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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 IASA-YIPSAEK(ABA)I and variants by  cloned S14 CTL. (A) 51Cr-labeled P815 cells were incubated with S14  cells in the presence of the indicated concentrations of IASA-YIPSAEK(ABA)I (•), P255A (○), P255S (▴), P255D (♦), P255L (▾) and  P255H (▪) at an E/T ratio of 3:1. After 4 h of incubation the specific lysis was determined from the relased chomium. (B) The relative antigenic  activities calculated from A were normalized with the relative Kd competitor activities (Table 1) (see Materials and Methods). By definition, the  normalized antigenic activity of IASA-YIPSAEK(ABA)I is 1.
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Figure 1: Recognition of IASA-YIPSAEK(ABA)I and variants by cloned S14 CTL. (A) 51Cr-labeled P815 cells were incubated with S14 cells in the presence of the indicated concentrations of IASA-YIPSAEK(ABA)I (•), P255A (○), P255S (▴), P255D (♦), P255L (▾) and P255H (▪) at an E/T ratio of 3:1. After 4 h of incubation the specific lysis was determined from the relased chomium. (B) The relative antigenic activities calculated from A were normalized with the relative Kd competitor activities (Table 1) (see Materials and Methods). By definition, the normalized antigenic activity of IASA-YIPSAEK(ABA)I is 1.

Mentions: The recognition of the different conjugates by cloned CTL was assessed in a 51Cr release cytolytic assay. As shown for a representative experiment in Fig. 1 A, half-maximal lysis of P815 cells by S14 CTL was observed at about 8 × 10−12 M IASA-YIPSAEK(ABA)I. The variant P255A showed the same efficiency of recognition, but serine substitution in this position (P255S) resulted in an ∼7-fold decrease and aspartic acid substitution (P255D) in a 2,000-fold decrease. Histidine or leucine substitution (P255H and P255L) obliterated recognition by S14 CTL. For sake of comparison and as shown in Fig. 1 B, the normalized antigenic activities were calculated for the different compounds from the antigenic activities (concentrations required for half-maximal lysis) and the corresponding relative Kd competitor activities (Table 1) (see Materials and Methods).


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 IASA-YIPSAEK(ABA)I and variants by  cloned S14 CTL. (A) 51Cr-labeled P815 cells were incubated with S14  cells in the presence of the indicated concentrations of IASA-YIPSAEK(ABA)I (•), P255A (○), P255S (▴), P255D (♦), P255L (▾) and  P255H (▪) at an E/T ratio of 3:1. After 4 h of incubation the specific lysis was determined from the relased chomium. (B) The relative antigenic  activities calculated from A were normalized with the relative Kd competitor activities (Table 1) (see Materials and Methods). By definition, the  normalized antigenic activity of IASA-YIPSAEK(ABA)I is 1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196149&req=5

Figure 1: Recognition of IASA-YIPSAEK(ABA)I and variants by cloned S14 CTL. (A) 51Cr-labeled P815 cells were incubated with S14 cells in the presence of the indicated concentrations of IASA-YIPSAEK(ABA)I (•), P255A (○), P255S (▴), P255D (♦), P255L (▾) and P255H (▪) at an E/T ratio of 3:1. After 4 h of incubation the specific lysis was determined from the relased chomium. (B) The relative antigenic activities calculated from A were normalized with the relative Kd competitor activities (Table 1) (see Materials and Methods). By definition, the normalized antigenic activity of IASA-YIPSAEK(ABA)I is 1.
Mentions: The recognition of the different conjugates by cloned CTL was assessed in a 51Cr release cytolytic assay. As shown for a representative experiment in Fig. 1 A, half-maximal lysis of P815 cells by S14 CTL was observed at about 8 × 10−12 M IASA-YIPSAEK(ABA)I. The variant P255A showed the same efficiency of recognition, but serine substitution in this position (P255S) resulted in an ∼7-fold decrease and aspartic acid substitution (P255D) in a 2,000-fold decrease. Histidine or leucine substitution (P255H and P255L) obliterated recognition by S14 CTL. For sake of comparison and as shown in Fig. 1 B, the normalized antigenic activities were calculated for the different compounds from the antigenic activities (concentrations required for half-maximal lysis) and the corresponding relative Kd competitor activities (Table 1) (see Materials and Methods).

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