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Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus.

Campos-Lima PO, Levitsky V, Imreh MP, Gavioli R, Masucci MG - J. Exp. Med. (1997)

Bottom Line: The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT).Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis.Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77, Stockholm, Sweden.

ABSTRACT
The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

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Fine specificity of the various IVT and AVF-specific TCR  types defined by alanine scanning mutagenesis. IVT and AVF-specific  CTL clones were tested for lysis of A11+ PHA blasts pulsed with 10−9  and 10−10 M of the indicated peptide analogue. The sequence of the wild-type peptide and the predicted orientation of each residue relative to the  A11 groove (20) are indicated above each Ala replacement set. Residues  pointing towards the groove are shown below the peptide backbone and  residues pointing towards the TCR are shown above. The putative accessory anchors are indicated crossing the backbone. Boxes below each residue indicate the corresponding Ala substituted analogue tested with  clones expressing the indicated TCR type (left). When the wild-type residue is shown in the box the corresponding analogue was not tested.  White boxes indicate that the analogue was recognized as efficiently as the  wild-type peptide at both peptide concentrations, black boxes indicate no  recognition at either peptide concentration and gray boxes correspond to  at least 50% of the control lysis at a concentration of 10−9 M. Each assay  was performed three times and the results obtained with individual clones  were highly reproducible. All clones listed in Table 2 were tested. In several cases the screening was performed before the TCR-α/β sequences  became available. Later results confirmed the absolute correlation between each TCR type and a given pattern of reactivity.
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Figure 2: Fine specificity of the various IVT and AVF-specific TCR types defined by alanine scanning mutagenesis. IVT and AVF-specific CTL clones were tested for lysis of A11+ PHA blasts pulsed with 10−9 and 10−10 M of the indicated peptide analogue. The sequence of the wild-type peptide and the predicted orientation of each residue relative to the A11 groove (20) are indicated above each Ala replacement set. Residues pointing towards the groove are shown below the peptide backbone and residues pointing towards the TCR are shown above. The putative accessory anchors are indicated crossing the backbone. Boxes below each residue indicate the corresponding Ala substituted analogue tested with clones expressing the indicated TCR type (left). When the wild-type residue is shown in the box the corresponding analogue was not tested. White boxes indicate that the analogue was recognized as efficiently as the wild-type peptide at both peptide concentrations, black boxes indicate no recognition at either peptide concentration and gray boxes correspond to at least 50% of the control lysis at a concentration of 10−9 M. Each assay was performed three times and the results obtained with individual clones were highly reproducible. All clones listed in Table 2 were tested. In several cases the screening was performed before the TCR-α/β sequences became available. Later results confirmed the absolute correlation between each TCR type and a given pattern of reactivity.

Mentions: To determine how the TCR structural differences related to target recognition, the fine specificity of clones representing the two AVF-specific and nine IVT-specific TCR types was determined using peptide analogues in which each residue of IVT or AVF was sequentially substituted by Ala. Analysis of the HLA A11 binding motif (19) and molecular modeling of the IVT- and AVF-containing complexes (20) indicate that Val at position 2 (P2) and the carboxy-terminal Lys serve as the main anchors. In accordance, the IVT-A9 and AVF-A10 analogues failed to stabilize A11 expression in transporter associated with antigen presentation (TAP) mutant cells (not shown) and were not included in this study. Residues at P1, P4, P5, P7, and P8 of IVT are predicted to point away from the binding groove and be accessible to TCR while the side chains of residues in P3 and P6 are likely to face the cleft, serving as accessory anchors. Sequence variations between IVT-specific TCR types were reflected in unique fine specificity patterns (Fig. 2). Whereas all clones were affected by substitutions at P4 and P5, that may represent important TCR contact sites, the type-IX TCR interacted stringently only with the central residues of the peptide and the remaining TCRs appeared to scan the complex with preferential recognition shifting from the COOH terminus (IVT types I, VI, II) to the NH2 terminus (IVT type VIII). The full array of potential contact residues was recognized by TCR type III, V, VII, and IV, albeit with different stringency. The effect of substitutions in P2, P3, and P6 suggests that they may induce conformational changes that indirectly affect the interaction with certain TCRs. It should be noted that all clones recognized equally well the wild-type peptide with half-maximal lysis observed at concentrations between 5 and 10 pM, confirming that the different recognition of the analogues is not an artifact due to different efficiency of the CTLs (not shown). As predicted by the conserved TCR usage, AVF-specific clones showed a homogeneous pattern of interaction with the Ala replacement set. All type I TCRs were sensitive to substitution of the solvent exposed residues in P4, P6, and P8 of the AVF peptide, and were also affected by replacement of the Phe in P3, probably due to significant conformational changes. Differences were observed when clones expressing BV3S1D2J2S2 chains containing Arg, Val, Pro, or Leu at position 96 were compared for recognition of the wild-type peptide presented by other members of the HLA A11 family (A1, A3, and Aw68, not shown), suggesting a possible role of this residue in contacting the α-helix. The Thr(Ala)-Ser-Ala motif may interact with the peptide since the type II clones, that lack the motif, were not affected by substitution of the Asp in P4.


Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus.

Campos-Lima PO, Levitsky V, Imreh MP, Gavioli R, Masucci MG - J. Exp. Med. (1997)

Fine specificity of the various IVT and AVF-specific TCR  types defined by alanine scanning mutagenesis. IVT and AVF-specific  CTL clones were tested for lysis of A11+ PHA blasts pulsed with 10−9  and 10−10 M of the indicated peptide analogue. The sequence of the wild-type peptide and the predicted orientation of each residue relative to the  A11 groove (20) are indicated above each Ala replacement set. Residues  pointing towards the groove are shown below the peptide backbone and  residues pointing towards the TCR are shown above. The putative accessory anchors are indicated crossing the backbone. Boxes below each residue indicate the corresponding Ala substituted analogue tested with  clones expressing the indicated TCR type (left). When the wild-type residue is shown in the box the corresponding analogue was not tested.  White boxes indicate that the analogue was recognized as efficiently as the  wild-type peptide at both peptide concentrations, black boxes indicate no  recognition at either peptide concentration and gray boxes correspond to  at least 50% of the control lysis at a concentration of 10−9 M. Each assay  was performed three times and the results obtained with individual clones  were highly reproducible. All clones listed in Table 2 were tested. In several cases the screening was performed before the TCR-α/β sequences  became available. Later results confirmed the absolute correlation between each TCR type and a given pattern of reactivity.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198955&req=5

Figure 2: Fine specificity of the various IVT and AVF-specific TCR types defined by alanine scanning mutagenesis. IVT and AVF-specific CTL clones were tested for lysis of A11+ PHA blasts pulsed with 10−9 and 10−10 M of the indicated peptide analogue. The sequence of the wild-type peptide and the predicted orientation of each residue relative to the A11 groove (20) are indicated above each Ala replacement set. Residues pointing towards the groove are shown below the peptide backbone and residues pointing towards the TCR are shown above. The putative accessory anchors are indicated crossing the backbone. Boxes below each residue indicate the corresponding Ala substituted analogue tested with clones expressing the indicated TCR type (left). When the wild-type residue is shown in the box the corresponding analogue was not tested. White boxes indicate that the analogue was recognized as efficiently as the wild-type peptide at both peptide concentrations, black boxes indicate no recognition at either peptide concentration and gray boxes correspond to at least 50% of the control lysis at a concentration of 10−9 M. Each assay was performed three times and the results obtained with individual clones were highly reproducible. All clones listed in Table 2 were tested. In several cases the screening was performed before the TCR-α/β sequences became available. Later results confirmed the absolute correlation between each TCR type and a given pattern of reactivity.
Mentions: To determine how the TCR structural differences related to target recognition, the fine specificity of clones representing the two AVF-specific and nine IVT-specific TCR types was determined using peptide analogues in which each residue of IVT or AVF was sequentially substituted by Ala. Analysis of the HLA A11 binding motif (19) and molecular modeling of the IVT- and AVF-containing complexes (20) indicate that Val at position 2 (P2) and the carboxy-terminal Lys serve as the main anchors. In accordance, the IVT-A9 and AVF-A10 analogues failed to stabilize A11 expression in transporter associated with antigen presentation (TAP) mutant cells (not shown) and were not included in this study. Residues at P1, P4, P5, P7, and P8 of IVT are predicted to point away from the binding groove and be accessible to TCR while the side chains of residues in P3 and P6 are likely to face the cleft, serving as accessory anchors. Sequence variations between IVT-specific TCR types were reflected in unique fine specificity patterns (Fig. 2). Whereas all clones were affected by substitutions at P4 and P5, that may represent important TCR contact sites, the type-IX TCR interacted stringently only with the central residues of the peptide and the remaining TCRs appeared to scan the complex with preferential recognition shifting from the COOH terminus (IVT types I, VI, II) to the NH2 terminus (IVT type VIII). The full array of potential contact residues was recognized by TCR type III, V, VII, and IV, albeit with different stringency. The effect of substitutions in P2, P3, and P6 suggests that they may induce conformational changes that indirectly affect the interaction with certain TCRs. It should be noted that all clones recognized equally well the wild-type peptide with half-maximal lysis observed at concentrations between 5 and 10 pM, confirming that the different recognition of the analogues is not an artifact due to different efficiency of the CTLs (not shown). As predicted by the conserved TCR usage, AVF-specific clones showed a homogeneous pattern of interaction with the Ala replacement set. All type I TCRs were sensitive to substitution of the solvent exposed residues in P4, P6, and P8 of the AVF peptide, and were also affected by replacement of the Phe in P3, probably due to significant conformational changes. Differences were observed when clones expressing BV3S1D2J2S2 chains containing Arg, Val, Pro, or Leu at position 96 were compared for recognition of the wild-type peptide presented by other members of the HLA A11 family (A1, A3, and Aw68, not shown), suggesting a possible role of this residue in contacting the α-helix. The Thr(Ala)-Ser-Ala motif may interact with the peptide since the type II clones, that lack the motif, were not affected by substitution of the Asp in P4.

Bottom Line: The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT).Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis.Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77, Stockholm, Sweden.

ABSTRACT
The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

Show MeSH
Related in: MedlinePlus