation with which TCR can interact with ligands that are not required of Ig. Davis and Bjorkman (1) first proposed a model of this interaction in which complementarity-determining regions (CDRs)3 1 and 2 of the TCR interacted with the MHC α helices while the CDR3 regions interacted with peptide. A key feature of this model was that it allowed for the most diverse portions of the TCR (CDR3s) to bind the most diverse portion of its complex ligand, the peptide. The predicted interaction of CDR3 residues with peptide has been verified by direct mutagenesis studies of TCRs (2, 3, 4, 5, 6, 7, 8, 9), by cross-linking with a photoactivatable peptide (10), and by the in vivo selection of compensatory TCR mutations with variant peptides (11, 12). Additional studies have demonstrated that mutation of CDR1 or CDR2 can abrogate or alter recognition (4, 5, 6, 13, 14, 15, 16, 17). Framework residues must also have a role in recognition because CDR grafting experiments do not consistently transfer specificity between TCRs (6, 18).

Based on mutagenesis studies in which class I MHC mutants were selected with a CTL clone, Sun et al. (19) proposed that TCRs interacted with the complex of class I MHC and peptide in a standard conformation. In this conformation, the TCR was oriented parallel to the β-pleated strands in the floor of the class I molecule and diagonal relative to the α helices. Several x-ray crystallographic structures of TCRs bound to peptide-MHC class I complexes have been solved and yield support for this diagonal orientation of recognition (20, 21, 22, 23). The emerging picture from these structures is that peptide interacts with both CDR3s but also with both CDR1s. CDR3α and CDR3β form a pocket that accepts a side chain from a specific, centrally located peptide residue. The CDR2 of the TCR α-chain is located over the MHC α2 helix while the TCRβ CDR2 interacts with the class I α1 helix. At present, a class II-peptide-TCR crystal structure has not been reported. Furthermore, the static picture afforded by a crystal structure alone cannot resolve the relative contributions of different elements of the TCR in shaping its structure, allowing or preventing recognition of its ligand.

We have previously described site-specific mutagenesis of TCRs derived from two human T cell clones specific for the immunodominant influenza virus hemagglutinin (HA) peptide, HA307–319 (16). In these studies, mutagenesis of CDR2α residues altered the HLA-DR restriction of the TCRs while maintaining peptide specificity. These results are consistent with a TCR orientation in which CDR2α interacts with the DRβ molecule. Further, they point out an advantage of mutagenesis studies of TCRs from human HA307–319-specific T cell clones. Because the HA peptide binds different allelic DR molecules, DR1, DR2, DR4w4, DR5, and DR7, with similar conformations and affinities (24, 25), mutagenesis of appropriate TCRs should facilitate the separate identification of TCR sites that contact specific peptide or HLA residues. In the current study, this mutagenesis approach has been applied to the CDR3 regions of HA307–319-specific TCRs. Most mutations in CDR3α or CDR3β resulted in loss of recognition. However, a modest change of Gly to Ala at one position in CDR3α dramatically broadened the specificity of the TCR such that HA variant peptides with substitutions at any TCR contact residue as well as multiple different DR presenting molecules now elicited responses. The effect of this mutation on DR recognition was dominant when paired with CDR2α mutations. These results suggest that, despite a standard orientation of TCR relative to its ligand, there is substantial flexibility in this interaction such that minor differences in CDR2 residues may have dramatic effects on TCR specificity and that simple one to one correspondences between TCR and peptide or MHC residues may be difficult to generalize across TCRs.

The specificities of the TCRs from T cell clones JS515.11 (JS) and 3BC6.6 (3BC) have been described previously (16). When transfected into Jurkat cells, the TCR from 3BC recognizes the HA307–319 peptide only in the context of DR4. The TCR from JS responds strongly to the HA peptide in the context of DR7 but also displays a weaker, promiscuous response to the peptide presented by DR1, DR4, and DR5. Both clones use products of the TCR BV3S1 gene and allelic forms of the TCR AV1S2 gene, which differ by only two residues in CDR2α (16). J.RT3-T3.5 (JRT3), a Jurkat cell line derivative defective in endogenous TCRβ expression (26), was obtained from the American Type Culture Collection (Manassas, VA). B lymphoblastoid cell lines (B-LCL) that were homozygous for HLA-DR alleles, HOM2 (DR1), MGAR (DR2), Priess (DR4), Sweig (DR5), and DBB (DR7), were used as APCs. Cells were grown in RPMI 1640 supplemented with sodium pyruvate, penicillin, streptomycin, and 10% calf serum.

Chimeric TCRα and TCRβ constructs were created by PCR amplification using primers that spanned the positions where sequences exchanged from one receptor to the other. Point mutations were introduced by making the desired change in the appropriate primer. Constructs were cloned initially in Bluescript (Stratagene, San Diego, CA), and their nucleotide sequences were confirmed. Subsequently, the mutant constructs were transferred to the episomal expression vectors pREP8 and pREP9 (Invitrogen, San Diego, CA).

Episomal vectors containing in vitro mutagenized TCR genes were introduced into JRT3 cells by electroporation as previously described (16). Briefly, electroporation was performed at 250 V, 960 μF using a Gene Pulser (Bio-Rad, Hercules, CA). Cells were allowed to recover for 24 h, the medium was changed, and cells were grown for a further 3 days under nonselective conditions. Cells were then transferred to medium containing 600 μg/ml active geneticin (Life Technologies, Gaithersburg, MD) and 0.5 mM histidinol (Sigma, St. Louis, MO). After 2–3 wk of selection, cells were stained with an Ab to Vβ3 (βV3a; T Cell Diagnostics, Cambridge, MA) and analyzed on a FACSort (Becton Dickinson, San Jose, CA). All transfectants were strongly positive for Vβ3 expression. For experiments in which the responses of different mutagenized TCR constructs were compared, surface Vβ3 expression levels on all transfectants were measured by FACS to ensure that there were no significant differences. Retrospective examination of the expression data revealed no cases in which increased or decreased responsiveness of a given transfectant correlated with high or low expression, respectively, of the TCR. Cells were always stained within 48 h of their use in stimulation assays.

Transfected T cells were stimulated with the influenza virus (A/PR/8/34) HA peptide 307–319 (PKYVKQNTLKLAT) and single position variants presented by B-LCL. Transfected JRT3 cells (5 × 104) and APCs (5 × 104) were cultured in the presence of various concentrations of peptide and 3 ng/ml PMA in triplicate in volumes of 150 μl in 96-well plates. At 16 h, 1 μCi of [3H]thymidine (5 Ci/mmol) was added. Cells were harvested 8 h later, and [3H]thymidine incorporation was measured by scintillation counting. Controls for all experiments included stimulation of T cells with 3 ng/ml PMA and stimulation in microtiter wells coated with α-CD3.

To evaluate the effect of differences in the CDR3 regions on peptide recognition and/or HLA restriction, the CDR3α and β regions of the TCRs from the T cell clones JS and 3BC were systematically mutagenized by exchanging analogous sequences between the two receptors. The Jα regions of the 3BC and JS TCRs are of identical length and vary at 12 of 20 residues between positions 95 and 114. Chimeric TCR α-chain genes were created in which all sequences to the N-terminal side (left in Fig. 1,A) were derived from one TCR α-chain while all sequences to the C-terminal side (right in Fig. 1,A) were derived from the other chain. TCRα chimeras in which the “crossover” point was located in the middle of CDR3, between positions 98 and 99 (constructs JSα98 and 3BCα98), could not be activated when paired with either the 3BC TCRβ or JS TCRβ, regardless of whether the HA307–319 peptide was presented by DR4 or DR7 (Figs. 2 A and 3A). Similarly, no response was observed when the crossover point was between positions 96 and 97 (JSα96 and 3BCα96). In these latter constructs, only the two N-terminal residues of CDR3α are altered relative to the cognate receptor. In TCRα chimeras where the crossover point was between positions 94 and 95 (constructs JSα94 and 3BCα94), the expected peptide recognition and HLA restriction was maintained, as previously described (16).

FIGURE 1.

A, Sequences of TCR α-chain mutants. Shown are sequences from the conserved 90C position through CDR3 and the J region. JS and 3BC are parental TCR sequences. In all mutants, sequence derived from the JS chain is shaded and sequence derived from the 3BC chain is unshaded. Mutant constructs are named by the transition point and the identity of the sequence to the left of that point. Thus, JSα94 has JS sequence up to and including position 94 and 3BC sequence after position 94. JSα105P and 3BCα105K are point mutations. B, Sequences of TCR β-chain mutants. Shown are sequences from the conserved 91C position through CDR3 and including the D and J regions. Nomenclature and shading are as described for the α constructs. The two parental β-chains are identical in the areas not shown in the figure.

FIGURE 1.

A, Sequences of TCR α-chain mutants. Shown are sequences from the conserved 90C position through CDR3 and the J region. JS and 3BC are parental TCR sequences. In all mutants, sequence derived from the JS chain is shaded and sequence derived from the 3BC chain is unshaded. Mutant constructs are named by the transition point and the identity of the sequence to the left of that point. Thus, JSα94 has JS sequence up to and including position 94 and 3BC sequence after position 94. JSα105P and 3BCα105K are point mutations. B, Sequences of TCR β-chain mutants. Shown are sequences from the conserved 91C position through CDR3 and including the D and J regions. Nomenclature and shading are as described for the α constructs. The two parental β-chains are identical in the areas not shown in the figure.

Close modal
FIGURE 2.

Response of TCR α- and β-chain constructs. A and B, TCR α-chain mutants were transfected into JRT3 cells along with the 3BC β-chain or JS β-chain as indicated. Transfectants were challenged with 50 μg/ml HA307–319 presented by DR4 (Priess) or DR7 (DBB) cells. Supernatants from these cultures were applied to IL-2-sensitive HT-2 cells, and proliferation was measured by the incorporation of [3H]thymidine. Values presented were obtained by subtracting the cpm obtained with no peptide from the raw cpm. C, TCR β-chain mutants were transfected into JRT3 cells along with the 3BC α-chain or the JS α-chain as indicated. Transfectants were stimulated and proliferation was measured as above.

FIGURE 2.

Response of TCR α- and β-chain constructs. A and B, TCR α-chain mutants were transfected into JRT3 cells along with the 3BC β-chain or JS β-chain as indicated. Transfectants were challenged with 50 μg/ml HA307–319 presented by DR4 (Priess) or DR7 (DBB) cells. Supernatants from these cultures were applied to IL-2-sensitive HT-2 cells, and proliferation was measured by the incorporation of [3H]thymidine. Values presented were obtained by subtracting the cpm obtained with no peptide from the raw cpm. C, TCR β-chain mutants were transfected into JRT3 cells along with the 3BC α-chain or the JS α-chain as indicated. Transfectants were stimulated and proliferation was measured as above.

Close modal

The highly conserved J segment sequence FGXG and residues further C-terminal in Jα are outside of the structurally defined CDR3 region. Nevertheless, there is significant sequence variation at these positions. For example, the two TCR α-chains studied here differ in five of these 12 residues. Therefore, TCRα chimeras were made with the crossover point between residues 104 and 105, immediately N-terminal to the nonconserved residue in the FGXG sequence. In contrast to results obtained from chimeras with breakpoints within the structurally defined CDR3α region, both of these constructs (JSα104 and 3BCα104), when paired with the cognate β-chain, had responses characteristic of the TCR from which the α-chain sequences N-terminal to the FGXG sequence were derived (Figs. 2 B and 3B). In addition, single point mutations at the nonconserved, “X”, position in the FGXG sequence had no effect on TCR recognition (constructs JSα105P and 3BCα105K).

Chimeric TCR β-chain constructs analogous to those made for TCRα were also generated, paired with each normal TCR α-chain, and tested for recognition of the HA peptide presented by HLA-DR4 and by DR7. Breakpoints in the TCRβ constructs were located in the middle of CDR3β (constructs JSβ98 and 3BCβ98, Fig. 1,B). When these chimeric TCRβs were introduced into cells, no activation was observed regardless of which TCR α-chain they were paired with or whether the HA peptide was presented by DR4 or DR7 (Figs. 2 C and 3C). As was observed for CDR3α, sequences within or C-terminal to the conserved FGXG sequence in Jβ could be exchanged without loss of responsiveness or specificity (constructs JSβ105 and 3BCβ105).

Because chimeric TCR α-chains with crossover points between positions 94 and 95 functioned normally while those with crossover points between positions 96 and 97 were nonresponsive, the role of specific residues between these crossover points was further explored by site-specific mutagenesis. The most significant difference between the JS and 3BC TCR α-chains at these sites is at position 95 where the JS TCRα has a small neutral residue, glycine, while the 3BC TCRα has an aspartic acid residue. Site-directed mutagenesis was used to create TCR α-chains in which the D residue at position 95 in the 3BC TCRα was changed to A, E, and G. Substitution of the 95D residue in the 3BC TCRα with either A or even the similarly charged E residue abrogated recognition (data not shown). The substitution of residue 95D with G, the analogous amino acid from the JS TCRα, retained the 3BC receptor’s response to peptide presented by DR4 (data not shown).

Mutations were also made at position 95 of the JS TCR α-chain where the G residue was changed to A, E, and D. The JS TCRα mutant in which the 95G residue was substituted with D was unresponsive to peptide presented by either DR4 or DR7. The JS TCRα 95G to E mutant responded to higher concentrations of peptide presented by DR7, but the promiscuous response in the presence of DR4 was lost (Fig. 4 A). The JS TCRα 95G to A mutant, which differed only by the addition of a single methyl group to the wild-type JS receptor, had a significantly enhanced response to the HA peptide presented by DR4 such that this promiscuous response was as sensitive as the primary response in the context of DR7. Thus, four different residues at position 95 of the TCR α-chain of the JS receptor yield four very different responses to peptide and MHC.

FIGURE 4.

Response of transfectants with mutations at position 95 of the JS α-chain. A, Transfectants expressing the JS β-chain and either the JS α-chain (JS95G) or a construct with a single change at position 95 of the JS α-chain were challenged with HA307–319 as in Fig. 2. B, Transfectants expressing the JS β-chain and either the JS α-chain (JSα95G) or a construct with alanine at position 95 of the α-chain (JSα95A) were challenged with peptide presented by DR1 (HOM2), DR2 (MGAR), DR4 (Priess), DR5 (Sweig), and DR7 (DBB) cells.

FIGURE 4.

Response of transfectants with mutations at position 95 of the JS α-chain. A, Transfectants expressing the JS β-chain and either the JS α-chain (JS95G) or a construct with a single change at position 95 of the JS α-chain were challenged with HA307–319 as in Fig. 2. B, Transfectants expressing the JS β-chain and either the JS α-chain (JSα95G) or a construct with alanine at position 95 of the α-chain (JSα95A) were challenged with peptide presented by DR1 (HOM2), DR2 (MGAR), DR4 (Priess), DR5 (Sweig), and DR7 (DBB) cells.

Close modal

In addition to its normally sensitive response to the HA307–319 peptide presented by DR7, the TCR from the JS T cell clone also exhibits a modest response to high concentrations of the HA peptide (≥10 μg/ml) when presented by DR1, DR4, and DR5. No response is seen with the DR2 homozygous APC, where the HA peptide is presented by the DRB5 molecule rather than DRB1. To look more broadly at the effect of the 95G to A mutation on the HLA restriction of the JS TCR, the mutant TCR was transfected into cells and challenged with the HA peptide presented by DR1, DR2, DR4, DR5, and DR7 (Fig. 4 B). The JS TCRα with the alanine substitution at position 95 was significantly more sensitive (>2 orders of magnitude) to peptide presented by DR1, DR4, and DR5. Response to peptide presented by DR2 was largely unaffected except for a slight increase in responsiveness at the highest peptide concentration. Despite the increased sensitivity in the promiscuous response seen with this mutant TCRα, the dose-response curve for DR7 was unchanged.

Five amino acids in the HA307–319 peptide have been demonstrated to act as T cell contact residues when presented by the various DR alleles studied here (25). A panel of HA variant peptides incorporating residues varying in charge and size at each of these T cell contact positions was tested for recognition by the JS TCR with the 95A mutation (Fig. 5). There was no clear one to one correspondence between substitutions at a specific position in the peptide and either increased or decreased responsiveness seen with the TCR mutant. Although cell-surface expression of the normal and the mutant TCR was similar, the mutant TCR consistently exhibited stronger responses to peptide variants at every T cell contact position. In some cases (e.g., V310L or N313T), robust responses were seen with the mutant TCR but were undetectable with the normal receptor. In no case did a peptide variant elicit a significantly weaker response with the 95A mutant TCR.

FIGURE 5.

Response of the JSα95A mutant to peptide variants. Transfectants expressing the JS β-chain and either the JS α-chain (JSα95G) or the JSα95A mutant were challenged with 10 μg/ml HA307–319 or the indicated HA variants with single changes at positions 308, 310, 311, 313, and 316.

FIGURE 5.

Response of the JSα95A mutant to peptide variants. Transfectants expressing the JS β-chain and either the JS α-chain (JSα95G) or the JSα95A mutant were challenged with 10 μg/ml HA307–319 or the indicated HA variants with single changes at positions 308, 310, 311, 313, and 316.

Close modal

In previous studies, we have demonstrated that amino acid substitutions at one or both of two positions in the CDR2α region of the JS TCR still allows recognition of the HA peptide when presented by DR7, but eliminates the promiscuous response seen in the context of DR4 (16). These CDR2α mutations, which depress responsiveness in the context of DR4, and the CDR3α mutant, which increases responsiveness in the context of multiple DR alleles, including DR4, were incorporated into a single JS TCRα construct and introduced into cells with the cognate JS TCRβ. The resulting transfected cells, along with appropriate transfectants of the single mutants and the wild-type JS TCRα, were challenged with the HA peptide presented by DR4 and by DR7 (Fig. 6). The single mutant transfectants reproduced their previously demonstrated specificities. The double mutant responded similarly to the JSα95A single mutant. The dose-response curve for DR7 was shifted to the left relative to the CDR2α single mutant, and the DR4-restricted response was significantly increased relative to the wild-type JS TCRα.

FIGURE 6.

Interaction of CDR2 and CDR3 mutations in the TCR α-chain. Transfectants expressing the JS β-chain and either the JS α-chain, the 3BCα94 chain (two changes in CDR2), the JSα95A chain, or a double mutant α-chain incorporating the CDR2 and position 95 changes together, were challenged with HA307–319 presented by DR4 or DR7.

FIGURE 6.

Interaction of CDR2 and CDR3 mutations in the TCR α-chain. Transfectants expressing the JS β-chain and either the JS α-chain, the 3BCα94 chain (two changes in CDR2), the JSα95A chain, or a double mutant α-chain incorporating the CDR2 and position 95 changes together, were challenged with HA307–319 presented by DR4 or DR7.

Close modal

In this study, chimeric TCRs have been constructed using two receptors that differ in only three of six CDRs (CDR2α, CDR3α, and CDR3β). Previously, we have shown that changes in CDR2α of the HLA-promiscuous receptor, JS, modulate promiscuity in this system (16). This current study examines the specificity of TCRs chimeric in the CDR3 region and compares the relative strength of the effects of substitutions in CDR2 and CDR3 through the construction of double mutants.

In the class I-restricted TCRs whose structures when complexed with ligand have been solved, CDR3α and CDR3β pair to form a pocket that accepts protruding amino acid side chains from the MHC-bound peptide (20, 21, 22, 23). Despite the degree of sequence divergence in the CDR3α regions in the two TCRs studied here, there are enough general similarities to suggest that one receptor might be capable of functionally substituting for the other. The CDR3α sequences from these two TCRs are exactly the same length. As in the TCRα motif described by Kieber-Emmons et al. (27), both of these CDR3α regions have the same negatively and positively charged residues at positions 5 and 11 (counting from the conserved C residue in the Vα), which potentially can form an ionic pair and stabilize the CDR3α loop. Nevertheless, chimeras with a breakpoint in the middle of CDR3α (constructs JSα98 and 3BCα98), whether paired with cognate or the alternative β-chain, did not respond to the HA peptide presented by DR4 or DR7. Indeed, chimeras with as little as two amino acids of the “incorrect” CDR3α region (JSα96 and 3BCα96) were not able to recognize ligand. In contrast, chimeras with a breakpoint at position 94 still supported recognition; the TCR constructed from JSα94 and 3BCβ behaves like the 3BC receptor and a 3BCα94, JSβ TCR retained recognition of peptide in the context of DR7, although the promiscuous recognition in the context of other DRs, characteristic of the native JS receptor, was lost. These mutants differ from the native JS or 3BC α-chains at only two residues in CDR2α.

CDR3 loops are formally considered to be confined to the N-terminal side of the conserved FGXG sequence. Nevertheless, the positions in Jα and Jβ between FGXG and the constant region are highly variable in TCRs. The α-chains of the JS and 3BC receptors differ at five positions in this region. Sequences to the C-terminal side of the conserved F of the FGXG sequence could be exchanged between these receptors without loss of recognition (JSα104, 3BCβ104). Similar results were obtained with β-chain chimeras. Breakpoints in the middle of CDR3β, JSβ98, and 3BCβ98, did not allow recognition regardless of the α-chain they were paired with or the class II allele presenting the HA peptide. However, sequences to the C-terminal side of the conserved F residue in CDR3β could, again, be exchanged without loss of recognition (constructs JSβ105 and 3BCβ105). A previous study of the TCRβ peripheral repertoire in humans concluded that the identity of the “X” residue in the conserved FGXG sequence played a crucial role in thymic selection of T cells in the context of DR4 (28). Although the in vitro specificity of only two TCRs was examined here, no evidence of a role for this residue was observed in either DR4- or DR7-restricted recognition. Given the frequency with which site-specific amino acid substitutions in this region, and, indeed, in TCRs in general, result in loss of recognition, the maintenance of function in the presence of these nonconservative substitutions argues against a significant role for residues in this position in TCR recognition.

The exchange of as few as the first two amino-terminal residues in CDR3α (3BCα96 or JSα96) in the TCRs studied led to a loss of recognition. Site-specific mutants were constructed to look more closely at requirements for recognition in this region. Substitutions at position 95 of the JS α-chain yielded a diverse array of outcomes when stimulated with the HA peptide in the context of DR4 or DR7. The JS receptor normally has G at position 95 of the α-chain. Change to either negatively charged residue reduced responsiveness; 95D did not allow recognition while 95E responded only at higher peptide concentrations. Even then, a response was only observed in the context of DR7; the promiscuous response of the wild-type receptor in the context of DR4 was lost. In contrast, the modest change of 95G to 95A created a more sensitive receptor in which the response to the “promiscuous” alleles was as sensitive as the response to DR7.

If the enhanced responsiveness observed with the JS TCRα 95G to A mutant is mediated by a specific interaction with a residue in the HA peptide, then it should be possible to augment or dampen this response by substitutions at TCR contact residues in the peptide sequence. However, of 22 variant peptides with substitutions at one of the five T cell contact residues in the HA peptide, none evoked a significantly weaker response from the 95A mutant. Some variant peptides (V310L, N313T) elicited strong responses in 95A-transfected cells while responses in 95G-transfected cells were undetectable.

The broader specificity observed with TCRs incorporating the 95A mutant is inconsistent with any simple one to one correspondence between the side chain at position 95 of the TCR α-chain and a side chain of either peptide or HLA DRβ. The 95G to A change appears to affect receptor affinity globally and allows variants at many positions on the compound ligand of DR and peptide to be recognized. One possible explanation for this observation is that the 95A mutation facilitates a TCR conformation in which the threshold for activation is significantly lower than in the wild-type JS receptor and allows for more flexibility in the acceptable amino acids at TCR contact positions in the peptide or HLA presenting molecule.

A study of the energy of binding for peptide/MHC recognition by single-chain TCRs suggests that CDRs 1 and 2 contribute significantly more energy to binding than CDR3 (29). We have previously identified substitutions in CDR2α of the JS TCR that depress the promiscuous response of this TCR to HA peptide presented by DR4, while in this current study we have identified a mutation in CDR3α (95A) that enhances this response. When both of these mutations were paired together in the same TCR α-chain, the response of this double mutant to the HA peptide presented by either DR7 or DR4 was significantly more sensitive than that for the CDR2α single mutant. These results suggest that, at least in the context of this particular TCR, interactions involving CDR3 predominate in determining specificity.

In previous studies of TCR/MHC/peptide interactions by systematic mutagenesis of CDR residues, most mutations resulted in nonresponsive TCRs. Where success has been achieved in identifying contact residues in CDR3, it has been through an elegant genetic approach in which single-chain TCR transgenic mice are challenged with peptides that have substitutions at putative TCR contact residues. The powerful selection afforded by this approach allows the isolation and identification of TCRs with “compensating” amino acid substitutions in CDR3 and hence the definition of one to one side-chain interactions between specific residues in peptide and TCR. If such localized interactions are a common feature of TCR recognition, then it should be possible to identify analogous examples by the reverse approach of mutagenizing CDR3 residues and testing for compensation either with variant peptides or by presentation on different allelic DR molecules. Further, it might be expected that CDRs would tolerate at least conservative amino acid substitutions at some positions without loss of responsiveness. However, our results, and those from previous TCR mutagenesis studies (4, 5, 9, 13), indicate that TCR recognition is exquisitely sensitive to even modest substitutions in CDR3 and suggest caution in correlating CDR sequences with similarity in recognition in the absence of a known specificity for a TCR. Many CDR3 substitutions may have significant effects that are not localized to interaction with a specific side chain on a specific ligand. These effects could include alterations in the shape of the CDR3 loop or abrogation of the interaction with the cognate CDR3 of the other TCR chain. Where we have been able to identify substitutions that retain some responsiveness, as in the 95A mutation described in this study or previous CDR2α mutations (16), the nature of the substituted amino acids does not suggest obvious side-chain interactions, and the effect of the substitution appears to be global, altering interactions with multiple DR alleles and/or multiple peptide variants. Thus, while the general orientation of the interaction between TCRs and their peptide/MHC ligands appears to be standard, the specific details of side-chain interactions involving TCR CDR3 residues may not be generalizable. The ability of a single CDR3 substitution, such as the JSα 95A variant, to govern a broad specificity phenotype has implications for the selection and maintenance of specific T cells capable of promiscuous and alloreactive recognition.

FIGURE 3.

Dose-response curves for α- and β-chain constructs. TCR α-chain mutants with a native β-chain (A–B) or TCR β-chain mutants with a native α-chain (C) were transfected into JRT3 cells and assayed for response to the HA307–319 peptide presented by DR4 (Priess) or DR7 (DBB) cells. The dose-response curves are shown only for those constructs that gave positive results (as illustrated in Fig. 2). Values presented are means of triplicates without subtraction of background values obtained with no peptide.

FIGURE 3.

Dose-response curves for α- and β-chain constructs. TCR α-chain mutants with a native β-chain (A–B) or TCR β-chain mutants with a native α-chain (C) were transfected into JRT3 cells and assayed for response to the HA307–319 peptide presented by DR4 (Priess) or DR7 (DBB) cells. The dose-response curves are shown only for those constructs that gave positive results (as illustrated in Fig. 2). Values presented are means of triplicates without subtraction of background values obtained with no peptide.

Close modal

We thank K. Snoke (Epimmune Corporation) for the gift of variant peptides and G. Nepom for careful review of the manuscript.

1

This work was supported by Grant AI39636 from the National Institutes of Health.

3

Abbreviations used in this paper: CDR, complementarity-determining region; HA, hemagglutinin; JS, JS515.11; 3BC, 3BC6.6; JRT3, J.RT3-T3.5; B-LCL, B lymphoblastoid cell line.

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