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Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

Kessler BM, Bassanini P, Cerottini JC, Luescher IF - J. Exp. Med. (1997)

Bottom Line: In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold.The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity.There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland.

ABSTRACT
We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

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TCR–ligand binding assessed by TCR photoaffinity labeling and a direct binding  assay. (A) Cloned S14 CTL were  incubated with equal amounts of  radiolabeled soluble covalent complexes of Kd and peptide derivatives IASA-YIPSAEK(ABA)I (lane  1 without and lane 2 with antiKd mAb 20-8-4S), P255A (lane  3), P255S (lane 4), P255D (lane  5), P255L (lane 6), and P255H  (lane 7) at 0–4°C, and after UV  irradiation, the immunoprecipated TCR–ligand complexes  were analyzed by SDS-PAGE  and autoradiography. (B) The gels  were evaluated by phospho- imaging and represented as bar  graphs. (C) Alternatively, the  UV irradiation was omitted and  cell-associated and free ligand  were separated by centrifugation  through oil gradients. The cellassociated radioactivity of wildtype ligand was defined as 100%  and those of the variant ligands  are expressed in percent. The  mean values and standard deviations were calculated from triplicates.
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Figure 2: TCR–ligand binding assessed by TCR photoaffinity labeling and a direct binding assay. (A) Cloned S14 CTL were incubated with equal amounts of radiolabeled soluble covalent complexes of Kd and peptide derivatives IASA-YIPSAEK(ABA)I (lane 1 without and lane 2 with antiKd mAb 20-8-4S), P255A (lane 3), P255S (lane 4), P255D (lane 5), P255L (lane 6), and P255H (lane 7) at 0–4°C, and after UV irradiation, the immunoprecipated TCR–ligand complexes were analyzed by SDS-PAGE and autoradiography. (B) The gels were evaluated by phospho- imaging and represented as bar graphs. (C) Alternatively, the UV irradiation was omitted and cell-associated and free ligand were separated by centrifugation through oil gradients. The cellassociated radioactivity of wildtype ligand was defined as 100% and those of the variant ligands are expressed in percent. The mean values and standard deviations were calculated from triplicates.

Mentions: The same peptide derivatives were radiolabeled and photocross-linked to soluble monomeric Kd. These complexes were incubated with cloned S14 cells and after photoactivation of the ABA group, the immunoprecipitated TCR were analyzed by SDS-PAGE under reducing conditions and autoradiography. As shown for a representative experiment in Fig. 2 A, the trimolecular complexes (MHC–peptide–TCR α or β chain) migrated with an apparent Mr of ∼90 kD. Intense TCR photoaffinity labeling was observed in the case of the wild-type ligand (Fig, 2 A, lane 1), which was abolished in the presence of the anti-Kd mAb 20-8-4S, which blocks Kd–TCR interactions (lane 2) (29). Strong labeling was also observed with the ligand variants Kd-P255A and Kd-P255S (Fig. 2 A, lanes 3 and 4), whereas the variants Kd-P255D and KdP255L produced only trace labeling (lanes 5 and 6). No labeling was detectable with the ligand Kd-P255H (Fig. 2 A, lane 7).


Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

Kessler BM, Bassanini P, Cerottini JC, Luescher IF - J. Exp. Med. (1997)

TCR–ligand binding assessed by TCR photoaffinity labeling and a direct binding  assay. (A) Cloned S14 CTL were  incubated with equal amounts of  radiolabeled soluble covalent complexes of Kd and peptide derivatives IASA-YIPSAEK(ABA)I (lane  1 without and lane 2 with antiKd mAb 20-8-4S), P255A (lane  3), P255S (lane 4), P255D (lane  5), P255L (lane 6), and P255H  (lane 7) at 0–4°C, and after UV  irradiation, the immunoprecipated TCR–ligand complexes  were analyzed by SDS-PAGE  and autoradiography. (B) The gels  were evaluated by phospho- imaging and represented as bar  graphs. (C) Alternatively, the  UV irradiation was omitted and  cell-associated and free ligand  were separated by centrifugation  through oil gradients. The cellassociated radioactivity of wildtype ligand was defined as 100%  and those of the variant ligands  are expressed in percent. The  mean values and standard deviations were calculated from triplicates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196149&req=5

Figure 2: TCR–ligand binding assessed by TCR photoaffinity labeling and a direct binding assay. (A) Cloned S14 CTL were incubated with equal amounts of radiolabeled soluble covalent complexes of Kd and peptide derivatives IASA-YIPSAEK(ABA)I (lane 1 without and lane 2 with antiKd mAb 20-8-4S), P255A (lane 3), P255S (lane 4), P255D (lane 5), P255L (lane 6), and P255H (lane 7) at 0–4°C, and after UV irradiation, the immunoprecipated TCR–ligand complexes were analyzed by SDS-PAGE and autoradiography. (B) The gels were evaluated by phospho- imaging and represented as bar graphs. (C) Alternatively, the UV irradiation was omitted and cell-associated and free ligand were separated by centrifugation through oil gradients. The cellassociated radioactivity of wildtype ligand was defined as 100% and those of the variant ligands are expressed in percent. The mean values and standard deviations were calculated from triplicates.
Mentions: The same peptide derivatives were radiolabeled and photocross-linked to soluble monomeric Kd. These complexes were incubated with cloned S14 cells and after photoactivation of the ABA group, the immunoprecipitated TCR were analyzed by SDS-PAGE under reducing conditions and autoradiography. As shown for a representative experiment in Fig. 2 A, the trimolecular complexes (MHC–peptide–TCR α or β chain) migrated with an apparent Mr of ∼90 kD. Intense TCR photoaffinity labeling was observed in the case of the wild-type ligand (Fig, 2 A, lane 1), which was abolished in the presence of the anti-Kd mAb 20-8-4S, which blocks Kd–TCR interactions (lane 2) (29). Strong labeling was also observed with the ligand variants Kd-P255A and Kd-P255S (Fig. 2 A, lanes 3 and 4), whereas the variants Kd-P255D and KdP255L produced only trace labeling (lanes 5 and 6). No labeling was detectable with the ligand Kd-P255H (Fig. 2 A, lane 7).

Bottom Line: In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold.The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity.There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland.

ABSTRACT
We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

Show MeSH
Related in: MedlinePlus