Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life

Activation of CD8(+) cytolytic T lymphocytes (CTLs) by antigen is triggered by the interaction of clonotypic alpha beta T cell receptors (TCRs) with a ntigenic peptides bound to MHC class I molecules (pMHC complexes). Fluoresc ent multimeric pMHC complexes have been shown to specifically stain antigen -specific CTLs by directly binding the TCR. In tumor-infiltrating lymphocyt es from a melanoma patient we found a high frequency of tyrosinase(368-376) peptide-specific cells as detected by IFN-T ELISPOT, without detectable st aining with the corresponding A2/peptide multimers. Surprisingly, these T c ells were able to lyse tyrosinase(368-376) peptide-pulsed target cells as e fficiently as other specific T cells that were stained by multimers. Analys is of the staining patterns under different conditions of incubation time a nd temperature revealed that these results were explained by major differen ces in TCR-multimeric ligand interaction kinetics among the clones. Whereas no direct quantitative correlation between antigenic peptide concentration required for CTL effector functions and equilibrium multimer binding was o bserved interclonally, the latter was profoundly affected by the kinetics o f TCR-ligand interaction. More importantly, our data indicate that similar levels of T cell activation can be achieved by independent CD8(+) T cell cl onotypes displaying different TCR/pMHC complex dissociation rates.