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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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Recognition of target cells expressing covalent Kd-“IASA”- YIPSAEK(ABA)I complexes by S14 CTL is inhibited by variants P255L  and K259(BA). Kd molecules on 51Cr-labeled P815 target cells were photocross-linked with a suboptimal concentration of IASA-YIPSAEK(ABA)I.  After extensive washing the target cells were incubated with CTL S14 (A  and B) or S15 (C) in the presence of the indicated concentrations of peptide PbCS 252-260 (open circles), or peptide variants K259(BA) (closed circle  in A), or P255L (closed circle in B) or K259(ASA) (closed circle in C). The  recognition of the respective conjugate variants on normal 51Cr-labeled  target cells is shown (closed squares). After 4 h, of incubation 51Cr-release  was measured and the specific lysis calculated as described for Fig. 1.
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Figure 5: Recognition of target cells expressing covalent Kd-“IASA”- YIPSAEK(ABA)I complexes by S14 CTL is inhibited by variants P255L and K259(BA). Kd molecules on 51Cr-labeled P815 target cells were photocross-linked with a suboptimal concentration of IASA-YIPSAEK(ABA)I. After extensive washing the target cells were incubated with CTL S14 (A and B) or S15 (C) in the presence of the indicated concentrations of peptide PbCS 252-260 (open circles), or peptide variants K259(BA) (closed circle in A), or P255L (closed circle in B) or K259(ASA) (closed circle in C). The recognition of the respective conjugate variants on normal 51Cr-labeled target cells is shown (closed squares). After 4 h, of incubation 51Cr-release was measured and the specific lysis calculated as described for Fig. 1.

Mentions: To find out whether the partial recognition of variant K259(ASA) by S15 CTL (Fig. 3 A) was accounted for by Fas-mediated cytotoxicity, antigen recognition experiments were performed by using as target cells either A20 B lymphoma cells or an A20 variant that lacked functional Fas (A20 Fas−). As shown for a representative experiment in Fig. 4, A20 cells were efficiently lysed by S15 CTL in the presence of the wild-type peptide derivative, with half-maximal lysis reached at ∼2 × 10−12 M of IASA-YIPSAEK(ABA)I. Conversely, the variant K259 (ASA) was recognized only partially, with half-maximal lysis of only 13% observed at about × 10−10 M. Similar results were obtained on P815 target cells (see Figs. 3 A and 5 C). However, A20 Fas− target cells were not detectably lysed in the presence of this variant, although they were efficiently killed in the presence of the wild-type epitope (half-maximal lysis at about 2 × 10−11 M) (Fig. 4 B). These results strongly suggest that the low lysis of K259(ASA) sensitized A20 cells was accounted for essentially by Fas-mediated cytotoxicity, similarly as has been described in other systems (18, 19).


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)

Recognition of target cells expressing covalent Kd-“IASA”- YIPSAEK(ABA)I complexes by S14 CTL is inhibited by variants P255L  and K259(BA). Kd molecules on 51Cr-labeled P815 target cells were photocross-linked with a suboptimal concentration of IASA-YIPSAEK(ABA)I.  After extensive washing the target cells were incubated with CTL S14 (A  and B) or S15 (C) in the presence of the indicated concentrations of peptide PbCS 252-260 (open circles), or peptide variants K259(BA) (closed circle  in A), or P255L (closed circle in B) or K259(ASA) (closed circle in C). The  recognition of the respective conjugate variants on normal 51Cr-labeled  target cells is shown (closed squares). After 4 h, of incubation 51Cr-release  was measured and the specific lysis calculated as described for Fig. 1.
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Related In: Results  -  Collection

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Figure 5: Recognition of target cells expressing covalent Kd-“IASA”- YIPSAEK(ABA)I complexes by S14 CTL is inhibited by variants P255L and K259(BA). Kd molecules on 51Cr-labeled P815 target cells were photocross-linked with a suboptimal concentration of IASA-YIPSAEK(ABA)I. After extensive washing the target cells were incubated with CTL S14 (A and B) or S15 (C) in the presence of the indicated concentrations of peptide PbCS 252-260 (open circles), or peptide variants K259(BA) (closed circle in A), or P255L (closed circle in B) or K259(ASA) (closed circle in C). The recognition of the respective conjugate variants on normal 51Cr-labeled target cells is shown (closed squares). After 4 h, of incubation 51Cr-release was measured and the specific lysis calculated as described for Fig. 1.
Mentions: To find out whether the partial recognition of variant K259(ASA) by S15 CTL (Fig. 3 A) was accounted for by Fas-mediated cytotoxicity, antigen recognition experiments were performed by using as target cells either A20 B lymphoma cells or an A20 variant that lacked functional Fas (A20 Fas−). As shown for a representative experiment in Fig. 4, A20 cells were efficiently lysed by S15 CTL in the presence of the wild-type peptide derivative, with half-maximal lysis reached at ∼2 × 10−12 M of IASA-YIPSAEK(ABA)I. Conversely, the variant K259 (ASA) was recognized only partially, with half-maximal lysis of only 13% observed at about × 10−10 M. Similar results were obtained on P815 target cells (see Figs. 3 A and 5 C). However, A20 Fas− target cells were not detectably lysed in the presence of this variant, although they were efficiently killed in the presence of the wild-type epitope (half-maximal lysis at about 2 × 10−11 M) (Fig. 4 B). These results strongly suggest that the low lysis of K259(ASA) sensitized A20 cells was accounted for essentially by Fas-mediated cytotoxicity, similarly as has been described in other systems (18, 19).

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