ANTIGEN PRESENTATION AND CYTOTOXIC T-LYMPHOCYTE KILLING STUDIED IN INDIVIDUAL, LIVING CELLS

Interactions between individual, living fibroblasts and cytotoxic T ly mphocyte (CTL) clones were analyzed by using video-enhanced differenti al interference contrast and fluorescence microscopy in a multimode co nfiguration. Fibroblasts expressing known major histocompatibility com plex I alleles (MC57: H-2b; Balb: H-2d) were sensitized for killing by incubating or microinjecting them with peptide fragments of lymphocyt ic choriomeningitis virus. Previous determination of the CTL clones' s pecificity for these peptides and MHC-I alleles enabled us to study CT L killing of fibroblasts, and nonlethal CTL interaction with targets d ue to ''mismatches'' of the CTL, target, and/or peptide. During viral peptide-specific MHC-restricted CTL killing, distinct morphological al terations were observed (CTL shape changes, movements of granules in C TL cytoplasm, and target cell contraction and blebbing). When no killi ng occurred, CTL engaged in prolonged, nonrandom movement on the targe t cells. Alloreactive and virus-specific CTL displayed the same morpho logy during killing. To study antigen presentation further within indi vidual, living cells, a LCMV glycoprotein peptide (aa 272-286, LSDSSGV ENPGGYCL) was covalently labeled with tetramethylrhodamine. In Cr-51 r elease assays, the labeled peptide specifically induced potent CTL kil ling, but neither labeled nor unlabeled peptide proved toxic for unsen sitized targets. Microinjection of the labeled peptide into the cytopl asm of fibroblast cells]ed to CTL killing of those cells, yet nearby u ninjected cells contacted by CTL were not killed, indicating that kill ing was due to presentation of microinjected peptide rather than bindi ng of extracellular peptide to cell surface MHC. Peptide-injected targ et cells were killed only when combined with CTL specific for the pept ide and for the MHC allele of the injected cell. (C) 1994 Academic Pre ss, Inc.