The efficiency of antigen recognition by CTL is determined by the frequency of serial TCR engagement

To investigate the relation between the efficiency of antigen recognition and TCR-ligand binding, we utilized H-2-Kd-restricted CD8+CTL clones that permit assessment of TCR-ligand interactions by TCR photoaffinity labeling. The clones were specific for a derivative of the Plasmodium berghei cirumsporozoite peptide PbCS 252-260 (SYIPSAEKI) containing photoreactive iodo-4-azidosalicylic acid (IASA) in place of PbCS S-252 and 4- azidobenzoic acid (ABA) on PbCS K-259. Selective photoactivation of IASA permitted crosslinking to Kd and photoactivation of ABA to TCR. By assessing antigen recognition (cytotoxicity and IFNγ production) and TCR-ligand binding (TCR photoaffinity labeling) for 12 peptide derivative variants on seven CTL clones, several cases were found, for which the functional response was either ≥ 5-fold lower (antagonists and weak agonists) or higher (strong agonists) than TCR-ligand binding. The efficiency of antigen recognition correlated with the rates of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. Strong agonist exhibited faster TCR-ligand complex dissociation than weak agonists, suggesting that CTL activation depends on the frequency of serial TCR engagement. Consistent with this, we observed that acceleration of TCR-ligand complex dissociation, brought about by blocking of CD8, can increase the efficiency of antigen recognition. On the other hand intercellular TCR-ligand photocrosslinking abrogated CTL function by an active, probably phosphatase mediated, inhibition of T cell activation. Our results indicate that CTL activation is driven by the frequency of serial TCR engagement.