The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR α- and β-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vβ8.2 chains, but differ in all three CDR of their related Vα2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2α, and an antagonist residue at the C terminus, which interacts with the CDR3β, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR α- and β-chains.

CD4 T cell responses are generated following engagement of the TCR with a foreign peptide bound in the groove of an appropriate MHC class II molecule. These bound peptides vary in amino acid length and can contain from 8 to more than 20 residues (1, 2), although crystal structures of both the I-Ak and I-Ek show that only 9 residues are involved in direct contacts with the murine MHC class II molecule or the TCR (3, 4). The peptide sits in the MHC groove in a proline twist orientation resulting in five residues that are pointed up and out toward the TCR (3, 4). A thorough mapping of the TCR contacts of the D10 T cell clone restricted to the hen egg conalbumin (CA)3 peptide from residues 134–146 (HRGAIEWEGIESG) in the context of I-Ak in our laboratory revealed molecular interactions between the second amino acid of the CA134–146 peptide (R) and the TCR α-chain complementarity-determining region 2 (CDR2) (5). The fifth residue (I) was shown to interact with both the TCR α- and the TCR β-chains via their CDR3 (5). In addition, the C-terminal E in the eighth position of the CA peptide was shown to interact with the TCR β CDR3 (5). The crystal structure exhibiting interactions between two different human TCRs and HLA-A2 was recently determined and showed that the TCR binds the MHC class I molecule in a diagonal pattern (6) consistent with the motif predicted by Sant’Angelo and colleagues (5) for MHC class II molecules. However, the crystal structure containing a complex of the D10 TCR interacting with the MHC class II molecule I-Ak bound with cognate Ag showed that the D10 TCR crossed the peptide in an orthogonal manner (7). The D10 TCR:I-Ak/CA complex crystal structure demonstrates interactions between the CA peptide residue 2 and the D10 TCR CDR1α and CDR3α domains, the CA peptide residue 5 with the D10 TCR CDR3α domain, and the CA peptide residue 8 with the D10 TCR CDR3α and CDR3β domains, slightly modified from the interaction we previously predicted (5, 7).

Single amino acid changes in the peptide TCR contacts have been shown to alter the nature of the biochemical signal received through the TCR, changing the response from an agonist to a nonagonist, partial agonist, or antagonist T cell response (8, 9, 10). Each type of signal has a unique biochemical signature pattern with agonist peptides eliciting a signal characterized by TCR ζ-chain tyrosine phosphorylation to the p21 and p23 forms of equal intensity (11, 12, 13, 14, 15). In addition, the tyrosine kinase Zap-70 is recruited to the TCR complex and is activated following tyrosine phosphorylation (16). Partial agonist peptides have been described that alter the nature of the biochemical signal that leads to T cell activation and cytokine production, but no progression through the cell cycle (17, 18). Antagonist peptides lead to a reduction in cellular responses associated with T cell activation and are characterized biochemically by an alteration in the ratio of phospho-ζ p21:p23 and a lack of phosphorylation of the recruited Zap-70 (8, 10, 11, 12, 19, 20) possibly by rapid recruitment of the tyrosine phosphate Src homology protein 1 (SHP-1) to the molecular complex involved in T cell Ag recognition (15). Nonagonist peptides do not elicit any cellular responses or biochemical signals (15).

In this study, we use two well-characterized T cell clones, D10 and AK8, that were obtained in a similar fashion from an H-2k mouse immunized with the Ag CA, and recognizing the same I-Ak-binding peptide CA134–146. The D10 and AK8 T cell clones have an identical Vβ8.2 chain and two similar Vα2 chains, with amino acid differences in all three CDRs. The sequence differences in the Vα2 chain alter how the two TCRs interact with and respond to CA134–146 analogue peptides containing single amino acid changes in the TCR contact residues. The D10 TCR is antagonized with peptides containing substitutions of the E at position 8 (20), while AK8 is antagonized by peptides with substitutions of the R at position 2 (21). Even though the TCR β-chains are identical, AK8 is not antagonized by position 8 analogue peptides, demonstrating a role for both TCR chains in determining the nature of the TCR signal. We also identified analogue peptides at positions 2, 5, and 8 that resulted in differential agonist responses when D10 and AK8 were compared. In addition, using a peptide with two different substitutions, the first at position 2 that enhances D10 responsiveness and the other at position 8 that mediates D10 antagonism, we show that the overall nature of the D10 response is a combination of the two responses. Thus, the TCR α- and β-chains contribute similarly and function cooperatively in mediating T cell signaling leading to cellular responses.

Mice were either purchased from The Jackson Laboratory (Bar Harbor, ME) or reared in our colony at Yale University (New Haven, CT). All mice were between 5 and 12 wk of age upon use.

The CA134–146 synthetic peptide (HRGAIEWEGIESG) coding for residues 134–146 of CA and the CA134–146 analogue peptides R2H, R2G, I5A, I5G, I5N, I5L, I5M, I5V, W7L, W7M, E8A, E8D, E8N, R2HE8A, and R2GE8A were synthesized and HPLC purified by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. CA134–146 synthetic peptides with substitutions of all 20 aa at positions R2, G3, I5, W7, and E8 were synthesized using the multipin cleavable peptide synthesis kit from Chiron Mimotopes Peptide Systems (San Diego, CA) using GAP linker pins, as previously described (20). The substituted peptides were screened for stimulatory activity and production of IL-4 by incubating 5 or 10 μl of the peptide mixture with D10 or AK8 cells and APC, as described below.

The D10.G4.1 (D10)- and AK8-cloned Th2 lines were developed in this laboratory and have been previously described (21, 22). D10 and AK8 were maintained by restimulation in Click’s Eagle’s-Hanks’ amino acids medium containing 5% FCS (Click’s 5%) and 100 μg/ml CA (Sigma, St. Louis, MO) in the presence of inactivated spleen cells every 2–4 wk.

D10 and AK8 were stimulated as above and allowed to rest for at least 9 days before use. Cells were washed four times, and 1 × 104 cells were cocultured with B10.BR splenocytes (I-Ak) as APC in the presence of the CA134–146 Chiron peptide library diluted 1/10 or 1/20. For proliferation assays conducted with HPLC-purified peptides, the peptides were diluted 1/10 from 0.001 to 10 μM. For the antagonist assay, the spleen cells were prepulsed in Click’s 0% containing various doses of CA134–146 for 2 h at 37°C. The APC were washed four times, and 2 × 105 cells were added to each well. Chiron peptides were added at a 1/10 or 1/20 dilution, and the HPLC-purified peptides were diluted 1/3 from 1.2 to 100 μM. Cultures were pulsed after 48 h with 1 μCi [3H]TdR and harvested after 15–18 h.

Secretion of IL-4 by D10 and AK8 was detected in culture supernatants collected 24 h following stimulation of D10 and AK8 with CA134–146 analogue peptides presented by B10.BR spleen cells and assayed by ELISA for the presence of IL-4 using anti-IL-4 mAb purchased from PharMingen (San Diego, CA), as previously described (20).

We have previously described and extensively studied the cellular responses of the CD4 Th2 D10 clone after TCR ligation (22). The D10 clone bears a TCR consisting of AV2S5 and BV8S2 restricted to I-Ak specific for a peptide of CA encompassing residues 134–146 (CA134–146) (HRGAIEWEGIESG) (23). In addition, we have previously described the AK8 Th2 clone with the same MHC restriction and peptide specificity as D10 (21). AK8 and D10 share an identical Vβ-chain, but differ in the Vα2 gene segment use that alters CDR1 and CDR2, as well as differing markedly in the CDR3, resulting in several changes in specificity. D10 is alloreactive to a variety of MHC class II molecules (22, 24, 25), but AK8 has no alloreactivity (26). In addition, the position of the single amino acid change rendering the CA134–146 wild-type (wt) peptide antagonist differs for the D10 and AK8 clones (20, 21).

We have previously described an orientation of the D10 TCR to the peptide/MHC complex demonstrating critical molecular contacts for TCR function between the TCR CDRα and β domains and amino acid residues at positions R2, I5, and E8 of the CA134–146 peptide (5). This identification of the important TCR contact residues in CA134–146 led us to further investigate the cellular responses invoked by CA134–146 analogue peptides containing amino acid substitutions in the TCR contacts to more accurately define the TCR:peptide:MHC interaction. Specifically, we were interested in identifying and comparing TCR contact residues that were critical for mediating cellular responses leading to cell proliferation and cytokine secretion in D10 and AK8. To accomplish this goal, we generated a peptide library containing CA134–146 analogue peptides with substitutions at the TCR contact residues: R at position 2 (R2), G at position 3 (G3), I at position 5 (I5), W at position 7 (W7), and E at position 8 (E8) with all 20 aa (Fig. 1). We then used D10 and AK8 to screen our peptide library for the ability of individual analogue peptides to induce cell proliferation and IL-4 secretion (Fig. 1). When positions R2 and G3 were substituted with all 20 aa, the resulting 40 peptides retained the ability to stimulate D10 (Fig. 1, A and B). Similarly, when the D10 culture supernatants were examined 24 h after stimulation with R2- and G3-substituted peptides for the production of IL-4 by ELISA, measurable amounts of IL-4 were detected with all 40 peptides (Fig. 1, F and G). Although the level of cell proliferation was similar for all 40 R2- and G3-substituted peptides, the level of IL-4 production varied a great deal. Thus, these data indicate that the production of IL-4 by D10 is a more sensitive means to identify alterations in biological responses invoked by a stimulating peptide than is cell proliferation. These data are in accordance with our previous finding and with a recent study demonstrating the importance of position R2 contacts with the TCR for TCR functionality (5, 7), and although differences in the level of IL-4 secretion were detected in the supernatants of D10 stimulated with R2 and G3 analogue peptides, these two residues do not appear to be critical for cellular proliferation mediated through the D10 TCR. The disparity between proliferation and IL-4 production, however, suggests that some peptides deliver a greater or lesser signal via the TCR (R2G, G3Y; Fig. 1, F and G).

FIGURE 1.

Proliferation and IL-4 secretion detected in D10 and AK8 responding to a CA134–146 peptide library containing substitutions in the TCR contacts. D10 (A–J) or AK8 (K–T) (1 × 104 cells) was cocultured with 1 × 106 inactivated B10.BR splenocytes as APC in the presence of a 1/10 dilution of the Chiron peptide library containing all 20 aa at positions R2 (A, F, K, P), G3 (B, G, L, Q), I5 (C, H, M, R), W7 (D, I, N, S), and E8 (E, J, O, T). Proliferation was detected by the addition of 1 μCi [3H]TdR after 48 h culturing for an additional 15–18 h and depicted as cpm (A–E, K–O). For the detection of IL-4 production, one-half the culture supernatant was removed after 24 h and used in an ELISA to detect IL-4, shown as pg/ml (F–J, P–T). Background cpm in the absence of added peptide are as follows: A, B, and D, 1916 cpm; C and E, 114 cpm; K, L, and N, 117 cpm; and M and O, 99 cpm. Each assay was performed at least twice as single data points.

FIGURE 1.

Proliferation and IL-4 secretion detected in D10 and AK8 responding to a CA134–146 peptide library containing substitutions in the TCR contacts. D10 (A–J) or AK8 (K–T) (1 × 104 cells) was cocultured with 1 × 106 inactivated B10.BR splenocytes as APC in the presence of a 1/10 dilution of the Chiron peptide library containing all 20 aa at positions R2 (A, F, K, P), G3 (B, G, L, Q), I5 (C, H, M, R), W7 (D, I, N, S), and E8 (E, J, O, T). Proliferation was detected by the addition of 1 μCi [3H]TdR after 48 h culturing for an additional 15–18 h and depicted as cpm (A–E, K–O). For the detection of IL-4 production, one-half the culture supernatant was removed after 24 h and used in an ELISA to detect IL-4, shown as pg/ml (F–J, P–T). Background cpm in the absence of added peptide are as follows: A, B, and D, 1916 cpm; C and E, 114 cpm; K, L, and N, 117 cpm; and M and O, 99 cpm. Each assay was performed at least twice as single data points.

Close modal

In comparison, AK8 is very sensitive to amino acid substitutions at R2, as shown by the lack of substantial cell proliferation and IL-4 production to all R2 substitutions except M and C (Fig. 1, K and P). Like D10, substitutions at G3 had little effect on AK8 with only two substitutions, L and E, resulting in decreased proliferation and IL-4 secretion and P resulting in a loss of effector function (Fig. 1, L and Q). Thus, these data show that AK8 cellular responses are dependent upon contacts between the TCR and position R2 of CA134–146, with G3 contacts with either TCR being of minimal importance. The lack of effect with G3-substituted peptides is consistent with the D10 TCR:CA/I-Ak complex crystal structure that shows the G3 residue buried with no exposure to the TCR or MHC (7).

Substitutions at position I5 with A, S, T, Q, K, M, and P led to a complete loss of proliferation by D10 (>98%), while substitutions with G, E, and F led to partial loss in the proliferation of D10 (90–98%) (Fig. 1,C). Similarly, residues G, E, Q, K, F, and P led to a complete loss in IL-4 production (>98%), while residues S, T, H, R, M, and Y led to a partial loss of IL-4 production (90–98%) (Fig. 1,H). However, there was not a direct correlation in the loss of cell proliferation and reduction in IL-4 production. For example, the residues R, L, and C resulted in >86% reduction in secreted IL-4, while maintaining cell proliferation at the same level as the wt peptide I5I (Fig. 1, C and H). This indicates that proliferation of the D10 T cell clone is very sensitive to IL-4. AK8 cellular responses were very sensitive to I5 substitutions with all substitutions except V, L, T, H, M, F, and Y, reducing cell proliferation by >90% (Fig. 1,M). Likewise, only substitutions V, L, and M led to IL-4 secretion that was >90% of that to the control I5I peptide (Fig. 1,R). The loss of both cell proliferation and IL-4 production by I5 substitutions confirms the major role of this amino acid in mediating both D10 and AK8 cellular responses (5). Because the interaction of the D10 TCR with the CA/I-Ak complex is thought to center on position I5 (7), our findings demonstrate both common and disparate effects of I5 substitutions. Strong responses of D10 to the I5L and I5N substitutions are mimicked by AK8 only in the strong response to I5L, but not I5N, while AK8 responds strongly to I5V and I5M, which do not stimulate D10 (Fig. 1, H and R). This is most likely due to differences in CDR3α residues GSFNKLTFGAG in D10 and PNTDVVT in AK8.

The substitution of amino acids at position W7 had a more dramatic effect on responsiveness of D10 than the I5 substitutions. All substitutions except V, S, F, and Y decreased D10 cell proliferation by more than 98% (Fig. 1,D). When the loss of IL-4 production was examined, all substitutions except S, F, and Y resulted in >80% reduction in the production of measurable IL-4 by D10 (Fig. 1,I). Similarly, when AK8 cell proliferation and IL-4 production were measured, all substitutions except S and Y resulted in a >90% reduction (Fig. 1, N and S). A study by Reinherz et al. (7) showed the W7 residue, which has a bulky indole ring, partially exposed to the TCR-binding surface and interacting with the TCR CDR3α domain residue Phe101, which also contacts the I5 residue. Thus, it is conceivable that amino acid substitutions in the W7 residue could alter TCR CDR3α interactions with residue I5, altering recognition due to the pivotal role of I5 in TCR recognition (7). Although substitutions at position 7 were not tested with D10 single chain transgenic mice, it appears from these data that the shared β-chains may account for this similarity in response of D10 and AK8 to the set of position 7 substitutions.

Finally, the E8 substitutions G, A, V, S, T, K, C, M, F, and Y resulted in a >98% loss in D10 proliferation, and the I substitution resulted in a >95% loss (Fig. 1,E). When IL-4 secretion was examined, all substitutions except L, D, N, and H resulted in a >90% reduction in the level of IL-4 detected (Fig. 1,J). AK8 cell proliferation was reduced by >95% with the G, A, V, L, I, S, T, H, K, C, M, F, Y, W, and P E8 substitutions (Fig. 1,O), and all E8 substitutions resulted in a >95% loss in IL-4 secretion (Fig. 1 T).

Because the multipin cleavable peptide synthesis kit from Chiron Mimotopes Peptide Systems generated crude preparations of peptides that did not allow for control of peptide quality or concentration, a direct comparison of the level of stimulatory activity and peptide concentration was not possible for any given peptide or between peptides. Thus, the peptides were titrated over a 100-fold range to confirm their potency (data not shown). The data shown in Fig. 1 are representative of the titrations performed.

The lack of D10 cell proliferation and subsequent IL-4 production induced by peptides with substitutions at positions I5, W7, and E8, all of which contact the CDR3 in the D10 α-chain (7), demonstrates that critical contacts leading to D10 activation are mediated through the α-chain contacting the C-terminal half of the CA134–146 peptide. In addition, contacts between residue E8 and the CDR3 of the D10 β-chain were found to be equally important for effector functions, demonstrating a critical role for the TCR β-chain in D10 TCR signaling as well. The importance of the C-terminal residues was further reinforced by the effects of substitutions at position R2, which had little impact on D10 cellular responses. In comparison, like D10, positions I5, W7, and E8 were important for AK8 cellular responses. One major difference between D10 and AK8 was the importance of position R2 in AK8 responsiveness. As previously discussed, the AK8 α- and D10 α-chains have amino acid variability in all three CDRs. These differences render the contacts between the CDRα domains with the CA134–146 peptide essential for AK8 TCR signaling.

To further confirm the unique specificities of the D10 and AK8 TCR, we chose those peptides examined in Fig. 1 that exhibited opposing cellular responses in the two clones for further study, and had them resynthesized and HPLC purified. This allowed for a direct comparison of the stimulatory activity of the individual peptide analogues based on known protein concentrations. Using IL-4 as the more sensitive readout of T cell activation, we chose to examine the peptides R2H, R2G, E8D, E8N, and I5N for the ability to stimulate D10, but not AK8 activation, and peptides I5L, I5M, and I5V for the ability to stimulate AK8, but not D10 activation. Shown in Fig. 2,A is a comparison of the proliferative response of D10 and AK8 to the CA134–146 wt peptide, with both T cell clones responding to 0.1 μM peptide with a similar stimulation profile. Interestingly, while the D10 antagonist E8A (20) is a nonagonist for AK8 (Fig. 1,O), the AK8 antagonist peptide R2H (21) induced a proliferative response in D10 similar to the CA134–146 peptide (Fig. 2,B). Thus, the sequence differences in the CDRα domains of AK8 and D10 determine how the N-terminal residues of the peptide are recognized. Further confirmation of this is the proliferative response to the R2G peptide. AK8 is nonresponsive to R2G, while this peptide is a superagonist for the D10 TCR. D10 responds to the R2G peptide at a concentration of 0.01 μM, 10-fold less peptide than required for the response observed with the CA134–146 peptide (Fig. 2, A and C). In addition to position 2-substituted peptides, differential recognition of peptides by D10, but not AK8, is also observed with the position 8-substituted peptides E8D (Fig. 2,D) and E8N (Fig. 2,E) and the position 5-substituted peptide I5N (Fig. 2,F). By comparison, the only peptide we could identify that resulted in AK8 responsiveness, but not D10 responsiveness, was the I5M peptide (Fig. 2,H). In addition, we identified the I5L and I5V peptides, which are weak D10 agonists requiring a peptide dose of 1–10 μM to observe a proliferative response, while proliferation of AK8 was observed at 0.1 μM (Fig. 2, G and I). Further analysis of the I5V peptide showed that the peptide is a partial agonist based on tyrosine-phosphorylation patterns following engagement of the D10 TCR with I5V (our unpublished observations). We confirmed this by performing semiquantitative RT-PCR to identify IL-4 transcripts following engagement with I5V (our unpublished observations). Thus, the high level of D10 proliferation observed in Fig. 1,C, not observed in Fig. 2,I, is most likely due to a low level of IL-4 production induced by a high level of peptide concentration that could not be measured due to the nature of the crude peptide preparation used in Fig. 1.

FIGURE 2.

Differential recognition of CA134–146-substituted peptides by D10 and AK8. D10 (•) or AK8 (○) was cocultured with B10.BR splenocytes in the presence of 1/10 dilutions of CA134–146 (A), R2H (B), R2G (C), E8D (D), E8N (E), I5N (F), I5L (G), I5M (H), or I5V (I) from 0.001 to 10 μM. Proliferation was detected as described for Fig. 1. The data presented for D10 and AK8 were performed in the same experiment in duplicate at least three times.

FIGURE 2.

Differential recognition of CA134–146-substituted peptides by D10 and AK8. D10 (•) or AK8 (○) was cocultured with B10.BR splenocytes in the presence of 1/10 dilutions of CA134–146 (A), R2H (B), R2G (C), E8D (D), E8N (E), I5N (F), I5L (G), I5M (H), or I5V (I) from 0.001 to 10 μM. Proliferation was detected as described for Fig. 1. The data presented for D10 and AK8 were performed in the same experiment in duplicate at least three times.

Close modal

To determine which TCR contact residue(s) of CA134–146 could mediate TCR antagonism of D10 and AK8, we screened our peptide library for the ability to inhibit D10 and AK8 cell proliferation. We have previously demonstrated antagonism of AK8 cell proliferation with the position 2-substituted peptide R2H (21). Thus, to confirm the specificity of our peptide library, we screened the R2-substituted peptides for the ability to inhibit AK8 cell proliferation in an antagonist assay. As shown in Fig. 3,E, the R2 substitutions G, A, S, Q, and H inhibited AK8 cell proliferation to prepulsed APC by 80, 85, 75, 80, and 78%, respectively. In comparison, D10 was not antagonized by any of the R2-substituted peptides (Fig. 3,A). The suppression observed with the G and H substitutions in Fig. 3,A most likely is not due to antagonism, as indicated by the robust IL-4 production shown in Fig. 1,F, but rather may be explained by the presence of peptide in greater concentration than the concentration required for a maximal proliferative response, which is known to be inhibitory. No antagonism was observed for either D10 or AK8, with G3-substituted peptides as predicted from the proliferative responses to these peptides shown in Fig. 1 (data not shown). The examination of I5 substitutions showed no D10 antagonism (Fig. 3,B) and only peptide I5A-antagonized AK8, inhibiting proliferation by 77% (Fig. 3,F). Similar results were observed with the W7 substitutions with no antagonism of D10 (Fig. 3,C) and antagonism of AK8 with only the W7L peptide leading to a 75% inhibition of proliferation (Fig. 3,G). We have previously reported that the D10 TCR is antagonized with E8-substituted peptides (20), and this result is confirmed in Fig. 3,D, showing antagonism with the G, A, V, S, and T substitutions leading to inhibition of proliferation by 92, 93, 95, 92, and 89%, respectively. Because D10 and AK8 share an identical TCR β-chain, we reasoned that AK8 would also be antagonized by substitutions at position E8. However, the data shown in Fig. 3 H show no antagonism of AK8 with the E8-substituted peptides and demonstrate that the TCR β-chains of D10 and AK8 must interact differently, as the identical peptide:MHC molecule leads to distinct cellular responses.

FIGURE 3.

Antagonism of D10 and AK8 proliferative responses induced by CA134–146 and antagonized by a peptide library containing substitutions in the TCR contacts. B10.BR splenocytes (2 × 106 cells) were prepulsed with CA134–146 for 2 h before coculture with D10 (A–D) or AK8 (E–H) (2 × 104 cells) in the presence of the R2 (A, E)-, I5 (B, F)-, W7 (C, G)-, and E8 (D, H)-substituted peptides. Proliferation was detected as described for Fig. 1. The spleen cells were prepulsed with CA134–146 at 10 μg/ml for B and D; at 5 μg/ml for E, F, G, and H; and at 1 μg/ml for A and C. The peptides were diluted 1/10 for B and D; 1/20 for E, F, G, and H; and 1/100 for A and C. The prepulse level corresponds to proliferation of D10 or AK8 in the presence of the appropriate dilution of a mock peptide synthesis. Background cpm in the absence of added peptide are as follows: A and C, 5500 cpm; B and D, 446 cpm; E and G, 689 cpm; and F and H, 332 cpm. Each assay was performed at least twice as single data points.

FIGURE 3.

Antagonism of D10 and AK8 proliferative responses induced by CA134–146 and antagonized by a peptide library containing substitutions in the TCR contacts. B10.BR splenocytes (2 × 106 cells) were prepulsed with CA134–146 for 2 h before coculture with D10 (A–D) or AK8 (E–H) (2 × 104 cells) in the presence of the R2 (A, E)-, I5 (B, F)-, W7 (C, G)-, and E8 (D, H)-substituted peptides. Proliferation was detected as described for Fig. 1. The spleen cells were prepulsed with CA134–146 at 10 μg/ml for B and D; at 5 μg/ml for E, F, G, and H; and at 1 μg/ml for A and C. The peptides were diluted 1/10 for B and D; 1/20 for E, F, G, and H; and 1/100 for A and C. The prepulse level corresponds to proliferation of D10 or AK8 in the presence of the appropriate dilution of a mock peptide synthesis. Background cpm in the absence of added peptide are as follows: A and C, 5500 cpm; B and D, 446 cpm; E and G, 689 cpm; and F and H, 332 cpm. Each assay was performed at least twice as single data points.

Close modal

Candidate antagonist peptides identified in the studies shown in Fig. 3 were resynthesized, HPLC purified, and screened for their ability to inhibit proliferation of D10 and AK8. As shown for D10 in Fig. 4,A, only the position 8 substitutions resulted in antagonism of D10, with E8A resulting in complete antagonism. The E8-substituted peptides E8T, E8S, E8V, and E8G are also antagonist for D10 (20 and data not shown). Upon further examination, the I5G and I5M peptides were found to be weak agonists of D10 proliferation, thus not inhibiting proliferation in an antagonist assay (Fig. 4,A and data not shown). The W7L and W7M peptides were found to be nonagonist peptides failing to induce D10 proliferation or IL-4 production (Ref. 15 ; Fig. 2, D and I), and having no inhibitory effect in an antagonist assay (Fig. 4,A). Using R2H to confirm antagonist activity on AK8 (Fig. 4,B) (21), we found that the I5G, W7M, and E8A peptides, although nonagonist (Fig. 1, M, N, and O), did not demonstrate any antagonist activity (Fig. 4,B). In contrast, the peptides R2G, I5A, and W7L, which exhibited antagonism in Fig. 2, resulted in a dose-dependent inhibition of AK8 proliferation (Fig. 4 B). These data confirm our previously published findings describing antagonist peptides for D10 and AK8 and clearly demonstrate differences in recognition of peptides by D10 and AK8. Of particular interest is the antagonism of AK8 with position 2-, 5-, and 7-substituted peptides, while D10 is only antagonized with position 8 analogue peptides. Thus, the nature of the cellular response stimulated is influenced by interactions of both the TCR α- and β-chains with the peptide:MHC complex, as the presence of an identical TCR β-chain in the two T cell clones is not sufficient to render the signal delivered through the TCR identical. The α-chain sequence of the two distinct T cell clones makes a difference both in response to N-terminal substituted peptides that interact with the TCR α-chain and to C-terminal substituted peptides that interact with the TCR β-chain.

FIGURE 4.

Dose-dependent antagonism of D10 and AK8 with selected peptides from the TCR contact peptide library. B10.BR splenocytes (2 × 106 cells) were prepulsed with 0.5 μg/ml CA134–146 for 2 h before coculture with D10 (2 × 104 cells) in the presence of 1/3 dilutions of peptides I5G (•), I5M (○), W7L (▪), W7M (□), or E8A (▴) from 1.2 to 100 μM. For AK8, the 5 μg/ml CA134–146 was used for prepulsing, and peptides R2G (•), R2H (○), I5A (▪), I5G (□), W7L (▴), W7M (▵), and E8A (+) were used. Proliferation was detected as described for Fig. 1 in duplicate and performed at least three times.

FIGURE 4.

Dose-dependent antagonism of D10 and AK8 with selected peptides from the TCR contact peptide library. B10.BR splenocytes (2 × 106 cells) were prepulsed with 0.5 μg/ml CA134–146 for 2 h before coculture with D10 (2 × 104 cells) in the presence of 1/3 dilutions of peptides I5G (•), I5M (○), W7L (▪), W7M (□), or E8A (▴) from 1.2 to 100 μM. For AK8, the 5 μg/ml CA134–146 was used for prepulsing, and peptides R2G (•), R2H (○), I5A (▪), I5G (□), W7L (▴), W7M (▵), and E8A (+) were used. Proliferation was detected as described for Fig. 1 in duplicate and performed at least three times.

Close modal

To examine the contribution that the α- and β-chains make to the overall TCR signal received in D10, we generated peptides with two amino acid substitutions at positions 2 and 8. As previously shown in Fig. 2, the R2G peptide is a superagonist peptide requiring 10-fold less peptide to induce the same response as the CA134–146 wt peptide (Fig. 5) (23). In contrast, the R2H peptide requires ∼10-fold more peptide than CA134–146 to generate a similar response (Fig. 5). Because the E8A antagonist peptide does not induce cell proliferation, we generated peptides with the E8A substitution and the R2G or R2H substitution to determine whether the nonstimulatory E8A residue would affect the level of D10 activation observed with R2G or R2H alone. The dual substituted peptide R2GE8A resulted in a proliferative response that required 100-fold more peptide than the R2G peptide to induce the same level of proliferation (Fig. 5). Similarly, the R2H residue in combination with E8A rendered the peptide a nonagonist, leading to no D10 proliferation (Fig. 5). These data demonstrate that the overall strength of the signal through the D10 TCR is contributed to by contacts between peptide:MHC complexes with both the TCR α-chain CDRs and the TCR β-chain CDR3 domain. It also shows that the nature of the signal received through both chains determines the overall quality of signal leading to agonist, nonagonist, superagonist, or antagonist responses.

FIGURE 5.

D10 proliferation induced by dual substituted peptides with a highly stimulatory and a nonstimulatory substitution. D10 was cocultured with B10.BR splenocytes in the presence of 1/10 dilutions of CA134–146 (•), R2G (▪), R2H (▴), E8A (○), R2HE8A (□), and R2GE8A (▵) from 0.001 to 10 μM. Proliferation was detected as described for Fig. 1 in duplicate performed twice.

FIGURE 5.

D10 proliferation induced by dual substituted peptides with a highly stimulatory and a nonstimulatory substitution. D10 was cocultured with B10.BR splenocytes in the presence of 1/10 dilutions of CA134–146 (•), R2G (▪), R2H (▴), E8A (○), R2HE8A (□), and R2GE8A (▵) from 0.001 to 10 μM. Proliferation was detected as described for Fig. 1 in duplicate performed twice.

Close modal

Peptide-based therapies using analogue peptides containing amino acid substitutions that alter T cell responses from that observed with the original antigenic peptide have been shown to affect a T cell response and thereby prevent disease. Most notable is the prevention or reversal of the CD4 T cell-mediated disease experimental autoimmune encephalomyelitis (EAE), the rodent model for the human autoimmune demyelinating disease multiple sclerosis (27). Using cellular based assays, peptides with antagonist activity (28, 29, 30) and peptides with the capacity to alter cytokine profiles (30, 31, 32) were used successfully to prevent or alter the EAE disease course. Of particular interest are the reports by Kuchroo et al. (28) and Franco et al. (29), in which they describe prevention of proteolipid protein-induced EAE by either a single antagonist peptide or a mixture of two antagonist peptides, respectively, even though the T cell response is known to involve the usage of diverse TCR genes. Whether the antagonist peptides directly inhibit all responding T cells or indirectly inhibit T cell responses by altering the phenotype of the immune response is an important consideration, because our data suggest that at least for some Ags, even minimal TCR diversity could render therapeutic protocols with antagonist peptides useless.

We thank the W. M. Keck Foundation Biotechnology Resource Laboratory for peptide synthesis and Charles Annicelli III for assistance with mice.

1

Supported by National Institutes of Health Grant AI/AR 36529 and by the Howard Hughes Medical Institute.

3

Abbreviations used in this paper: CA, hen egg conalbumin; CDR, complementarity-determining region; EAE, experimental autoimmune encephalomyelitis; wt, wild type.

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