CYTOTOXIC T-LYMPHOCYTE (CTL) ADHERENCE ASSAY (CAA) - A NONRADIOACTIVEASSAY FOR MURINE CTL RECOGNITION OF PEPTIDE-MHC CLASS-I COMPLEXES

Cytotoxic T lymphocytes (CTL) form an important immune surveillance sy stem against intracellular pathogens. Here we describe a simple, visua l assay for identifying peptides specifically recognized by CTL, based on the discovery that CTL develop increased adhesive properties upon TCR triggering. Several CTL lines were shown to pellet to the bottom o f a round bottom 96-well plate in the absence of peptide. In contrast, these same CTL lines incubated with their cognate peptide, allowing t hem to present peptide to each other, adhered to the sides of the well and were readily distinguished by macroscopic visual examination of t he plate after 4-5 h or overnight incubation. This CTL adherence assay (CAA) demonstrated peptide specificity and MHC restriction, and was t itratable with peptide concentration. With this technique, a minimal-s ized, malaria CTL epitope was correctly identified from a panel of ove rlapping nonamers, although the adherence pattern of two mono-substitu ted, variant peptides was less predictive of lytic activity. Also, sub stitutions in an HIV-1 envelope CTL epitope that reduced lytic activit y were correctly predicted. Inhibitors of RNA and protein synthesis, u pon preincubation, abrogated the adherence, indicating, at minimum, a need for live cells. Wortmannin, a PI-3 kinase inhibitor, inhibited th e peptide specific adherence, consistent with a role for TCR or integr in signal transduction in CAA. Other cytoskeletal and metabolic inhibi tors had no effect. Adherence of the T cells may involve low affinity, nonspecific interactions since wells coated with FCS, BSA or milk pow der all produced an effective CAA in the presence of peptide under ser um foe conditions. Consequently, CAA may represent a rapid, simple met hod for screening large numbers of peptides to find cytolytic epitopes for a given CTL line and may identify additional epitopes causing T c ell activation and adherence but not cytolytic activity.