Cytotoxic T Lymphocyte Recognition 
totoxic T lymphocytes by RMA-S. After viral infec- 
tion or transfection of the nucleoprotein gene 
alone, this epitope appears on the cell surface. By 
moving this sequence of eight residues around, 
we hope to be able to discover what makes these 
escape peptides special — is it the peptides them- 
selves or surrounding sequences? The leakiness 
in this cell line also suggests that a Ham- 1 homo- 
dimer may function inefficiently in this mutant 
cell line. 
Cytotoxic Responses 
to Intracellular Bacteria 
Listeria monocytogenes is a frequent food- 
borne pathogen that causes severe disease in 
immunocompromised individuals. It is a gram- 
positive facultative anaerobe. Following uptake 
by macrophages. Listeria escapes from the hos- 
tile environment of the phagolysosome by secret- 
ing a hemolysin, listeriolysin, which ruptures the 
phagosome membrane. Once into the cytoplasm, 
the bacterium replicates and can push itself into a 
neighboring cell by polymerizing actin. Previous 
workers had demonstrated a cytotoxic T cell re- 
sponse in mice to Listeria infection. Our main 
priority was to determine which of the approxi- 
mately 4,000 proteins made by Listeria were 
providing class I-associated peptide epitopes for 
cytotoxic T lymphocytes. 
We determined that the secreted listeriolysin 
molecule itself provided a strong class I-re- 
stricted epitope in the mice we studied. To pin- 
point the presumed nonamer within the 600 
amino acid residues of the listeriolysin molecule, 
we used class I peptide-binding motifs that had 
predicted tyrosine at position 2 and leucine at 
position 9. The listeriolysin contained three non- 
amers that fit this criteria. These and others were 
made as synthetic peptides, one of which tar- 
geted our cytotoxic T lymphocytes at picomolar 
concentration. Thus we have used these motifs 
for the first time and shown that they are valid in 
pinpointing cytotoxic T lymphocyte epitopes. 
In more recent studies we have been able to 
show that cytotoxic T lymphocyte immunity to 
this short 9-residue peptide presented by a class I 
molecule is sufficient to confer adoptive immu- 
nity to the whole bacterium. Lines of cytotoxic T 
lymphocytes with specificity only to this epi- 
tope, when transferred into syngeneic mice, 
confer protection against lethal doses of Listeria 
monocytogenes. 
The listeriolysin epitope of Listeria that in- 
duces conventional cytotoxic T cell immunity is 
restricted by a classical class I molecule. "Classi- 
cal" refers here to the highly polymorphic H-2K, 
D, or L loci in mice and their equivalent HLA-A, B, 
and C loci in humans. We have evidence that 
other peptides derived from this bacterium are 
presented by nonclassical, nonpolymorphic class 
I molecules. We know that class I is involved in 
presenting these peptides, because cell lines that 
lack j82"r"icrogIobulin are unable to present the 
peptides. Furthermore, we know that the class I 
molecule maps just outside the MHC, as revealed 
by experiments with congenic mice differing 
only in this region. 
But unlike the classical class I molecules, this 
I'estricting element is rather nonpolymorphic: 
i.e., cells from most mouse strains are able to 
bind and present the peptide. We have yet to 
identify the peptide derived from Listeria or the 
class I molecule involved. It will be interesting in 
the future to determine whether cytotoxic T lym- 
phocytes directed to these peptides, seen in the 
context of nonpolymorphic MHC molecules, 
confer protective immunity. 
38 
