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The Journal of Immunology, 2000, 165: 280-285.
Copyright © 2000 by The American Association of Immunologists

Altered Peptide Ligand-Mediated TCR Antagonism Can Be Modulated by a Change in a Single Amino Acid Residue Within the CDR3ß of an MHC Class I-Restricted TCR1

Alexis M. Kalergis* and Stanley G. Nathenson2,*,{dagger}

Departments of * Microbiology and Immunology and {dagger} Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8–10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the ß-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3ß loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The {alpha}ß TCR recognizes Ags as short peptides bound to MHC molecules present on the surface of APCs (1, 2). The interaction between the TCR and MHC/peptide complex usually leads to the activation of the T cell, which involves its proliferation and cytokine release. In addition, for CD8+ CTL, the recognition of an antigenic MHC class I/peptide complex causes the lysis of the target cell through the release of granules containing perforin and proteases. This process is tightly regulated by the specificity of the TCR for its ligand, which is determined by interactions occurring between amino acid residues on the TCR and residues on the MHC class I/peptide surface. Recent data obtained from studies on TCR transgenic mice (3, 4, 5, 6), as well as crystallographic analyses of TCR/MHC/peptide complexes (7, 8, 9), have begun to reveal the molecular interactions occurring when the TCR engages its cognate ligand. These interactions involve residues from both the MHC and the peptide on the ligand side, and residues from the complementarity-determining regions (CDRs)3 1, 2, and 3 present on the {alpha} and ß variable domains of the TCR (10). While the diversity of the germline-encoded CDRs 1 and 2 is restricted by the number of V{alpha} and Vß genes, the CDR3s can be much more diverse because they are generated by the genetic rearrangement of V{alpha}/J{alpha} and Vß/Dß/Jß genes, occurring during T cell development (1).

Based on their ability to activate T cells, antigenic peptides can be grouped into different categories (11, 12). Peptides that lead to complete activation of T cells are called full TCR agonists. Peptide variants derived from the full agonist that carry substitutions at TCR-contact residues are known as altered peptide ligands (APLs). APLs can either be ignored (null ligand), partially activate (partial agonists), or antagonize/inhibit (antagonists) the TCR signaling cascade. While neither partial agonists nor null peptides interfere with the signaling promoted by the full agonist, antagonist peptides are able to inhibit TCR activation induced by the agonist peptide. Biochemical studies have recently correlated the dissociation rate (koff) of the TCR-MHC/peptide interaction with the biological activity of APLs (13, 14, 15, 16, 17, 18). Thus, while partial agonists dissociate faster from the TCR than the full agonists, antagonist peptides have even faster dissociation rates than partial agonists. Null peptides show the fastest dissociation rates from the TCR. However, these findings are by no means an absolute rule, for a number of exceptions have been reported in which partial agonists or antagonist peptides show more stable interactions with the TCR than full agonists (19, 20).

The molecular mechanism involved in TCR antagonism is still an unresolved issue. It has been shown that antagonist peptides can reduce the activity of TCR-associated Lck and induce an altered phosphorylation of the CD3 {zeta}-chain (21, 22, 23, 24). As result, the recruitment and activation of ZAP-70 and LAT is abrogated, preventing the completion of the signal transduction cascade that leads to the formation of TCR clusters on the cell membrane required for T cell activation (21, 22, 25, 26). In addition to the alteration of TCR signaling, it has been proposed that, due to their faster dissociation rates, antagonist peptides can block TCR serial engagement by the agonist peptide (27, 28), resulting in the inhibition of the T cell activation.

The molecular interactions between the MHC/peptide and the TCR that are responsible for TCR antagonism have not been defined. In an early attempt to approach this issue, Ostrov et al. analyzed the antagonistic properties of a panel of APLs derived from the hemagglutinin 307–319 peptide on two DR5-restricted T cell clones (29). The authors observed that those T cell clones were differentially antagonized by one of the peptide variants tested and suggested this difference was due to the TCR CDR3{alpha} composition of the clones. However, these data did not conclusively identify the TCR residues involved in antagonism.

In the present work, we describe the identification and characterization of APLs derived from the VSV peptide that specifically antagonize an H-2Kb/VSV-specific TCR. By site-directed mutagenesis of the CDR3ß of this TCR, we were able to show an association between a single amino acid residue on the TCR CDR3ß and the antagonism caused by an APL. Thus, modification of that CDR3ß residue can abolish the antagonism caused by the APL. Our results provide evidence for an active role for the CDR3 loops in the specificity and biological outcome of the TCR/ligand interaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Peptide synthesis and purification

The wild-type vesicular stomatitis virus (VSV) peptide (RGYVYQGL) and peptide variants carrying substitutions at position 1 and/or position 6 were synthesized by solid-phase method using F-moc chemistry on an automated 433A peptide synthesizer (Applied Biosystems, Foster City, CA) at the Peptide Synthesis Facility of the Albert Einstein College of Medicine. All peptides were purified to >98% homogeneity by reversed-phase HPLC on a Vydac C-18 column (2.1 or 4.6 mm x 25 cm, 300 Å) using HP-1090 M HPLC (Hewlett Packard, Palo Alto, CA). The identity of the purified peptides was determined by a tandem quadrupole mass spectrometer (TSQ700, Finnigan MAT, San Jose, CA).

Activation of T hybridomas by VSV peptide

The preparation of T cell hybridomas expressing the wild-type N30.7 TCR has been previously described (30). Briefly, stable transfectants were made by introducing DNA encoding for the N30.7 TCR {alpha}- and ß-chains into the TCR-deficient 58{alpha}-ß- cell line. The site-directed mutagenesis of the N30.7 CDR3ß leading to the S96A, S96T, G97A, and E101A TCR mutants was done by cassette mutagenesis as described by Goyarts et al. (30). The T cell hybridoma expressing the N15 TCR was kindly provided Dr. H.-C. Chang (31).

For the Ag activation assays, 1 x 105 R8 cells (H-2Kb/d) pulsed with various concentrations of VSV peptide were incubated with the same number of T cell hybridomas for 20 h at 37°C. N30.7 and the CDR3ß mutants S96A, S96T, G97A, and E101A, as well as the N15 hybridomas, secreted IL-2 in response to Ag stimulation. The amount of IL-2 secreted was determined by cytokine ELISA as previously described (30).

For activation through CD3{epsilon} cross-linking, T cell hybridomas (2 x 105/well) were added to anti-CD3{epsilon}-coated plates (0.25 µg/well). For PMA/Ca2+ ionophore activation, T cell hybridomas were incubated in presence of PMA (100 ng/ml; Life Technologies, Grand Island, NY) and A23187 (200 ng/ml; Sigma, St. Louis, MO). After 20 h incubation, IL-2 release was measured by cytokine ELISA (30).

In vitro antagonism assays

TCR antagonism was determined by performing Ag prepulse assays (32, 33, 34). R8 cells (H-2Kb/d) were prepulsed during 2 h with a suboptimal amount of VSV peptide (10 nM). After this time, the cells were washed to remove any unbound VSV peptide, and the antagonist peptides were added at various concentrations. After 2 h, the R8 cells were washed and plated at 1 x 105 cells/well in 96-well plates. The same number of T hybridoma cells were added to the wells. The amount of IL-2 released was measured after 20 h as described previously (30).

H-2Kb up-regulation assays

RMA/s cells were incubated overnight at 31°C to promote accumulation of surface H-2Kb molecules. These RMA/s cells (2 x 105) were incubated with VSV or VSV peptide variants (100 µM) for 2 h at room temperature. After this time, cells were transferred to 37°C for 5 h and stained with 0.25 µg of FITC-conjugated anti-H-2Kb mAb (AF6-88.5; PharMingen, San Diego, CA).

Flow cytometry analyses

T cell hybridomas were routinely screened for CD8{alpha} and TCR expression by flow cytometry using anti-CD8{alpha} (53-6.7) and anti-TCRß (H57) Abs (PharMingen), respectively, and analyzed by FACScan (Becton Dickinson, Mountain View, CA) as previously described (30).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Position 6 of VSV peptide is critical for recognition by N30.7 TCR

T cell hybridomas expressing the wild-type N30.7 TCR were tested for recognition of VSV peptide variants carrying single amino acid substitutions either at position 1 or position 6. Amino acid side chains at position 1 and 6 of VSV peptide are solvent exposed and make contact with H-2Kb/VSV-specific TCRs (35, 36, 37). As shown in Fig. 1Go, most of the position 1 variants were recognized by the N30.7 TCR with similar efficiency as the wild-type VSV peptide. The E1 peptide variant was the only one that was not recognized by N30.7. This peptide carries a nonconservative substitution in which arginine at position 1 was changed to glutamic acid (Fig. 2Go).



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  		FIGURE 1. Position 6 of the VSV peptide is critical for recognition by the N30.7 TCR. Hybridomas expressing the N30.7 TCR (1 x 105) were incubated with R8 cells (H-2Kb/d, 1 x 105) pulsed with VSV peptide or the indicated peptide variant (1 µM). After 20 h, supernatants were collected and IL-2 was determined by ELISA.

 


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  		FIGURE 2. Sequence of VSV and peptide variants. For peptide variants, only residues different from those of the VSV peptide are shown. The figure shows the TCR contacting residues of the VSV peptide. The biological activity of each peptide on hybridomas expressing the N30.7 TCR is indicated (NR, not recognized by N30.7 but not included in antagonism assays).

 
In contrast, none of the position 6 VSV peptide variants tested was recognized by N30.7 TCR (Figs. 1Go and 2Go). This observation would be consistent with a more important role for the TCR-exposed residue at position 6 of the VSV peptide for the specificity of N30.7 recognition, as compared with position 1 that can be widely modified without altering recognition by this specific TCR.

VSV-derived altered peptide variants E6 (Q->E mutation at position 6) and E1 (R->E mutation at position 1) are weak antagonists for N30.7 TCR

The observation that position 6 of VSV was critical for activation of N30.7 TCR led us to evaluate the ability of a panel of position 6 peptide variants to antagonize N30.7. As shown in Fig. 3GoA, most of the singly substituted VSV position 6 variants did not antagonize the recognition of wild-type VSV by N30.7 TCR (data summarized in Fig. 2Go). Only the E6 peptide variant, in which E replaced Q at position 6, showed some antagonistic activity (Fig. 3GoA). This peptide variant behaved as a weak antagonist as an ~1000-fold molar excess over the agonist peptide was required to achieve 50% inhibition of IL-2 release.



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  		FIGURE 3. I1E6 peptide variant is a strong antagonist, while E6, E1, and I1I6 peptide variants only weakly antagonize N30.7. The ability of position 6 singly substituted (A) or position 1 and 6 doubly substituted (B) VSV-derived peptide variants to antagonize the N30.7 TCR was evaluated by Ag prepulse assays. R8 cells (H-2Kb/d) were pulsed with a suboptimal concentration of VSV peptide (10 nM), washed to remove the excess of agonist peptide, and then incubated with different concentrations of the indicated peptide variants. The peptide-pulsed R8 cells were then mixed with N30.7 hybridomas and incubated for 20 h. IL-2 release was determined by ELISA. Data are expressed as percentage of inhibition of IL-2 release as compared with the amount of IL-2 induced by R8 cells pulsed only with 10 nM VSV peptide.

 
In addition, the E1 peptide variant, the only position 1 variant not recognized by N30.7 TCR, was also a weak antagonist for this TCR (Fig. 3GoA). The strength of the antagonism caused by the E1 peptide was comparable to the E6 peptide, as similar molar excess (1000-fold) over the agonist peptide was required to achieve 50% inhibition of IL-2 release.

R->I mutation at position 1 of E6 peptide improves its antagonistic activity by increasing binding efficiency to H-2Kb.

With the aim to improve the antagonistic activity of the E6 peptide, we made a second mutation, now at position 1 of the peptide, that could increase binding to the H-2Kb molecule. The R->I replacement at position 1 of VSV peptide resulted in an increase of the ability of the peptide to stabilize H-2Kb on the surface of RMA/s cells (Fig. 4Go). As shown in Fig. 4Go, whereas 100 µM VSV peptide causes 40% up-regulation of H-2Kb, the same concentration of I1 peptide causes 85% up-regulation. In contrast, the E6 peptide was even less efficient than the wild-type VSV peptide in stabilizing H-2Kb, as 100 µM E6 peptide caused only 17% MHC up-regulation on RMA/s cells. Thus, the Q->E mutation at position 6 of VSV seems to reduce its binding affinity for H-2Kb.



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  		FIGURE 4. Isoleucine at position 1 of the VSV peptide enhances binding to H-2Kb, and overcomes the loss of binding due to the Q->E replacement at position 6 of VSV peptide. RMA/s cells were incubated overnight at 31°C and then incubated with VSV or VSV peptide variants (100 µM) for 2 h at room temperature. After this time cells, were transferred to 37°C for 5 h and stained with 0.25 µg of FITC-conjugated anti-H-2Kb mAb (AF6-88.5; PharMingen). Data shown represent the mean of two independent experiments ± SD.

 
The introduction of the R->I replacement at position 1 in the E6 peptide (I1E6, Fig. 2Go) increased significantly the efficiency of H-2Kb stabilization, as incubation with 100 µM I1E6 peptide resulted in 58% MHC up-regulation by RMA/s cells (Fig. 4Go). Thus, it seems likely that the R->I replacement at position 1 is able to overcome the loss of H-2Kb-binding efficiency due to the Q->E mutation at position 6.

When this double-substituted VSV peptide variant, I1E6, was tested for antagonism on N30.7 TCR-expressing hybridomas, it was found to be a stronger antagonist than E6 peptide, as 50% inhibition of IL-2 release was obtained with <1 µM I1E6 peptide (Fig. 3GoB). The requirement for Q->E mutation at position 6 for efficient antagonism of N30.7 is underscored by the observation that the doubly substituted I1I6 peptide is only a weak antagonist (Fig. 3GoB), even though it stabilizes H-2Kb with higher efficiency than I1E6 (Fig. 4Go). Moreover, I1K6, another doubly substituted peptide variant, does not antagonize N30.7 TCR (Fig. 3GoB).

This experimental evidence would suggest that the E6 peptide is a weak antagonist for N30.7 due to its inefficient H-2Kb binding properties. However, it is remarkable that despite its low MHC binding efficiency, the E6 peptide is still able to antagonize N30.7 at similar levels as I1I6 (Fig. 3Go), a very strong MHC binding peptide (Fig. 4Go). This observation highlights the importance of position 6 of the VSV peptide for recognition by N30.7 and, more importantly, the specificity of the Q->E mutation for antagonizing the N30.7 TCR.

Antagonism mediated by I1E6 peptide is specific for N30.7 and not due to competition for H-2Kb.

The antagonistic properties of I1E6 were specific for N30.7, as another H-2Kb/VSV-specific TCR, N15, was not antagonized by this peptide variant (compare Fig. 5Go, A and B). This result is consistent with the observation that E6 peptide is not an antagonist for the N15 CTL clone, as recently described by Ghendler et al. (38)



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  		FIGURE 5. I1E6 peptide-induced antagonism is specific for the N30.7 TCR, as this peptide variant does not antagonize N15, an H-2Kb/VSV-specific TCR. The ability of I1E6 peptide variant to antagonize the N30.7 and N15 TCRs was evaluated by Ag prepulse assays. R8 cells (H-2Kb/d) were pulsed with a suboptimal concentration of VSV peptide (10 nM), washed to remove the excess of agonist peptide, and then incubated with different concentrations of I1E6 peptide (•). The peptide-pulsed R8 cells were then mixed either with N30.7 (A) on N15 (B) T cell hybridomas and incubated for 20 h. IL-2 release was determined by ELISA. Data are expressed as percentage of inhibition of IL-2 release as compared with the amount of IL-2 induced by R8 cells pulsed only with 10 nM VSV peptide. As H-2Kb-competition control, the Sendai virus nucleoprotein-derived peptide (FAPGNYPAL, {circ}) was also evaluated for antagonism of N30.7 and N15 TCR. Data shown represent the mean of two independent experiments ± SD.

 
In addition, I1E6 antagonism of the N30.7 TCR was unlikely to be due to MHC competition as an unrelated peptide derived from Sendai virus nucleoprotein (FAPGNYPAL) that binds to H-2Kb with similar efficiency as I1E6 (Fig. 4Go) was not able to antagonize N30.7 TCR (Fig. 5GoA).

A single amino acid replacement in the CDR3ß of N30.7 TCR can partially abolish antagonism by I1E6 peptide.

To test whether alterations in the CDR3 sequence of N30.7 TCR could influence the TCR antagonism mediated by the I1E6 peptide variant, point mutations were introduced at amino acid residues encompassing residues 96–102 of N30.7 TCR (30) (Fig. 6Go). The rationale for mutating the TCR CDR3ß of N30.7 was based on previous studies that have shown interactions between the CDR3ß and residues flanking the C terminus of the antigenic peptide bound to MHC class I (3, 5, 10, 39).



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  		FIGURE 6. CDR3ß sequences of wild-type N30.7 TCR (Vß13, Jß1.1) and CDR3ß mutants S96A, S96T, G97A, and E101A. The predicted N30.7 TCR wild-type CDR3ß sequence is enclosed in the box. Only the amino acid residue that was altered is indicated for each of the CDR3ß mutants.

 
As we have previously described, while mutations at position 98, 99, 100, and 102 of N30.7 CDR3ß completely abolished recognition of VSV peptide bound to H-2Kb, mutations at positions 96 (S->A), 96 (S->T), 97 (G->A), and 101 (E->A) of N30.7 CDR3ß retained recognition of H-2Kb/VSV (Figs. 6Go and 7GoA) (30). However, while the S96A, S96T, and E101A mutants recognized VSV with similar efficiency as wild-type N30.7, the G97A mutant showed an increased IL-2 release in response to VSV (Fig. 7GoA). Consistently, while the N30.7 wild-type TCR and CDR3ß mutants S96A, S96T, and E101A showed comparable surface expression levels for TCR and CD8, the G97A mutant showed equivalent TCR expression levels but an increased CD8 surface expression (Table IGo). This difference could contribute to the increased VSV peptide sensitivity shown by the G97A CDR3ß mutant. Furthermore, all the T cell hybridoma lines secreted IL-2 in response to CD3{epsilon} cross-linking or to PMA/Ca2+ ionophore (Table IGo), indicating that the TCR signaling machinery associated with the wild-type N30.7 TCR, as well with the mutant TCRs, was functional for each of the T cell lines tested.



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  		FIGURE 7. The E101A mutation on the CDR3ß of N30.7 partially abolishes antagonism by I1E6 peptide. A,. The wild-type N30.7 TCR ({circ}) as well as the CDR3ß point mutants S96A ({square}), S96T (•), G97A ({blacksquare}), and E101A (*) recognize R8 cells loaded with VSV peptide. R8 cells (1 x 105) pulsed with different concentrations of VSV peptide were mixed with an equal number of T hybridomas expressing either the N30.7 TCR or one of the CDR3ß mutants. After 20 h incubation, supernatants were collected and the amount of IL-2 released was determined by cytokine ELISA. The ability of I1E6 (B) or E1 (C) peptide variants to antagonize the N30.7 ({circ}) and the CDR3ß mutant TCRs S96A ({square}), S96T (•), G97A ({blacksquare}), and E101A (*) was evaluated by Ag prepulse assays. R8 cells (H-2Kb/d) were pulsed with a suboptimal concentration of VSV peptide (10 nM), washed to remove the excess of agonist peptide, and then incubated with different concentrations of I1E6 (B) or E1 (C) peptide. The peptide-pulsed R8 cells were then mixed with an equal number of T hybridomas expressing either the N30.7 TCR or one of the CDR3ß mutants and incubated for 20 h. IL-2 release was determined by ELISA. Two independent transfectant lines were tested for each TCR. Data are expressed as percentage of inhibition of IL-2 release as compared with the amount of IL-2 induced by R8 cells pulsed only with 10 nM VSV peptide. Data shown represent the mean of four independent experiments ± SD.

 

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  		Table I. CD8 and TCR surface expression and CD3- or PMA-mediated activation for T cell hybridomas transfected either with the wild-type N30.7 or any of the CDR3ß point mutants S96A, S96T, G97A, and E101A TCRs

 
When the ability of the S96A, S96T, G97A, and E101A CDR3ß mutants to be antagonized by I1E6 peptide was evaluated, we observed that the S96A, S96T, and G97A CDR3ß mutants, as well as the wild-type N30.7 TCR, were efficiently antagonized by the I1E6 peptide (Fig. 7GoB). In contrast, the E101A CDR3ß mutant was significantly less sensitive to the antagonism caused by the I1E6 peptide variant (Fig. 7GoB), as a very high concentration of this peptide (100 µM) was required to antagonize this mutant TCR.

The abolishment of TCR antagonism by the E101A mutation on the CDR3ß of N30.7 TCR was specific for the I1E6 peptide. As shown in Fig. 7GoC, T cell hybridomas expressing the E101A CDR3ß mutant were antagonized by the E1 peptide variant with similar efficiency as the wild-type N30.7 TCR and the CDR3ß mutant TCRs S96A and S96T. This observation indicates that the antagonism caused by the R->E replacement at position 1 (N terminus) of the VSV peptide was not affected by changes in the CDR3ß sequence of the N30.7 TCR.

These results would be consistent with a possible interaction occurring between E at position 6 of the I1E6 peptide and E at position 101 of the CDR3ß of the N30.7 TCR. This potential interaction could be important for the antagonism caused by the I1E6 peptide, as the electrostatic repulsion resulting from the interaction between those two negatively charged residues could lower the affinity of the N30.7 TCR for the ligand to a level where TCR antagonism is observed. However, it is also possible that other interactions resulting from the E->A mutation at position 101 of N30.7 CDR3ß could alter the antagonism mediated by I1E6, as long-distance effects due to point mutations in TCR have been previously observed (30).

Our findings are consistent with a recent crystallographic study by Ding et al. in which the structures of two antagonist peptides (P6A and Y8A) derived from the HTLV-1 Tax peptide bound to HLA-A2 complexed with the A6 TCR were solved (40). While the crystal structure of HLA-A2/P6A-A6 was identical with the HLA-A2/Tax-A6, the HLA-A2/Y8A-A6 structure showed some conformational rearrangements of the CDR3ß loop of A6 TCR. The major change observed was a 3.5 Å displacement of CDR3ß loop at position 101 of the TCR ß-chain, which breaks hydrogen bonds between the peptide/MHC complex and the CDR3ß loop (40). Notably, the E101 residue of N30.7-CDR3ß found to be involved in the antagonism caused by the H-2Kb/I1E6 complex occupies the same relative position as the residue of the A6-CDR3ß (G101) described by Ding et al. to undergo the most significant conformational change during interaction with the antagonist peptide Y8A (40, 41).

The results reported here are consistent with specific interactions occurring between the CDR3ß loop of N30.7 and position 6 of the VSV peptide bound to H-2Kb, specifically, the importance of the amino acid residue at position 101 of N30.7 CDR3ß for the antagonism caused by the altered peptide ligand I1E6. When such specific TCR/peptide interactions impair the complementarity of the TCR/MHC/peptide interface they could cause TCR antagonism by preventing the completion of TCR signaling. This could probably be the case for the N30.7-TCR when it interacts with the H-2Kb/I1E6 complex.

In conclusion, the findings described in this work indicate that the interactions occurring between TCR CDR3 loops and the antigenic peptide play a crucial role in determining the nature of the T cell response to a particular peptide. Furthermore, the identification of these interactions could allow the design of highly specific antigenic peptides for interfering with deleterious T cell responses.


   Acknowledgments
 
We thank Drs. Steve Almo, Anne Davidson, Betty Diamond, Teresa DiLorenzo, David Ostrov, and Steven Porcelli, as well as Matt Roden and Jean-Claude Schwartz, for critical reading of the manuscript. We also thank David Gebhard at the FACS facility for technical assistance and Marie Muranelli for secretarial assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants RO1 AI07289-32, 5T52CA09173-23, and RO1 AR42533-5. Back

2 Address correspondence and reprint requests to Dr. Stanley G. Nathenson, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Chanin Building, Room 407, Bronx, NY 10461. Back

3 Abbreviations used in this paper: CDR, complementarity-determining region; APL, altered peptide ligand; VSV, vesicular stomatitis virus. Back

Received for publication February 1, 2000. Accepted for publication April 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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