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but Requires TNF-
1
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Interdisciplinary Graduate Program in Immunology and
Department of Microbiology, University of Iowa, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
, and mediate target cell lysis
in vitro. Cytolysis mediated by the PO-derived CD8+ T cell
line is delayed relative to the B6-derived line and is completely
inhibited by anti-CD95 Abs. In vivo, PO-derived CD8+ T
cells provide specific antilisterial immunity in B6 hosts,
CD95-deficient hosts, and IFN--depleted hosts. However, PO-derived
CD8+ T cells fail to provide antilisterial immunity in
hosts depleted of TNF-. These results indicate that single
Ag-specific CD8+ T cells derived from PO mice can mediate
antilisterial immunity by a mechanism that is independent of CD95 or
IFN-, but requires TNF-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activated CD8+ T cells are capable of elaborating a diverse
array of effector functions. Following Ag-specific stimulation,
CD8+ T cells produce a broad range of cytokines including
IFN- and TNF-. These cytokines, which are also produced by other
cell types, have been shown to play critical roles in innate resistance
of mice to L. monocytogenes infection (2). In contrast, we
have used IFN- gene knockout mice to provide evidence that
CD8+ T cell immunity to L. monocytogenes can
occur in the absence of IFN- (8). These experiments suggested that
CD8+ T cell-mediated cytolysis may be important in specific
immunity to L. monocytogenes.
The ability of activated CD8+ T cells to carry out in vitro
cytolysis is well documented. Following ligation of the TCR-ß by
the appropriate MHC class I-peptide complex, the CD8+ T
cell induces its target to undergo programmed cell death
(PCD).3 Two independent
pathways account for the majority of in vitro target cell lysis by
CD8+ T cells (9). One is a perforin-dependent pathway,
mediated by granzymes, which are serine proteases found in the
cytoplasmic granules of activated CD8+ T cells that gain
access to the cytoplasm of the target cell and induce PCD by activation
of the caspase cascade. Although the mechanism(s) by which perforin and
granzymes are involved in cytolysis remain controversial (10, 11), the
dependence of some cytolytic activity on perforin is clear
(12, 13, 14, 15).
The other pathway by which CD8+ T cells are capable of efficient in vitro cytolysis is dependent upon interactions between CD95 ligand (CD95L, Fas ligand) on the activated CD8+ T cell and CD95 on the target cell. Ligation of CD95, in some cell types, leads to intracellular signaling events that also activate the caspase cascade and induce PCD (16).
The relevance of perforin-dependent killing in CD8+ T cell-mediated resistance to infectious disease was demonstrated by experiments using mice with a targeted disruption of the perforin gene (PO mice). These animals are susceptible to infection with lymphocytic choriomeningitis virus (LCMV) in spite of the ability of their CD8+ T cells to be specifically activated during the infection (12, 15). The role of perforin in CD8+ T cell-dependent immunity to other viruses has been subsequently explored (17, 18, 19, 20) and remains a topic of current investigation.
The role of perforin in resistance to L. monocytogenes was addressed by Kagi and colleagues using PO mice (21). In these experiments, it was demonstrated that CD8+ T cells from immune H-2b MHC PO mice are deficient, relative to CD8+ T cells derived from control mice, in their ability to transfer antilisterial immunity to naive hosts. These experiments suggest that perforin-dependent cytotoxicity plays an important role in antilisterial immunity mediated by CD8+ T cells. Unfortunately, little information exists regarding L. monocytogenes-derived Ags presented by H-2b MHC molecules. Thus, the activation status of the CD8+ T cell compartment of the PO donor mice could not be easily evaluated. Therefore, an alternative interpretation of these data is that the doses of L. monocytogenes used to immunize donor mice did not activate the CD8+ T cell compartment equivalently in the PO donors and the B6 donors. The finding that PO mice exhibit more resistance than B6 mice to primary challenge with L. monocytogenes is consistent with this alternative interpretation (21).
In the present study, we confirm the findings of Kagi and colleagues that splenocytes from immunized PO mice are deficient relative to splenocytes from B6 mice in the transfer of antilisterial immunity. We then make use of single Ag-specific CD8+ T cell lines that are uniformly restimulated in vitro to test the hypothesis that perforin is required for CD8+ T cell-mediated immunity to L. monocytogenes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57Bl/6 (H-2b MHC) mice were obtained from the National Cancer Institute (Frederick, MD). PO (H-2b MHC) mice were kindly provided by Dr. W. R. Clark (15) and then maintained by brother-sister mating. MRL lpr/lpr (H-2b MHC) mice were the kind gift of Dr. M. O. Dailey at the University of Iowa. All mice were housed under specific pathogen-free conditions at the University of Iowa and were generally used at 8 to 12 wk of age in an age- and sex-matched fashion.
Bacteria
L. monocytogenes strain 10403s (22) and Salmonella typhimurium strain SL1344 (23) are both resistant to streptomycin and were used as previously described (24). Briefly, bacteria were grown in tryptic soy broth to an OD600 of approximately 0.1, diluted in pyrogen-free 0.9% sodium chloride (Abbott Laboratories, North Chicago, IL) and injected i.v. in 0.2- to 0.5-ml vol per animal. Aliquots were plated onto tryptic soy agar containing 50 µg/ml of 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-2d MHC) (American Type Culture Collection (ATCC), Rockville, MD; TIB-64); EL4 is a B6-derived thymoma cell line (H-2b MHC) (ATCC TIB-39); EL4-LLO refers to EL4 cells stably transfected with a plasmid construct expressing the L. monocytogenes Ag listeriolysin O (LLO) and neo-resistance (25); L-929 is a C3H/An-derived fibroblast cell line (H-2k MHC) (ATCC CCL-1); L-Kb refers to L-929 cells stably transfected with the gene for H-2Kb (26) and neo-resistance; L-Kb-LLO refers to L-929 cells stably transfected with the LLO expression construct (27) and the H-2Kb gene. Electroporation and selection of transfectants with G-418 were performed as described (24). Parental cell lines were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS, antibiotics, L-glutamine, HEPES buffer, and 2-ME (RP10 (24)). Transfected cells were maintained in RP10 supplemented with G-418 at 400 µg/ml.
Expression of H-2Kb by L-Kb-LLO cells was confirmed in 51Cr release assays in which CD8+ T cells specific for the OVA peptide SIINFEKL bound to H-2Kb (28, 29) were observed to specifically lyse L-Kb-LLO cells in the presence (but not in the absence) of 2 nM of SIINFEKL.
mAbs, which were purified from culture supernatants and quantitated as
previously described (8), were: rat anti-mouse TNF- IgG1 (XT22
and XT3 (30) used in combination at a mass ratio of 1:1) and rat
anti-mouse IFN- IgG1 (XMG1.2 (31)). Control polyclonal rat IgG
was purchased from Sigma (St. Louis, MO). Armenian hamster
anti-mouse CD95 IgG (Jo2) and control Armenian hamster polyclonal
IgG were purchased from PharMingen (San Diego, CA). Flow cytometric
analysis of T cell subsets was performed as previously described (8)
using FITC-conjugated anti-CD8 (53.6–7; Sigma) and
phycoerythrin-conjugated anti-CD4 (H129.19; Sigma). T cell subset
depletion by mAb and complement was performed using rat anti-mouse
CD4 (RL172) and rat anti-mouse CD8 (3.168) as previously
described (8).
Generation and maintenance of CD8+ T cell lines
CD8+ T cell lines specific for LLO were derived
from B6 and PO mice using methods previously described (25). Briefly, 2
to 4 x 107 splenocytes from mice injected 7 to 10
days previously with 104 CFU of virulent L.
monocytogenes were incubated with 3 x 106
irradiated (150 Gy) EL4-LLO cells in RP10. Subsequent weekly
restimulations were conducted by combining 1 to 3 x
106 responder cells with 3 x 106
irradiated stimulator cells and approximately 4 x 107
irradiated (30 Gy) syngeneic splenocytes in RP10 supplemented with 5%
supernatant from Con A-stimulated rat spleen cells and 50 mM -methyl
mannoside.
In vitro characterization of CD8+ T cell lines
51Cr release assays were performed as
previously described (32, 8). Briefly, 104 labeled target
cells were combined with effector cells at the indicated ratios in 200
µl of RP10 per well in round-bottom 96-well plates. Following a 4- to
8.5-h incubation (as indicated), 100 µl per well of supernatant was
harvested and assayed for 51Cr release. Percent specific
release of 51Cr was calculated by the formula:
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TNF- was quantitated using WEHI 164 clone 13 cells, which die in the
presence of TNF (33). Supernatants (50 µl from each well) from the
indicated 51Cr release assays or analogous
(nonradioactive) co-incubations of CD8+ T cells and target
cells were added to 50 µl of WEHI 164 cells (6 x
105/ml in RP10 supplemented with 2 µg/ml of actinomycin D
and 40 mM LiCl2) in flat-bottom 96-well plates. Following
overnight incubation at 37°C and 7% CO2, 10 µl/well of
Alamar blue (Acumed, West Lake, OH) was added. The percent death of the
indicator cells, a relative measure of TNF production, was determined 2
to 6 h after addition of Alamar blue from the OD570
and OD600 according to the manufacturer’s protocol.
Production of TNF by target cells in the absence of CD8+ T
cells was not detected. In some experiments, mAb specific for TNF-
(a 1:1 mix of XT22 and XT3 at a final concentration of 50 µg/ml) or
control rat IgG were added to the target cells before addition of the
CD8+ T cells. Recombinant murine TNF- was used as a
control and to determine the detection limits of the WEHI bioassay.
Concentrations <1 pg/ml of rTNF- were routinely detected using this
assay.
IFN- was quantitated by ELISA. The 96-well flat-bottom plates were
coated with 600 ng/ml of XMG1.2 in bicarbonate buffer (pH 9.6) at 4°C
for 24 h, blocked with 1% BSA in PBS at 37°C for 1 h, and
washed with 0.05% Tween-20 in PBS. Supernatants (100 µl from each
well) from co-incubations of effector cells and target cells and
rIFN- controls were added and incubated at room temperature for 2 to
3 h. Following three rinses, 100 µl per well of rabbit
anti-mouse IFN- (a kind gift of Dr. John Cowdery, University of
Iowa; diluted 1:1500 in PBS-Tween) was added and incubated at room
temperature for 1 to 2 h. Following three rinses, 100 µl per
well of alkaline phosphatase-conjugated goat anti-rabbit Ig (Sigma;
diluted 1:20,000 in PBS-Tween) was added and incubated at 37°C for
1 h. Following three rinses, alkaline phosphatase substrate
(Sigma) in 1 M diethanolamine, 0.5 mM MgCl2 (pH 9.8) was
added and developed according to the manufacturer’s protocol. OD was
measured at 405 nm. Limit of detection was 10 U/ml.
Adoptive transfer experiments
The capacity of splenocytes derived from immunized animals and CD8+ T cell lines to mediate antilisterial immunity in vivo was quantitated using adoptive transfer assays as described previously (32, 8). Briefly, RBC-depleted splenocytes from donor mice immunized 7 to 10 days previously with approximately 104 virulent L. monocytogenes, or CD8+ T cells restimulated in vitro 7 to 9 days previously, were harvested, washed in antibiotic-free buffer, and resuspended in pyrogen-free 0.9% sodium chloride. Cells were delivered i.v. in 0.2 to 0.5 ml vol into naive B6 or MRL lpr/lpr host mice. Within 2 h, host mice, including uninjected or saline-injected naive controls, were challenged i.v. with the indicated dose of 10403s or SL1344. CFU/spleen and liver were determined 3 days postchallenge by homogenizing the spleens and livers in 0.2% IGEPAL (Sigma), plating 10-fold serial dilutions onto tryptic soy agar containing 50 µg/ml of streptomycin, and calculating colony count averages after overnight incubation at 37°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Perforin is a vital component in the protective
CD8+ T cell response to some but not all viruses
(17, 18, 19, 20, 34, 35, 36). Previous studies with perforin gene knockout (PO)
mice suggest a role for perforin-mediated cytolysis in CD8+
T cell-mediated immunity to L. monocytogenes (21).
Consistent with these studies, we found that splenocytes from immunized
B6 (H-2b MHC) mice were more effective than splenocytes
from immunized PO (H-2b MHC) mice at transferring
antilisterial immunity to B6 recipients as measured by the number of
L. monocytogenes recovered at 3 days postinfection from
control groups and splenocyte recipients (Fig. 1
). Depletion of the CD8+ T
cells from the immune PO splenocytes further reduced their ability to
transfer antilisterial immunity (Fig. 1), suggesting the presence of a
perforin-independent, CD8+ T cell-mediated pathway of
antilisterial immunity.
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and TNF- but are deficient in target cell lysis in vitro To further address the impact of perforin deficiency on CD8+ T cell immunity to L. monocytogenes, we generated CD8+ T cell lines, specific for the same L. monocytogenes Ag, from MHC-matched PO and B6 mice. Splenocytes from B6 and PO mice, immunized 7 days previously with approximately 104 CFU of virulent L. monocytogenes, were cultured in vitro with syngeneic irradiated stimulator cells that express the L. monocytogenes CD8+ T cell Ag, LLO (EL4-LLO cells) (25). Following 4 wk of in vitro restimulation, flow cytometric analysis demonstrated that >95% of the cells displayed a CD4-, CD8+ phenotype (data not shown).
To measure cytokine production and verify Ag-specificity, LLO-specific
CD8+ T cells were assayed for IFN- and TNF- release
in response to Ag-expressing and control target cells. Both B6- and
PO-derived LLO-specific CD8+ T cells produced IFN- when
incubated with EL4-LLO cells, but not in response to the parental,
nontransfected EL4 cells (Fig. 2A). Similarly, TNF-
was produced by both B6- and PO-derived LLO-specific CD8+ T
cells in response to EL4-LLO but not EL4 cells (Fig. 2B). Nontransfected EL4 cells, incubated with
HPLC-purified peptides derived from L.
monocytogenes-infected B6 spleens (27), also induced TNF-
production by both B6- and PO-derived LLO-specific CD8+ T
cells (data not shown). The same peptide fraction sensitized EL4 cells
for lysis by B6-derived LLO-specific CD8+ T cells (data not
shown).
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C). Short-term assays also revealed that
target cell lysis by B6-derived LLO-specific CD8+ T cells
was inhibited by EGTA, consistent with the Ca2+ dependency
of perforin-mediated cytolysis (37) (data not shown). These results are
consistent with a lack of perforin-dependent cytolysis by PO-derived
CD8+ T cells and a functional perforin-dependent pathway in
CD8+ T cells derived from B6 mice. Thus, we have generated
LLO-specific CD8+ T cell lines from B6 and PO mice that
produce IFN- and TNF- in an Ag-specific fashion but that differ
in their ability to perform perforin-dependent cytolysis. PO-derived LLO-specific CD8+ T cells mediate delayed target cell lysis and possess a functional CD95-dependent cytolytic pathway
CD8+ T cells can also kill target cells via the
CD95L/CD95 pathway (38). To test the ability of PO-derived LLO-specific
CD8+ T cells to mediate perforin-independent cytolysis, we
performed extended chromium release assays (Fig. 3A). This approach
revealed that PO-derived LLO-specific CD8+ T cells were
able to carry out Ag-specific target cell lysis that is delayed
compared with cytolysis mediated by B6-derived CD8+ T
cells. We then tested whether in vitro lytic activity mediated by
PO-derived LLO-specific CD8+ T cells was dependent upon
CD95L or TNF-. Extended assays revealed that specific target cell
lysis by PO-derived LLO-specific CD8+ T cells was not
inhibited by neutralizing mAb against TNF- (Fig. 3A). Control assays for TNF-, performed on
supernatants derived from the cytotoxicity assays in Figure 3A, verified the complete neutralization of soluble TNF-
by the anti-TNF- mAb (Fig. 3B). To test for
CD95L/CD95-dependent killing, we used CD95-expressing (data not shown)
murine fibroblasts as targets. PO-derived LLO-specific CD8+
T cell-mediated cytolysis of L929 murine fibroblasts (H-2k
MHC) transfected with expression constructs for H-2Kb and
LLO (L-Kb-LLO), but not L929 cells transfected with H-2Kb
alone (L-Kb) (Fig. 3C). Ag-specific lysis by
PO-derived LLO-specific CD8+ T cells was completely
inhibited in the presence of a mAb that binds CD95 (Fig. 3C). Six hours of exposure to the same concentration
of anti-CD95 mAb did not kill the targets nor the CD8+
T cells (data not shown). These experiments demonstrate that
LLO-specific CD8+ T cells from PO mice are capable of
CD95-dependent target cell lysis in vitro.
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B6-derived LLO-specific CD8+ T cells provide
antilisterial immunity in adoptive transfer assays (25). To determine
the requirement for perforin in CD8+ T cell-mediated
immunity against L. monocytogenes, LLO-specific
CD8+ T cells were transferred into naive B6 host mice,
which were subsequently challenged with approximately 10
LD50 of L. monocytogenes. B6- and PO-derived
LLO-specific CD8+ T cells provide antilisterial immunity in
both the spleen and liver (Fig. 4). In
the liver, B6- and PO-derived LLO-specific CD8+ T cells
transferred similar degrees of immunity, as measured by reduction in
CFU per organ (Fig. 4C). Over multiple experiments,
the degree of immunity in the spleen transferred by PO-derived
LLO-specific CD8+ T cells varied from intermediate
(approximately 2 log10 CFU reduction as seen in Fig. 4A) to equivalent (3–4 log10 CFU
reduction) to that typically seen with B6-derived LLO-specific
CD8+ T cells (Fig. 6A). In a survival
study, 100% of mice that received PO-derived LLO-specific
CD8+ T cells survived 1 wk postchallenge, while all mice
that did not receive T cells died within 4 days. These results
demonstrate that PO CD8+ T cells can mediate significant
antilisterial immunity in both the spleen and liver.
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). To determine whether B6- and PO-derived
LLO specific CD8+ T cells provide antilisterial immunity
via a CD95-dependent pathway, B6- and PO-derived LLO-specific
CD8+ T cells were transferred into naive H-2b
MHC MRL lpr/lpr host mice (genetically deficient in CD95
expression (39)). T cell recipients and control MRL lpr/lpr
mice were then challenged with virulent L. monocytogenes,
and CFU/organ were determined 3 days later. The results revealed that
both B6- and PO-derived LLO-specific CD8+ T cells provide
antilisterial immunity in the spleen (Fig. 4B) and
liver (Fig. 4D) in the absence of CD95. In these
experiments, the degree of immunity transferred by PO-derived
LLO-specific CD8+ T cells was consistently equivalent to
the degree of immunity transferred by B6-derived LLO-specific
CD8+ T cells. Thus, in the absence of both pathways
required for efficient in vitro cytolysis (perforin-dependent
and CD95-dependent (9)), LLO-specific CD8+ T cells
are capable of providing antilisterial immunity. In vivo immunity mediated by PO-derived CD8+ T cells is Ag-specific
The in vivo antilisterial immunity conducted by PO-derived
LLO-specific CD8+ T cells was Ag-specific. PO-derived
LLO-specific CD8+ T cells did not cause a reduction in CFU
in B6 mice challenged with the unrelated bacterium S.
typhimurium (Fig. 5, A
and C). Likewise, transfer of a large number of
PO-derived alloreactive (H-2b anti-H-2d)
CD8+ T cells into naive B6 mice did not result in a
significant reduction in CFU following challenge with L.
monocytogenes (Fig. 5, B and D).
Specificity of PO allospecific CD8+ T cells for
H-2d was verified in extended chromium release assays using
P815 target cells (data not shown). These results confirm previous
findings that CD8+ T cells provide Ag-specific immunity in
transfer assays (32) and extend this finding to immunity mediated by
PO-derived LLO-specific CD8+ T cells.
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Previous studies with in vivo Ab neutralization (40) and IFN-
knockout mice (8) revealed that CD8+ T cell-derived
IFN- was not required for antilisterial immunity but did not rule
out the possibility that CD8+ T cell-derived IFN-
normally contributes to antilisterial immunity or that it plays a major
role in the absence of perforin-dependent cytotoxicity (21). PO-derived
LLO-specific CD8+ T cells clearly produce IFN- in
response to specific target cells (Fig. 2). To determine whether
LLO-specific CD8+ T cells from PO mice require IFN- to
mediate antilisterial immunity, we performed transfer experiments into
naive B6 mice that had been injected 1 day previously with a high dose
(1 mg/animal) of neutralizing anti-IFN- mAb. Since IFN- plays
an important role in the innate response to L. monocytogenes
(8, 25), pretreatment of mice with neutralizing mAb to IFN- results
in a severe exacerbation of infection. Preliminary studies revealed
that doses of mAb as low as 500 µg/animal resulted in maximal
exacerbation of infection at 3 days postchallenge (data not shown).
Previous studies have shown that mAb neutralization of IFN- in
normal mice (40) exacerbates L. monocytogenes infection to a
similar degree as seen in IFN- gene knockout mice (8). To prevent an
increased bacterial load in IFN--depleted animals, which would make
it difficult to compare T cell function in depleted vs control animals,
the dose of L. monocytogenes used to challenge
IFN--depleted animals was reduced by 50% relative to the dose used
to challenge rat IgG-treated animals. Even with the reduced challenge
dose, L. monocytogenes infection in mice that received no T
cells was exacerbated in the spleens (>30-fold) and the livers
(>100-fold) by pretreatment with anti-IFN- mAb compared with
rat IgG-pretreated mice (Fig. 6). These
results, combined with studies of L. monocytogenes infection
in IFN- gene knockout mice (8) and mice injected with neutralizing
mAb against IFN- (40), indicate that the mAb treatment resulted in
effective neutralization of IFN- in vivo. Despite the severity of
the infection, PO-derived LLO-specific CD8+ T cells
provided immunity to L. monocytogenes infection in
anti-IFN--treated mice (Fig. 6, B and
D) that is at least equivalent to that seen in mice
treated with rat IgG (Fig. 6, A and C).
Thus, antilisterial immunity mediated by PO-derived LLO-specific
CD8+ T cells is not inhibited by neutralization of
IFN-.
LLO-specific CD8+ T cells from PO mice fail to
transfer antilisterial immunity to hosts depleted of TNF-
Besides IFN-, PO-derived LLO-specific CD8+ T
cells produce TNF- in response to specific target cells (Fig. 2). To
determine whether LLO-specific CD8+ T cells from PO mice
require TNF- to mediate antilisterial immunity, we performed
transfer experiments into naive B6 mice that had been injected 1 day
previously with mAb that neutralize TNF-. In contrast to the result
observed following depletion of IFN-, depletion of TNF- severely
abrogated the ability of PO-derived CD8+ T cells to mediate
antilisterial immunity in vivo (Fig. 7).
Polyclonal splenocytes from previously immunized PO mice also failed to
transfer immunity to naive animals that had been treated with
anti-TNF- mAb, but provided normal levels of protection in hosts
preinjected with control IgG (data not shown). As is the case with
IFN-, TNF- plays an important role in the innate response to
L. monocytogenes (8, 25). Consequently, the dose of L.
monocytogenes used to challenge TNF--depleted animals was
reduced relative to the dose used to challenge rat IgG-treated animals.
Reduction of the challenge dose by 100-fold resulted in approximately
equal degrees of infection in TNF--depleted animals compared with
rat IgG-treated mice that received no T cells (Fig. 7). Despite the
lower challenge dose, PO-derived LLO-specific CD8+ T cells
failed to mediate immunity to L. monocytogenes in
anti-TNF--treated mice (Fig. 7, B and
D), whereas the same T cells provided significant
immunity in mice treated with control rat IgG (Fig. 7, A and
C). In additional experiments, in which
TNF--depleted and control animals were challenged with equivalent
high doses of L. monocytogenes, PO-derived CD8+
T cells also failed to provide protection (data not shown). Thus,
antilisterial immunity mediated by PO-derived LLO-specific
CD8+ T cells is dependent on TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but does
require TNF-.
The use of Ag-specific CD8+ T cell lines from wild-type and
perforin gene knockout mice allowed us to extensively characterize the
effector functions of these cells in vitro before their analysis in
vivo. While the perforin-dependent pathway accounts for most short-term
in vitro cytolysis, CD8+ T cells can also kill target cells
via the CD95L/CD95 pathway (38) and perhaps by production of TNF-
(41, 42). Our in vitro studies demonstrated that both B6-derived and
PO-derived LLO-specific CD8+ T cells lyse target cells and
produce IFN- and TNF- in an Ag-specific fashion. Their cytolytic
mechanism, however, is clearly different. Whereas B6-derived
LLO-specific CD8+ T cells mediate rapid,
Ca2+-dependent cytolysis, PO-derived LLO-specific
CD8+ T cells mediate delayed cytolysis, which is
independent of soluble TNF- and dependent on CD95. These results
suggest that the only major difference between the wild-type and
PO-derived CD8+ T cells is the lack of perforin-dependent
cytolysis.
The level of immunity provided by PO-derived CD8+ T cells,
at least in the liver, is indistinguishable from that provided by
wild-type CD8+ T cells. These results are consistent with
experiments using immune polyclonal splenocytes from PO mice (Fig. 1
and (21)), which do not rule out the existence of a
perforin-independent CD8+ T cell-mediated pathway of
antilisterial immunity. However, while the lack of perforin has little
impact on immunity mediated by single Ag-specific PO-derived
CD8+ T cells, perforin deficiency dramatically impairs
antilisterial immunity mediated by polyclonal CD8+ T cells
from immunized PO mice (21). This difference may be a consequence of
the uniform activation of CD8+ T cells provided by in vitro
restimulation compared with the complex in vivo setting where other
immunologic parameters may decrease priming of L.
monocytogenes specific CD8+ T cells in PO mice. This
possibility is consistent with the finding that CD8+ T
cell-mediated antilisterial immunity is at least partially functional
in the absence of perforin (Fig. 1 and (21)). In addition, this notion
is consistent with the observation that the innate immune response in
PO animals is hyperactive, resulting in increased resistance to primary
L. monocytogenes infection (21). Since the innate immune
response functions to inhibit early bacterial replication (43), a
heightened innate response might decrease Ag levels and result in
suboptimal CD8+ T cell priming in PO mice. Thus, the lack
of perforin-dependent cytotoxicity may not be the sole reason for the
partial loss of CD8+ T cell-mediated antilisterial immunity
observed with PO-derived splenocytes. Our single Ag-specific
CD8+ T cell lines, which are uniformly restimulated in
vitro, provide a method to overcome the potential differences in
priming of specific CD8+ T cells which may occur in mice
with different levels of innate immunity to L.
monocytogenes. Alternatively, the results may be specific for
CD8+ T cell responses to LLO, an immunodominant L.
monocytogenes Ag (44), as a target for protective CD8+
T cells.
The observation that PO-deficient CD8+ T cells provide
antilisterial immunity in the presence of an IFN--specific
neutralizing mAb is consistent with our previous studies demonstrating
that perforin-expressing CD8+ T cells can provide immunity
to L. monocytogenes infection in the absence of IFN- (8).
It should be pointed out that our results do not rule out a
contribution from perforin or IFN- in CD8+ T
cell-mediated antilisterial immunity in wild-type mice. However, our
results do suggest that these effector functions are not required for
antilisterial immunity mediated by single Ag-specific CD8+
T cells. In addition, the current results suggest that IFN-
expression is not the only effector function that can compensate for
perforin deficiency, and vice versa. Despite the ability of the
neutralizing anti-IFN- mAb to dramatically exacerbate L.
monocytogenes infection, it is possible that this treatment is
insufficient to completely eliminate the contribution of
CD8+ T cell-derived IFN-.
Our experiments suggest a critical role for TNF- in PO
CD8+ T cell immunity to L. monocytogenes. These
results are consistent with the results of Samsom and colleagues (45),
who reported that the secondary response to L. monocytogenes
in wild-type mice is inhibited by depletion of TNF- but not IFN-.
Neither of these systems, however, addresses the cellular source of the
required TNF-. CD8+ T cell-derived TNF- could mediate
antilisterial immunity by activating macrophages (46), recruiting
accessory phagocytes to sites of infection (47), or by directly
inducing the death of infected target cells (41, 42). Whether
CD8+ T cells produce the required TNF-, or it is
elaborated by another cell type and is simply required for the
expression of immunity by CD8+ T cells, requires further
investigation.
A number of experiments have addressed the mechanisms by which CD8+ T cells provide immunity to various pathogens. These include L. monocytogenes (21), vaccinia virus (48, 17), LCMV, vesicular stomatitis virus, Semliki forest virus (17), murine rotavirus (19), hepatitis B virus (18, 49), ectromelia virus (50), mouse hepatitis virus (20), Mycobacterium tuberculosis (34, 35, 51), and Plasmodium berghei (36). While perforin is a vital component in the protective CD8+ T cell response to some viruses, such as LCMV (12, 15), it is not required for clearance of all intracellular pathogens (17, 18, 19, 20, 34, 35, 36). Collectively, these studies suggest that CD8+ T cells can mediate resistance in vivo to some pathogens that is independent of efficient cytolytic pathways. The mechanisms utilized by CD8+ T cells to mediate antimicrobial resistance may vary from pathogen to pathogen and vary in effectiveness. Understanding the character of an effective CD8+ T cell response against specific pathogens may improve our ability to immunize healthy as well as immunocompromised patients against infectious agents.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. T. Harty, 3–512 Bowen Science Building, Department of Microbiology, University of Iowa, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: PCD, programmed cell death; PO, perforin deficient; LLO, listeriolysin O; LCMV, lymphocytic choriomeningitis virus.
Received for publication August 7, 1997. Accepted for publication October 7, 1997.
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