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.