In contrast, we only found two TCR antagonists (2,4%) (Figs. recognition by conventional CTL, but Rabbit Polyclonal to KR1_HHV11 had the unique features that the peptide derivative can be covalently attached to Kd molecules by selective photoactivation of the group and that TCRCligand interactions can be assessed by TCRCphotoaffinity labeling (23, 29). The TCR photoaffinity labeling with soluble ligand directly reflected TCRC ligand binding and its dependence on CD8 (23, 27). In the present study, we tested 12 variants of this peptide derivative on seven CTL clones for antigen recognition (chromium release assay) and TCRCligand binding by TCR photoaffinity labeling with soluble ligand. In 80% of the cases TCRCligand binding and antigen recognition correlated well. Among the exceptions (?fivefold divergences between these two parameters), the most frequent cases were partial agonists for which TCRCligand binding was more efficient than antigen recognition. However, cases in which the recognition was more efficient than TCRC ligand binding were observed as well. Only two antagonists were found, e.g., derivatives that were not recognized and could inhibit the recognition of the wild-type epitope. Remarkably, the relative efficiency of recognition of epitope variant did not correlate with TCRCligand binding avidity. Data are also presented indicating that CD8-dependent clones are more susceptible to TCR antagonism than CD8independent ones, suggesting that CD8 can interefere with CTL activation. Materials and Methods Synthesis and Characterization of Photoreactive PbCS Peptide Derivatives. Chemicals for peptide and conjugate synthesis were obtained from Chemie (Buchs, Switzerland), Neosystems (Strasbourg, France), and Bachem Finechemical AG (Bubendorf, Switzerland). The synthesis, purification, and analysis of and Y(PO3H2) were iodinated with 125I iodine (test from data of at least three different experiments, each performed in triplicates. The detection limit of TCR photoaffinity labeling was 1% for clones S4, S14, S17, and T1, 5% for S18 and S1, and 10% for S15. Relative TCR photoaffinity labeling was calculated by dividing the labeling intensity of the ligand variant by the one of the wildtype ligand. The TCR binding of KdC125IASA-YIPSAEK(group, namely K259(were normalized with the relative Kd competitor activities (Table ?(Table1)1) (see Materials and Methods). By definition, the normalized antigenic activity of group, LY2812223 the immunoprecipitated TCR were analyzed by SDS-PAGE under reducing conditions and autoradiography. As shown for a representative experiment in Fig. ?Fig.22 and and without and LY2812223 lane with antiKd LY2812223 mAb 20-8-4S), P255A (lane and group, or alanine substitution obliterated antigen recognition and TCRCligand binding (reference 29; data not shown). Open in a separate window Open in a separate window Figure 3 Antigen recognition and TCRCligand binding of and ?and55 and in in in 100% refers to the highest degree of binding, as observed on CTL S4. The TCRCligand binding avidity of the different CTL clones was assessed by the TCRCligand binding assay described for Fig. ?Fig.22 As shown in the inserts in Fig. LY2812223 ?Fig.6,6, the highest binding was observed for S4 CTL and was defined as 100%. The second highest TCRCligand binding was observed on T1 CTL (80%), followed by clones S14 (40%) and S17 (30%). Intermediate binding was recorded on clones S14 and S17, and for clones S1 and S15, the specific binding was barely above the background. According to TCR photoaffinity labeling, the weakest binding was observed for S15 CTL (7%), followed by CTL S1 (22%) and S18 (25%). The bindings avidities correlated poorly with the observed CD8 dependence, but rather well with the ability of the CTL clones to recognize the different epitope modifications (see Figs. ?Figs.33 and ?and6).6). This is probably explained by that low avidity TCRCligand interactions are more likely to be reduced below a critical threshold required for T cell activation. Discussion The availability of CD8+ CTL clones that permit direct assessment of LY2812223 TCRCligand binding by TCR photoaffinity labeling, provided an unique opportunity to study in a systematic manner the correlation between CTL function and TCRCligand binding. Whereas correlations between T cell responses to altered peptide ligands and TCRCligand binding have been studied previously with purified recombinant TCR and ligands (13, 25, 26), the present study is novel in that TCRCligand binding was measured on living cells, which takes into account CD8 contributions (27). In addition, since the study was performed on a family of seven CTL clones of.