Perforin-Deficient CD8?T Cells: In Vivo Priming and
Antigen-Specific Immunity Against Listeria monocytogenes1
Douglas W. White,* Adam MacNeil,†Dirk H. Busch,‡Ingrid M. Pilip,‡Eric G. Pamer,‡and
John T. Harty2*†
CD8?T cells require perforin to mediate immunity against some, but not all, intracellular pathogens. Previous studies with H-2b
MHC perforin gene knockout (PO) mice revealed both perforin-dependent and perforin-independent pathways of CD8?T cell-
mediated immunity to Listeria monocytogenes (LM). In this study, we address two previously unresolved issues regarding the
requirement for perforin in antilisterial immunity: 1) Is CD8?T cell-mediated, perforin-independent immunity specific for a
single Ag or generalizable to multiple Ags? 2) Is there a deficiency in the priming of the CD8?T cell compartment of PO mice
following an immunizing challenge with LM? We used H-2dMHC PO mice to generate CD8?T cell lines individually specific for
three known Ags expressed by a recombinant strain of virulent LM. Adoptive transfer experiments into BALB/c host mice
revealed that immunity can be mediated by PO CD8?T cells specific for all Ags examined, indicating that perforin-independent
immunity is not limited to CD8?T cells that recognize listeriolysin O. Analysis of epitope-specific CD8?T cell expansion by MHC
class I tetramer staining and ELISPOT revealed no deficiency in either the primary or secondary response to LM infection in PO
mice. These results demonstrate that the perforin-independent pathway of antilisterial resistance mediated by CD8?T cells is
generalizable to multiple epitopes. Furthermore, the results show that reduced antilisterial resistance observed with polyclonal PO
CD8?T cells is a consequence of a deficiency in effector function and not a result of suboptimal CD8?T cell priming. The Journal
of Immunology, 1999, 162: 980–988.
defense against certain viruses (1, 2), intracellular bacteria (3), and
protozoan pathogens (4).
One such bacterial pathogen, Listeria monocytogenes (LM),3
has been studied extensively as a model organism to dissect the
cellular immune response (5). Following injection into a mouse,
LM is phagocytosed by macrophages in the spleen and liver. Some
virulent LM, primarily through the actions of a secreted hemolysin
known as listeriolysin O (LLO), escape from the vacuole to the
cytoplasm of the infected eukaryotic host cell. Once in the cyto-
plasm, LM multiplies and polymerizes host-derived F-actin,
thereby initiating movement and direct spread to neighboring host
cells (6–9). In this fashion, LM can spread from host cell to host
cell without leaving the intracellular space.
The course of listeriosis in mice is acute: injection of a lethal
dose results in uncontrolled bacterial replication in the spleen and
D8?T cells play an important role in the immune re-
sponse against intracellular pathogens. A number of stud-
ies have demonstrated an obligate role for these cells in
liver and death in 4 to 6 days. However, mice challenged with a
sublethal primary dose clear the infection within 2 wk and are
subsequently resistant to challenges 100-fold greater than the LD50
in naive animals (10). Studies in mice that lack CD8?T cells have
demonstrated the importance of these cells in the adaptive re-
sponse to LM (3, 11–13).
The effectiveness of CD8?T cells in defense against LM and
other intracellular pathogens is based on their ability, via clonally
distributed TCRs, to recognize peptide Ags bound to MHC class I
molecules on the surface of an infected cell. Peptides displayed by
MHC class I molecules preferentially derive from proteins in the
cytoplasm of the host cell (14, 15). Thus, pathogens such as LM
with a component of their life cycle in the host cell cytoplasm are
often susceptible to protective immunity mediated by CD8?T
Once a CD8?T cell is activated, it is capable of elaborating a
number of effector functions that aid the immune system in the
clearance of the pathogen. Previously activated CD8?T cells
readily produce IFN-? and TNF in an Ag-specific fashion. Both of
these cytokines, which are also produced by cells other than CD8?
T cells, are known to be important in the normal immune response
against LM (10) and in other infectious disease models in which
CD8?T cells are important mediators of resistance (16). Our re-
sults have demonstrated that IFN-? is not required for the devel-
opment nor expression of CD8?T cell-mediated immunity to LM
(17). Recently, it has been shown that IFN-?-deficient CD8?, but
not CD4?, T cells are also capable of clearing a chronic LM in-
fection in SCID mice (18).
Efficient Ag-specific lysis of a target cell harboring an intracel-
lular pathogen is a function largely limited to CD8?T cells. At
least two molecular pathways have been identified by which CD8?
T cells can mediate cytolysis in vitro (19, 20). The granule exo-
cytosis pathway requires the coordinated activity of perforin and
granzymes, both of which are found in the granules of activated
*Interdisciplinary Graduate Program in Immunology and†Department of Microbiol-
ogy, University of Iowa, Iowa City, IA 52242; and‡Sections of Infectious Diseases
and Immunobiology, Yale University School of Medicine, New Haven, CT 06520
Received for publication August 11, 1998. Accepted for publication October 7, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI36864 and
AI42767 (J.T.H.). D.W.W. is a trainee in the Medical Scientist Training Program.
A.M. was supported as an undergraduate fellow by the Howard Hughes Medical
Institute. D.H.B. is a research fellow of the Deutsche Forschungsgemeinschaft (DFG).
E.G.P. is a Pew Scholar in Biomedical Sciences.
2Address correspondence and reprint requests to Dr. J. T. Harty, 3-512 Bowen Sci-
ence Building, Department of Microbiology, University of Iowa, Iowa City, IA
52242. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: LM, Listeria monocytogenes; ELISPOT, enzyme-
linked immunospot assay; LCMV, lymphocytic choriomeningitis virus; LLO, list-
eriolysin O; NP, nucleoprotein; PE, phycoerythrin; PO, perforin deficient.
Copyright © 1999 by The American Association of Immunologists0022-1767/99/$02.00
CD8?T cells, to activate the caspase cascade of the target cell and
induce apoptosis (21–23). The absolute requirement for this path-
way in the clearance of lymphocytic choriomeningitis virus
(LCMV) has been demonstrated in mice with targeted disruption
of the gene for perforin (24, 25). Activated CD8?T cells also
express CD95 ligand, which can ligate CD95 (Fas, Apo-1) on a
target cell and induce apoptosis via the caspase cascade. This path-
way is probably most important in the elimination of self-reactive
T cells that are repeatedly exposed to Ag (26). Evidence for a
role of this pathway in CD8?T cell-mediated immunity against
viruses in vivo has been presented (27, 28), but its importance
as an effector function against most infectious agents remains
Two studies on the role of perforin in secondary resistance and
CD8?T cell-mediated immunity to LM have been reported pre-
viously (29, 30). The first report demonstrated that CD8?spleno-
cytes that lack perforin are deficient in their ability to transfer
antilisterial immunity (29). This finding indicates that perforin is
required for an optimal CD8?T cell response to LM. However, the
data presented in this report did not rule out the possibility that the
deficiency observed with PO splenocytes was due to suboptimal
priming of the CD8?compartment of PO donor mice.
The second report used H-2bPO CD8?T cells that had been
restimulated in vitro to identify a perforin-independent pathway by
which CD8?T cells are capable of mediating antilisterial immu-
nity (30). However, this report concentrated on CD8?T cells spe-
cific for a single LM Ag. It was therefore impossible to generalize
the findings regarding perforin-independent immunity to CD8?T
cells specific for other LM-derived Ags.
Both studies were performed using PO mice of the H-2bhap-
lotype in which precise LM-derived epitopes recognized by CD8?
T cells are unknown. The lack of known epitopes in the H-2b
system prevented the analysis of CD8?T cell priming in H-2bPO
mice as well as the analysis of CD8?T cells specific for more than
one LM Ag. To address these issues, we generated H-2dMHC PO
mice by backcross of H-2bPO mice with BALB/c. The CD8?T
cell response to LM is well characterized in the H-2dhaplotype
(31), allowing us to analyze multiple Ags as targets of PO CD8?
T cells, as well as the expansion of LM Ag-specific CD8?T cells
after immunization. We present data demonstrating that the per-
forin-independent pathway is not limited to CD8?T cells specific
for a single epitope, but rather functions in T cells specific for all
Ags tested. We also present data that rule out suboptimal priming
of the CD8?T cell compartment in perforin-knockout mice as an
explanation for the apparent deficiency of PO CD8?splenocytes in
mediating antilisterial immunity.
Materials and Methods
BALB/c (H-2dMHC) mice were obtained from the National Cancer In-
stitute (Frederick, MD) and crossed with PO (H-2bMHC) mice kindly
provided by Dr. W. R. Clark (25). F1mice were backcrossed to BALB/c
and H-2d/dperforin?/?mice were identified by flow-cytometric analysis of
PBL using Abs specific for H-2d(SF1-1.1.1) and H-2b(Y-3) and Southern
blot analysis for perforin genotype, as described (25). H-2d/dperforin?/?
mice were backcrossed to BALB/c three additional times and then inter-
crossed to generate H-2d/dperforin?/?(H-2dPO) mice. H-2dPO mice were
maintained by brother-sister mating and housed under specific pathogen-
free conditions at the University of Iowa (Iowa City, IA) animal care unit.
All mice were used at 8–16 wk of age in an age- and sex-matched fashion.
LM strain 10403s (32) and recombinant strain XFL303 (derived from
10403s), which expresses the LCMV NP 118–126 epitope as a secreted
fusion protein (33), are both resistant to streptomycin and were used as
previously described (33–36). Briefly, bacteria were grown in tryptic soy
broth to an OD600of approximately 0.1, diluted in pyrogen-free 0.9% so-
dium chloride (Abbott Laboratories, North Chicago, IL), and injected i.v.
in 0.2 ml per animal. Aliquots were plated onto tryptic soy agar containing
50 ?g/ml streptomycin to verify the number of CFU injected.
Cell lines, Abs, flow cytometry, and T cell depletion
P815 is a DBA/2-derived mastocytoma (H-2dMHC) (American Type Cul-
ture Collection (ATCC), Manassas, VA; ATCC TIB-64); P815-LLO refers
to P815 cells stably transfected with a plasmid construct expressing the LM
Ag LLO and neo-resistance (37); P815-p60 refers to P815 cells stably
transfected with a plasmid construct expressing the LM Ag p60 and neo-
resistance (34). P815-Fas is a derivative of P815 that expresses 10-fold
more surface CD95 than P815 (38); L1210F?(25) and L1210F?(39) are
derivatives of the lymphoblastic cell line L1210 (ATCC CCL-219) that
have been transfected with Fas antisense and sense cDNA, respectively.
Cell lines were maintained in RPMI 1640 (Life Technologies, Grand Is-
land, NY) supplemented with 10% FCS, antibiotics, L-glutamine, HEPES
buffer, and 2-ME (RP10 (34)). Transfected cells were maintained in RP10
supplemented with G-418 at 400 ?g/ml.
mAbs, which were purified from culture supernatants and quantitated as
previously described (17), were: rat anti-mouse TNF IgG (XT22 and XT3
(40) used in combination at a mass ratio of 1:1), rat anti-mouse IFN-? IgG
(XMG1.2 (41)), rat anti-mouse CD8 (2.43 (42)), and rat anti-mouse CD4
(GK1.5 (43)). Control polyclonal rat IgG was purchased from Sigma (St.
Louis, MO). Flow-cytometric analysis was performed as previously de-
scribed (17) using FITC-conjugated anti-CD8 (53.6-7; Sigma), PE-conju-
gated anti-CD4 (H129.19; Sigma), mouse anti-mouse H-2KbIgG2b (Y-3;
ATCC), and mouse anti-mouse H-2KdIgG2a (SF1-1.1.1; ATCC). T cell
subset depletion by mAb and complement was performed in vitro using rat
anti-mouse CD4 (RL172) and rat anti-mouse CD8 (3.168), as previously
described (17). T cell subset depletion in vivo was conducted as previously
described using 2.43 and GK1.5 (17).
Generation and maintenance of CD8?T cell lines
CD8?T cell lines specific for LLO 91–99, p60 217–225, or NP 118–126
were derived from BALB/c and H-2dPO mice using methods previously
described (37). Briefly, 2–4 ? 107splenocytes from mice injected 7–10
days previously with 103–4CFU of virulent LM strain 10403s or XFL303
were incubated in RP10 with 3 ? 106irradiated (150 Gy) stimulator cells
(P815-LLO cells or P815-p60 cells). Subsequent weekly restimulations
were conducted by combining 1–3 ? 106responder cells with 3 ? 106
irradiated stimulator cells and approximately 4 ? 107irradiated (30 Gy)
syngeneic splenocytes in RP10 supplemented with 5% supernatant from
Con A-stimulated rat spleen cells and 50 mM ?-methyl mannoside. In the
case of T cell lines specific for NP 118–126, P815-derived stimulator cells
were left out and 3 ? 107irradiated syngeneic splenocytes were incubated
with 100 nM synthetic NP 118–126 peptide for 1 h at 37°C and rinsed
three times before their addition to the T cell culture.
In vitro characterization of CD8?T cell lines
51Cr release assays were performed as previously described (44, 17, 30).
Briefly, labeled target cells were combined with effector cells at the indi-
cated ratios in RP10 in round-bottom 96-well plates. Following a 4–7.5-h
incubation (as indicated), supernatant was harvested and assayed for
51Cr release. Percent specific release of
formula: 100 ? (experimental cpm ? spontaneous cpm)/(total cpm ?
spontaneous cpm). Spontaneous release was less than 20% of total in all
TNF was quantitated using a WEHI 164 clone 13 bioassay (45), as
previously described (30). Briefly, supernatants from coincubations of
CD8?T cells and target cells were added to WEHI 164 cells in flat-bottom
96-well plates. Following overnight incubation, survival of incubator cells
was assayed by the addition of alamar blue (Acumed, West Lake, OH).
Death of the indicator cells, a relative measure of TNF production, was
determined 2–6 h after addition of alamar blue by measuring OD570-
OD600. Production of TNF by target cells in the absence of CD8?T cells
was not detected. Murine rTNF (Boehringer Mannheim, Indianapolis, IN)
was used as a control and to determine the detection limits of the WEHI
bioassay. Concentrations of 1–10 pg/ml of rTNF were routinely detected
using this assay.
IFN-? was quantitated by ELISA, as previously described (30). Briefly,
supernatants from overnight coincubations of effector cells and target cells
and rIFN-? controls were added to 96-well plates that had been previously
coated with rat anti-mouse IFN-? (XMG1.2) mAb. Rabbit anti-mouse
IFN-? (a gift from J. Cowdery at the University of Iowa), alkaline phos-
phatase-conjugated goat anti-rabbit Ig (Sigma), and alkaline phosphatase
51Cr was calculated by the
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988 PERFORIN-DEFICIENT CD8?T CELLS AND IMMUNITY TO L. monocytogenes