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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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Antigen recognition and TCR–ligand binding of IASA-YIPSAEK(ABA)I by seven CTL clones. Antigen recognition, as expressed in normalized relative antigenic activities were assessed as described for Fig. 1 and TCR–ligand binding, expressed as normalized TCR photoaffinity labeling, as  described for Fig. 2. The TCR binding of Kd-“125IASA”-YIPSAEK(BA) was assessed as described in Materials and Methods. Shown in gray shaded bars  are cases in which TCR-ligand binding was ⩾fivefold more efficient than antigen recognition; in vertical-striped bars cases in which the antigen recognition was ⩾fivefold more efficient than TCR ligand binding; in diagonal-striped bars cases in which TCR antagonists and in checker-striped bars a partial  agonist exhibiting low plateau (lp) of lysis. (A) Six IASA-YIPSAEK(ABA)I variants were examined, containing either single alanine substitutions of PbCS  residues or modifications of the K(ABA) side chain. (B) Six additional conjugate variants containing L, S, N, D, K, or H in place of PbCS P255 likewise  were tested. The indicated mean values and standard deviations were calculated from at least three independent experiments.
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Figure 3: Antigen recognition and TCR–ligand binding of IASA-YIPSAEK(ABA)I by seven CTL clones. Antigen recognition, as expressed in normalized relative antigenic activities were assessed as described for Fig. 1 and TCR–ligand binding, expressed as normalized TCR photoaffinity labeling, as described for Fig. 2. The TCR binding of Kd-“125IASA”-YIPSAEK(BA) was assessed as described in Materials and Methods. Shown in gray shaded bars are cases in which TCR-ligand binding was ⩾fivefold more efficient than antigen recognition; in vertical-striped bars cases in which the antigen recognition was ⩾fivefold more efficient than TCR ligand binding; in diagonal-striped bars cases in which TCR antagonists and in checker-striped bars a partial agonist exhibiting low plateau (lp) of lysis. (A) Six IASA-YIPSAEK(ABA)I variants were examined, containing either single alanine substitutions of PbCS residues or modifications of the K(ABA) side chain. (B) Six additional conjugate variants containing L, S, N, D, K, or H in place of PbCS P255 likewise were tested. The indicated mean values and standard deviations were calculated from at least three independent experiments.

Mentions: We tested the first six variants of IASA-YIPSAEK(ABA)I (Table 1) for recognition on seven IASA-YIPSAEK(ABA)I-specific CTL clones. As shown in Fig. 3 A, the normalized relative antigenic activities of the variants I254A, P255A, and S256A were in the range of 0.1 to 6 (e.g., were recognized between 10-fold less to 6-fold more efficiently than IASA-YIPSAEK(ABA)I), indicating that none of these substitutions dramatically affected the antigen recognition by these clones. Conversely, the variant E258A was efficiently recognized only by clones S1, S4, S15, and S18, but only inefficiently by clone S14 and not detectably by clones S17 and T1. Of the K259(ABA) variants, K259(ASA) was efficiently recognized by all clones, except by clone S15, which recognized this variant only partially (e.g., the specific lysis never exceeded 30%). An even lower plateau of lysis was observed for the variant IASA-YIPSAEK(IASA)I (data not shown). Conversely, the variant K259(BA) was efficiently recognized only by clones S4, S17, S18, inefficiently by clones S1, S15, and T1 clones, and not detectably by clone S14. More drastic modifications of the K(ABA) side chain, such as shortening by one methylene group, deletion of the ABA group, or alanine substitution obliterated antigen recognition and TCR–ligand binding (reference 29; data not shown).


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)

Antigen recognition and TCR–ligand binding of IASA-YIPSAEK(ABA)I by seven CTL clones. Antigen recognition, as expressed in normalized relative antigenic activities were assessed as described for Fig. 1 and TCR–ligand binding, expressed as normalized TCR photoaffinity labeling, as  described for Fig. 2. The TCR binding of Kd-“125IASA”-YIPSAEK(BA) was assessed as described in Materials and Methods. Shown in gray shaded bars  are cases in which TCR-ligand binding was ⩾fivefold more efficient than antigen recognition; in vertical-striped bars cases in which the antigen recognition was ⩾fivefold more efficient than TCR ligand binding; in diagonal-striped bars cases in which TCR antagonists and in checker-striped bars a partial  agonist exhibiting low plateau (lp) of lysis. (A) Six IASA-YIPSAEK(ABA)I variants were examined, containing either single alanine substitutions of PbCS  residues or modifications of the K(ABA) side chain. (B) Six additional conjugate variants containing L, S, N, D, K, or H in place of PbCS P255 likewise  were tested. The indicated mean values and standard deviations were calculated from at least three independent experiments.
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Figure 3: Antigen recognition and TCR–ligand binding of IASA-YIPSAEK(ABA)I by seven CTL clones. Antigen recognition, as expressed in normalized relative antigenic activities were assessed as described for Fig. 1 and TCR–ligand binding, expressed as normalized TCR photoaffinity labeling, as described for Fig. 2. The TCR binding of Kd-“125IASA”-YIPSAEK(BA) was assessed as described in Materials and Methods. Shown in gray shaded bars are cases in which TCR-ligand binding was ⩾fivefold more efficient than antigen recognition; in vertical-striped bars cases in which the antigen recognition was ⩾fivefold more efficient than TCR ligand binding; in diagonal-striped bars cases in which TCR antagonists and in checker-striped bars a partial agonist exhibiting low plateau (lp) of lysis. (A) Six IASA-YIPSAEK(ABA)I variants were examined, containing either single alanine substitutions of PbCS residues or modifications of the K(ABA) side chain. (B) Six additional conjugate variants containing L, S, N, D, K, or H in place of PbCS P255 likewise were tested. The indicated mean values and standard deviations were calculated from at least three independent experiments.
Mentions: We tested the first six variants of IASA-YIPSAEK(ABA)I (Table 1) for recognition on seven IASA-YIPSAEK(ABA)I-specific CTL clones. As shown in Fig. 3 A, the normalized relative antigenic activities of the variants I254A, P255A, and S256A were in the range of 0.1 to 6 (e.g., were recognized between 10-fold less to 6-fold more efficiently than IASA-YIPSAEK(ABA)I), indicating that none of these substitutions dramatically affected the antigen recognition by these clones. Conversely, the variant E258A was efficiently recognized only by clones S1, S4, S15, and S18, but only inefficiently by clone S14 and not detectably by clones S17 and T1. Of the K259(ABA) variants, K259(ASA) was efficiently recognized by all clones, except by clone S15, which recognized this variant only partially (e.g., the specific lysis never exceeded 30%). An even lower plateau of lysis was observed for the variant IASA-YIPSAEK(IASA)I (data not shown). Conversely, the variant K259(BA) was efficiently recognized only by clones S4, S17, S18, inefficiently by clones S1, S15, and T1 clones, and not detectably by clone S14. More drastic modifications of the K(ABA) side chain, such as shortening by one methylene group, deletion of the ABA group, or alanine substitution obliterated antigen recognition and TCR–ligand binding (reference 29; data not shown).

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