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1
*
Department of Microbiology and
Interdisciplinary Graduate Program in Immunology, 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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-deficient
(GKO) or perforin-deficient (PKO) mice provide substantial immunity
against murine infection with Listeria monocytogenes. To
address the potential for redundancy between perforin and IFN- as
CD8+ T cell effector mechanisms, we generated
perforin/IFN- (PKO/GKO) double-deficient mice. PKO/GKO-derived
CD8+ T cells specific for the immunodominant listeriolysin
O (LLO91–99) epitope provide immunity to LM infection
similar to that provided by Ag-matched wild-type (WT) CD8+
T cells in the liver but reduced in the spleen. Strikingly, polyclonal
CD8+ T cells from immunized PKO/GKO mice were 
100-fold
more potent in reducing bacterial numbers than the same number of
polyclonal CD8+ T cells from immunized WT mice. This result
is probably quantitative, because the frequency of the CD8+
T cell response against the immunodominant LLO91–99
epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days
after identical immunizations. Moreover, PKO/GKO mice can be immunized
by a single infection with attenuated Listeria to resist
>80,000-fold higher challenges with virulent organisms than naive
PKO/GKO mice. These data demonstrate that neither perforin nor IFN-
is required for the development or expression of adaptive immunity to
LM. In addition, the results suggest the potential for perforin and
IFN- to regulate the magnitude of the CD8+ T cell
response to infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Priming of naive CD8+ T cells results from their
encounter with professional APC, such as dendritic cells, that express
pathogen peptides in the context of self MHC class I molecules and also
express high levels of costimulatory molecules. Once a
CD8+ T cell is activated, it is capable of
recognizing any infected cell and elaborating a number of effector
functions that aid the immune system in the clearance of the pathogen.
Previously activated effector or memory 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 and in other infectious disease
models in which CD8+ T cells are important
mediators of the resistance (8). Moreover, efficient
Ag-specific lysis of a target cell harboring an intracellular pathogen
is a function largely limited to CD8+ T cells. At
least two molecular pathways have been identified by which
CD8+ T cells mediate cytolysis (9, 10). The granule exocytosis pathway requires the coordinated
activity of perforin and granzymes, both of which are found in the
granules of activated CD8+ T cells, to activate
the caspase cascade of the target cell and induce apoptosis
(11). Activated CD8+ T cells also
express CD95 ligand, which can ligate CD95 (Fas, Apo-1) on a target
cells and induce apoptosis via the caspase cascade.
Recent studies to address the roles of these effector mechanisms in resistance to infection rely primarily on two approaches: in vivo treatments with mAbs and application of specific gene knockout mice. These approaches are complicated by the fact that most, if not all, of the potential effector mechanisms are expressed by multiple cell types. In addition, it is important to note that many of these molecules have the potential to play regulatory roles in the immune response, ranging from T cell homeostasis to enhancement of Ag presentation. Thus, our current understanding of which effector mechanisms resist specific pathogens is lacking.
Early reports using perforin-deficient mice revealed that
CD8+ T cell immunity against LM was 10- to
100-fold reduced compared with that in wild-type (WT) mice
(12), while IFN--deficient mice developed
CD8+ T cell immunity to LM indistinguishable from
that of WT mice (13). Although studies with Ab
neutralization of IFN- have provided disparate results (14, 15), the demonstration by Unanue and coworkers (16)
that IFN--deficient CD8+ T cells, but not
IFN--deficient CD4+ T cells, are able to clear
chronic LM infection of mice with severe combined immunodeficiency
supports the contention that IFN- is not required for
CD8+ T cell immunity to LM (13).
More recently, experiments with perforin-deficient
CD8+ T cells that are specific for single
LM Ags demonstrated the existence of a pathway of resistance that
is independent of both perforin- and CD95-mediated cytolysis
(17).
The finding that no single effector mechanism studied to date can
account for all the anti-listerial resistance mediated by
CD8+ T cells has led to the hypothesis that
multiple mechanisms, each capable of functioning independently, may
provide anti-listerial immunity (1). In the present
study we address the potential for redundancy in major
CD8+ T cell effector mechanisms using the LM
experimental model and perforin/IFN- (PKO/GKO) double-deficient
mice. We present evidence that neither the major cytolytic pathway nor
IFN- production is required for the development or expression of
CD8+ T cell-dependent adaptive immunity to LM.
However, our results are consistent with a regulatory role for these
molecules in control of the CD8+ T cell response
to infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (H-2d MHC) mice were obtained from
the National Cancer Institute (Frederick, MD).
H-2d MHC perforin (PKO) and IFN- (GKO)
double-deficient (PKO/GKO) mice were generated by crossing
H-2d MHC PKO (18) and GKO
(13) mice (both on the BALB/c background). Because
perforin and IFN- are both on chromosome 10,
F1 mice were backcrossed to
H-2d PKO mice, and
PKO-/-/GKO+/- progeny,
resulting from meiotic recombination, were identified by screening for
perforin and IFN- deficiency as previously described (13, 18) and mated to generate PKO/GKO double-deficient mice. PKO/GKO
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 until the initiation of experiments with LM, at which
point the mice were transferred to standard housing. All mice were used
at 8–16 wk of age.
Cell lines and Abs
P815 cells are a DBA/2 (H-2d)-derived mastocytoma tumor cell line (American Type Culture Collection, Manassas, VA; ATCC TIB-64); P815-LLO refers to P815 cells stably transfected with a plasmid construct expressing the LM Ag listeriolysin O (LLO) (19) and the G-418 resistance gene (20). P815-p60 refers to P815 cells stably transfected with a plasmid construct expressing the LM Ag p60 (21) and the G-418 resistance gene (22). Cells were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS, antibiotics, L-glutamine, HEPES buffer, and 2-ME (RP10) (13). Transfected cells were maintained in RP10 supplemented with G-418 at 400 µg/ml.
In vivo CD8+ T cell subset depletion was performed with anti-CD8 mAb 2.43 (23), which was purified from culture supernatants using protein G affinity chromatography as recommended by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). In vivo CD8+ T cell depletion was conducted by i.p. injection of 0.3 mg of mAb 2.43 on days -1, -2, and -3 relative to infection as previously described (13). In vitro T cell subset depletion was conducted with IgM mAbs RL-172 (anti-CD4) or 3.168 (anti-CD8) and rabbit complement as previously described (24). Depletion of T cell subsets was monitored by staining with FITC-conjugated anti-CD8 mAb 53.6–7 (PharMingen, San Diego, CA) and PE-conjugated anti-CD4 mAb H129.19 (PharMingen). Cells were analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ) using Flow Jo (Tree Star, San Carlos, CA) software. Armenian hamster anti-mouse CD95 IgG (Jo2) and control Armenian hamster polyclonal IgG were purchased from PharMingen.
Bacteria and immunizations
Virulent LM strain 10403s (25), attenuated LM
strain DP-L1942 (ActA deficient) (26), and Y92F LM mutant
strain DP-L2528 (27) are resistant to streptomycin and
were used as previously described (13). Briefly, frozen
stocks of bacteria were grown in a bacterial shaker at 37°C in
tryptic soy broth to an OD600 of 0.1
(108 CFU/ml), diluted in pyrogen-free 0.9%
sodium chloride (Abbott Laboratories, North Chicago, IL), and injected
i.v. in a 0.2-ml volume/animal. Aliquots were plated onto tryptic soy
agar containing 50 µg/ml of streptomycin (TSB-Strep) to verify the
number of CFU injected.
Generation and maintenance of CD8+ T cell lines
Several H-2d MHC CD8+ T cell lines
specific for LLO91–99 or p60217–225 were
derived from individual LM-infected WT (BALB/c) and PKO/GKO mice and
were restimulated with P815-LLO or P815-p60 cells as previously
described (13). Briefly, 2–4 x
107 splenocytes from mice injected 7 days
previously with 103 CFU of virulent LM strain
10403s i.v. or 106 attenuated LM DP-L1942 (in the
case of PKO/GKO mice) i.v. were incubated in RP10 at 37°C in 7%
CO2 with 3 x 106
irradiated (150 Gy) P815-LLO or P815-p60 stimulator cells. Subsequent
weekly restimulations were conducted by combining 1–3 x
106 responder cells with 3 x
106 irradiated stimulator cells and 5 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.
51Cr release assays (CRA)
CRA were performed as previously described (13, 28). Briefly, 104 labeled target cells were combined with effector cells at the indicated ratios in 200 µl of RP10/well in round-bottom 96-well plates. Following a 3- to 9.5-h incubation (as indicated), 100 µl/well of supernatant was harvested and assayed for 51Cr release. The percent specific release of 51Cr was calculated by the formula: 100 x [(experimental cpm - spontaneous cpm)/(total cpm - spontaneous cpm)]. Spontaneous release was <20% of the total in all assays. Armenian hamster anti-mouse CD95 IgG (Jo2) and control Armenian hamster polyclonal IgG (PharMingen) were used in the CRA as indicated.
Adoptive transfer experiments
The capacity of splenocytes derived from immunized animals and
CD8+ T cell lines to mediate anti-listerial
immunity in vivo was quantitated using adoptive transfer assays as
described previously (13, 28). Briefly, RBC-depleted
splenocytes from donor mice immunized 7 days previously with
106 ActA-deficient LM strain DP-L1942 or
CD8+ T cells restimulated in vitro 7 days
previously were harvested, washed, and resuspended in pyrogen-free
0.9% sodium chloride. Cells were delivered i.v. in a 0.3-ml volume
into naive BALB/c host mice. Within 2 h host mice, including
saline-injected naive controls, were challenged i.v. with the indicated
dose of virulent LM strain 10403s. CFU per spleen and gram of liver
were determined 3 days postchallenge by homogenizing the spleens and
livers in 0.2% IGEPAL (Sigma, St. Louis, MO), plating 10-fold serial
dilutions onto TSB-Strep, and calculating colony count averages after
overnight incubation at 37°C.
Intracellular cytokine staining
Intracellular cytokine staining was performed using the
Cytofix/Cytoperm plus (with GolgiPlug) kit (PharMingen) as previously
described (29). Briefly, 20 x
106 splenocytes from an infected mouse were
treated with ACK lysis buffer for 5 min at room temperature to remove
RBC. Splenocytes were washed twice in RP10 and resuspended in the same
medium in 1 ml. Cells (200 µl) were incubated for 6 h at 37°C
with medium alone or with a synthetic peptide epitope consisting of aa
91–99 from the LM LLO molecule (LLO91–99, 200
nM), all in the presence of 1 µl/ml GolgiPlug (Brefeldin A). Cells
were washed twice in FACS buffer (PBS supplemented with 1% FCS and
NaN3) and were incubated with Ab directed against
the FcII/III receptors (2.4G2) and FITC-labeled anti-CD8
(53-6.7) on ice for 30 min. The cells were washed twice with FACS
buffer, then fixed and permeabilized by incubating for 15 min in 250
µl of Cytofix/Cytoperm solution. Then, cells were washed twice in
Perm/wash solution and stained with PE-conjugated anti-TNF
(MP6-XT22, PharMingen) for 30 min on ice. Cells were washed twice in
Perm/wash solution and resuspended in 350 µl of FACS buffer before
flow cytometry analysis.
The same procedure was followed for detection of intracellular TNF and
IFN- (XMG1.2, PharMingen) production by CD8+ T
cell lines, except that the CD8+ T cells were
incubated for 4 h with P815 cells in the presence or the absence
of synthetic LLO91–99 (200 nM) peptide at the
indicated E:T cell ratios.
Acquisition and analysis
List mode data were acquired on a FACScan flow cytometer (Becton Dickinson) using CYCLOPS software. Dead cells, monocytes, and tumor cells from cocultivation experiments were excluded by forward and side scatter gating. Typically, 25,000 events for CD8 T cell lines or 100,000 events for splenocytes were acquired for analysis with Flow Jo software (Tree Star).
The gate for TNF+ cells was selected such that the percentage of TNF+ cells in the unstimulated sample for each mouse was 0.5 ± 0.05% of the CD8+ splenocytes; this level has been subtracted from the peptide-stimulated splenocytes to determine the frequency of the response above the background.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been determined previously that IFN--deficient (GKO)
mice and IFN- receptor-deficient mice are extremely susceptible to
primary infection with virulent LM (13, 30). In contrast,
perforin-deficient (PKO) mice and WT mice are equally resistant to
primary infection with virulent LM (12, 17, 18). To
quantify the resistance of PKO/GKO double-deficient mice to primary
infection with virulent LM, groups of PKO/GKO mice were injected i.v.
with serially diluted aliquots of virulent LM 10403s and monitored for
survival. The WT mice were injected as controls. The estimated
LD50 of virulent LM 10403s in each strain of
mouse is displayed in Table I
. As
expected, based on their lack of IFN-, PKO/GKO mice were highly
susceptible to primary infection with virulent LM
(LD50, 10 organisms) and died 4–6 days
postchallenge at all lethal doses.
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) show that PKO/GKO and WT mice exhibit similar
resistance to high dose challenge (>106
organisms) with attenuated LM DP-L1942. PKO/GKO-derived LLO-specific CD8+ T cells mediate delayed, CD95-dependent cytotoxicity in vitro, and produce TNF in an Ag-specific fashion
LM Ag-specific CD8+ T cells from H-2d MHC GKO (13) or PKO (17, 18) single-knockout mice provide significant immunity to LM infection. To address the issue of redundancy in these effector mechanisms, we generated CD8+ T cell lines specific for the LM Ags LLO and p60 from DP-L1942-immunized PKO/GKO double-deficient mice by in vitro restimulation with P815-LLO or P815-p60 APC.
After several in vitro restimulations all lines were >95%
CD8+CD4-, as measured by
flow cytometry (data not shown). To verify the Ag specificity and
cytolytic activity of the CD8+ T cells we
performed CRA. Whereas WT-derived LLO-specific
CD8+ T cells were capable of high levels of
specific lysis of LLO-expressing target cells (Fig. 1) (13, 18)),
PKO/GKO-derived LLO-specific CD8+ T cells,
similar to LLO-specific CD8+ T cells from PKO
single-knockout mice (17, 18), mediated minimal levels of
specific lysis of the same target cells in a short term (3-h) assay
(Fig. 1). Similar results were obtained with WT- and PKO/GKO-derived
p60-specific CD8+ T cell lines (data not
shown).
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). Thus, we have generated LLO-specific
CD8+ T cell lines from perforin and IFN-
double-deficient mice that can mediate Ag-specific cytolysis through
CD95-CD95L interactions.
To measure cytokine production and verify Ag specificity, LLO-specific
CD8+ T cells from WT and PKO/GKO mice were
assayed for Ag-specific IFN- and TNF synthesis using intracellular
cytokine staining. Previous studies from our laboratory demonstrated
that detection of Ag-specific CD8+ T cells with
intracellular cytokine staining is clearly dependent on the E:T cell
ratio as well as the incubation time in the presence of the protein
transport inhibitor Brefeldin A (29). Under stimulation
conditions where LLO-specific WT CD8+ T cells
produce both TNF and IFN-, the PKO/GKO-derived LLO-specific
CD8+ T cell line produced TNF, but not IFN-
(Fig. 2). The response is specific,
because intracellular TNF was not detected after incubation with P815
cells in the absence of LLO91–99 peptide.
Similar results were obtained using a bioassay with WEHI 164 clone 13
cells that die in the presence of TNF (data not shown). Finally,
comparable results were obtained with the p60-specific PKO/GKO
CD8+ T cell line (data not shown).
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. PKO/GKO-derived LLO-specific CD8+ T cells provide specific immunity against LM infection
To determine the requirement for CD8+ T
cell-derived perforin and IFN- in adoptive transfer of
anti-listerial immunity, we analyzed the ability of LLO-specific
CD8+ T cell lines from PKO/GKO mice to transfer
immunity to naive WT host mice. The degree of immunity transferred was
estimated by infecting groups of mice with >10
LD50 of virulent LM strain 10403s in the presence
or the absence of LLO-specific CD8+ T cells and
determining the number of LM in organ homogenates at 3 days
postinfection.
PKO/GKO-derived CD8+ T cell lines specific for
LLO transferred significant immunity against LM infection. Immunity, as
estimated by CFU reduction, was more potent in the liver (3 log10)
than in the spleen (1.5 log10; Fig. 3,
A and B). We have shown previously that
Ag-specific CD8+ T cells from WT and GKO mice
reduced CFU to a similar degree in spleen and liver (13).
Similarly, PKO-derived CD8+ T cells reduced CFU
in the liver to the same extent as Ag-matched WT
CD8+ T cells (17, 18). In contrast,
the degree of immunity in the spleen mediated by PKO-derived
CD8+ T cells was typically less than that usually
observed with WT- or GKO-derived CD8+ T cells.
Thus, perforin-dependent organ-specific differences in immunity are
also observed with PKO/GKO-derived CD8+ T
cells.
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, C
and D). Thus, the immunity provided by PKO/GKO-derived
CD8+ T cells is Ag specific.
Finally, the CFU reductions mediated by PKO/GKO-derived-LLO-specific
CD8+ T cells are biologically significant, as
these cells confer dose-dependent survival against lethal LM infections
(Fig. 3E).
These experiments demonstrate that CD8+ T
cell-derived perforin and IFN- are not required for transfer of
anti-listerial resistance by single Ag-specific
CD8+ T cells.
PKO/GKO CD8+ T cells from LM-infected mice provide increased immunity to LM infection compared with WT CD8+ T cells
Perforin- and IFN--independent transfer of anti-listerial
immunity by relatively large numbers of in vitro-propagated single
Ag-specific CD8+ T cells provides evidence that
CD8+ T cell-derived perforin and IFN- are not
required for expression of immunity. However, this scenario does not
accurately reflect the in vivo situation, where
CD8+ T cell responses to multiple LM Ags would be
expected. To address this issue, we infected WT or PKO/GKO mice with LM
DP-L1942, and 7 days later equivalent numbers (5 x
107) of WT or PKO/GKO donor splenocytes were
transferred into naive WT host mice that were subsequently challenged
with a high dose (10 LD50) of virulent LM.
Bacterial counts in splenocyte recipients and control mice 3 days
postchallenge demonstrated that PKO/GKO-derived splenocytes provided
dramatic anti-listerial immunity in both spleens (Fig. 4A) and livers (Fig. 4B), reducing bacterial recovery >10,000-fold compared with
mice that did not receive immune splenocytes. Moreover, the degree of
immunity provided by the PKO/GKO immune splenocytes was significantly
(50- and 100-fold in the liver and spleen, respectively) higher than
provided by the same number of WT immune splenocytes (Fig. 4).
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). Depletion of the
majority of CD4+ T cells from immune PKO/GKO
splenocytes (>95% depletion as measured by CD8 vs CD4 staining; data
not shown) with Ab and complement did not significantly affect their
capacity to provide anti-listerial immunity compared with
mock-depleted splenocytes (Fig. 5, A and B). On
the other hand, depletion of >95% of the CD8+ T
cells (data not shown) eliminated >99.8%, based on CFU reduction, of
the protective immunity seen in mock-depleted splenocytes (Fig. 5, C and D). Immunity mediated by CD4-depleted
PKO/GKO splenocytes was dose dependent (Fig. 5, A and
B). Interestingly, immunity mediated by 2.5 x
107 PKO/GKO CD4+-depleted
immune splenocytes was still higher than that observed by twice as many
WT cells. Transfer of 5 x 106 CD4-depleted
PKO/GKO splenocytes did not result in significant immunity. Thus,
immunity provided by CD4-depleted immune PKO/GKO splenocytes is
>2-fold, but <10-fold, better than that of WT immune splenocytes on a
per cell basis. Together, these data are consistent with the idea that
elevated immunity provided by LM-immune splenocytes from PKO/GKO mice
is CD8+ T cell dependent.
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LLO-specific CD8+ T cell priming is significantly elevated in PKO/GKO double-deficient mice compared with WT mice
The increased efficiency of anti-listerial immunity provided by PKO/GKO CD8+ T cells compared with WT cells could result from quantitative or qualitative differences. To address this issue we analyzed the level of expansion of Ag-specific CD8+ T cells in WT and double-deficient PKO/GKO mice.
The WT and PKO/GKO mice were infected with DP-L1942, and at the peak of the primary response (day 7), the CD8+ T cell response against the immunodominant LLO91–99 epitope (31) was determined by intracellular TNF staining. Spleen cells freshly explanted from infected mice were cultured in vitro for 6 h either without stimulation (i.e., no peptide) or with LLO91–99 peptide. In some experiments the frequency of LLO-specific CD8+ T cell was determined in the same populations used in adoptive transfer assays.
Although intracellular IFN- staining appears to consistently detect
a higher frequency of Ag-specific CD8+ T cells
than detection of intracellular TNF (32, 33),
intracellular staining for TNF can be used to reliably detect
Ag-specific CD8+ T cell responses
(29), particularly in IFN- deficient mice.
Intracellular cytokine staining detects 2% (1 of 52)
LLO91–99-specific CD8+ T
cells on day 7 after immunization of WT BALB/c mice (Fig. 6, A and B). This
number is in good agreement with estimates of the frequency of
LLO-specific CD8+ T cells at the peak of the
primary response in WT mice, detected either by phenotypic (MHC class
I-peptide tetramer staining) or functional (IFN- enzyme-linked
immunospot) measures, after virulent LM infection (18, 34, 35, 36).
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, A and B). This increased Ag-specific response
was not completely dependent on delayed bacterial clearance, as only
three of six PKO/GKO mice had evidence of low levels (<2000 CFU) of
bacteria on day 7 postinfection (Fig. 6B).
Thus, in the absence of perforin and IFN-, priming of
LLO91–99-specific
CD8+ T cells is enhanced compared with that in WT
mice and may account for the increased immunity observed in adoptive
transfer assays.
Vaccination of PKO/GKO mice with attenuated LM induces specific resistance to virulent LM that is dependent on CD8+ T cells
The immunity mediated by PKO/GKO-derived
CD8+ T cells in the transfer experiments
described above occurred in WT recipients that are capable of
expressing IFN- in response to LM infection. To determine whether
secondary resistance to virulent LM challenge could be expressed in the
absence of perforin and IFN- we vaccinated PKO/GKO mice by infection
with 1 x 106 LM DP-L1942, and 35 days later
challenged the immunized mice with various doses of virulent LM 10403s.
Vaccinated PKO/GKO mice exhibited 80,000 times higher resistance
(LD50, 105.8 CFU; Table II) compared with naive PKO/GKO mice
(LD50, 10 CFU; Table I). Again, depletion of
CD8+ T cells before secondary challenge with
virulent LM revealed that CD8+ T cells are the
major effectors of acquired anti-listerial immunity in the PKO/GKO
mice (Table II).
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. In addition, the
difference in resistance in naive PKO/GKO mice and PKO/GKO mice
receiving immunization with an attenuated LM strain demonstrates that
vaccination-induced immunity can overcome the lack of a major cytolytic
pathway and the lack of a cytokine that is critical for resistance to
acute infection. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is required for the
development or expression of CD8+ T cell immunity
to LM infection. However, these molecules have a substantial
quantitative impact on the magnitude of the CD8+
T cell response to the immunodominant LLO91–99
epitope.
Lethal infection of H-2d PKO/GKO double-deficient
mice requires 1000-fold fewer LM than the number required for lethal
infection of mice with an intact IFN- structural gene. These data
are in line with data obtained with GKO single-deficient mice
(13) and confirm that IFN- is a critical mediator of
innate immunity to acute LM infection. In contrast, PKO/GKO mice resist
infection with an ActA-deficient LM strain at a level comparable to
that in WT mice. Although they are attenuated for virulence,
ActA-deficient LM enter the cytoplasm of infected cells and are
accessible to the endogenous MHC class I Ag presentation pathway.
Indeed, infection of WT and GKO mice with ActA-deficient LM elicits a
CD8+ T cell response that can transfer
anti-listerial immunity to naive WT mice (13, 37).
Because PKO/GKO mice survive infection with ActA-deficient LM, we used
this strain to elicit LM-specific CD8+ T
cells that are incapable of perforin-mediated cytolysis or IFN-
production.
PKO/GKO-derived CD8+ T cell lines specific for the single epitope derived from the secreted LLO molecule transferred Ag-specific anti-listerial immunity to naive WT mice. Similar to the results we obtained with CD8+ T cells from PKO single-deficient mice (17, 18), the level of immunity provided by the PKO/GKO-derived LLO-specific CD8+ T cell lines is greater in the liver than in the spleen. This organ-specific immunity is probably due to perforin deficiency, because LM-specific CD8+ T cell lines derived from GKO and WT mice transfer indistinguishable levels of immunity in both organs (13). On the other hand, adoptive transfer of polyclonal immune splenocytes from PKO/GKO mice reveals an unexpectedly high degree of CD8+ T cell-dependent anti-listerial immunity compared with immune splenocytes from WT mice.
The increased efficiency of anti-listerial immunity observed in adoptive transfer assays probably results from quantitative differences, because the PKO/GKO-derived immune splenocytes contain >4.5-fold more CD8+ T cells specific for the immunodominant LLO91–99 epitope than the WT populations. Further studies with ex vivo separated Ag-specific CD8+ T cells will be required to demonstrate whether qualitative differences in providing anti-listerial immunity exist between double-deficient and WT CD8+ T cells.
Taken together, the adoptive transfer experiments with
single-Ag-specific as well as polyclonal CD8+ T
cells showed that neither perforin nor IFN- is required as an
effector mechanism for CD8+ T cells to provide
substantial anti-listerial immunity. Thus, the capacity of PKO- or
GKO-derived single knockout CD8+ T cells to
provide anti-listerial immunity does not result from a paired
redundancy between these effector mechanisms. These findings raise
several important questions.
If CD8+ T cells can provide anti-listerial
immunity in the absence of two major effector mechanisms, what
CD8+ T cell effector mechanism(s) is responsible
for CD8+ T cell-mediated protection? PKO/GKO
CD8+ T cells produce TNF after specific Ag
recognition and TNF derived from CD8+ T cells
could participate in anti-listerial immunity by at least three
independent mechanisms. CD8+ T cell-derived TNF
might contribute to the maximal activation of listericidal capacities
in macrophages (38). Besides activation of proinflammatory
genes, signaling through TNFRI can activate the caspase cascade and
induce apoptosis (39), suggesting that
CD8+ T cells might deliver a TNF-dependent signal
resulting in the death of the infected cell. This would release LM into
the extracellular space, where they are susceptible to phagocytosis by
listericidal macrophages and neutrophils. Finally, TNF has been
demonstrated to up-regulate adhesion molecule expression on endothelial
cells, which could aid in the recruitment of accessory phagocytes to
the site of infection (40, 41, 42). Consistent with a role for
TNF in adaptive immunity against LM are our results showing that
Ab-mediated neutralization of TNF prevents anti-listerial
immunity mediated by PKO-derived CD8+ T cells
(17) and that similar treatments inhibited secondary
resistance to challenge infection of immune WT mice
(14). However, CD8+ T cells derived
from LM immune TNF-deficient (52)
and TNF receptor I-deficient4
animals were cytolytic, produced IFN- in an Ag-specific fashion, and
mediated substantial anti-listerial immunity when transferred to WT
mice. Although these results do not support an essential role for
CD8+ T cell-derived TNF in anti-listerial
immunity, they do not rule out the possibility that TNF is required as
a CD8+ T cell effector mechanisms in the absence
of perforin and IFN-.
Besides Ag-specific TNF production, PKO/GKO CD8+
T cells are also capable of mediating CD95L-CD95-dependent lysis of
APCs. The importance of CD95-dependent cytolysis in
CD8+ T cell control of LM was previously
addressed by determining the ability of LM Ag-specific
CD8+ T cells, from WT and PKO mice to provide
anti-listerial immunity to CD95-deficient
(B6.MRLlpr/lpr) hosts. It was shown that
LM Ag-specific CD8+ T cells, regardless of
whether they express perforin, are capable of providing
anti-listerial immunity that is independent of both cytolytic
pathways and is even independent of IFN- production
(17); however, immunity mediated by these
CD8+ T cells was inhibited by in vivo
neutralization of TNF, pointing out the possible requisite role for TNF
in settings completely devoid of perforin and IFN-.
Interestingly, recent work suggests that CD8+ T
cell-derived macrophage inhibitory protein-1 (MIP-1), a chemokine
produced by multiple cell types, including CD8+ T
lymphocytes, may be required for adaptive immunity to LM
(43). CD8+ T cells, derived from
LM-infected MIP-1-deficient mice, were capable of LM-specific
cytolysis in vitro, but failed to transfer anti-listerial immunity
to naive mice. These results suggest that MIP-1 and chemokine
production, in general, may represent another mechanism for
CD8+ T cell-mediated anti-listerial immunity.
Finally, CD8+ T cells may elaborate molecules
with direct microbicidal activity (44, 45). Purified
recombinant granulysin, a protein normally found in the granules of
activated human CD8+ T cells and NK cells,
mediates direct microbicidal activity against a range of pathogens,
including LM (45). It has been shown that the presence of
perforin is necessary for granulysin to inhibit the growth of
intracellular Mycobacterium tuberculosis (44).
Extension of these studies into the LM model is currently limited by
the absence of an identified mouse homologue of human granulysin.
Priming of LLO91–99-specific
CD8+ T cells in the absence of perforin and
IFN- is substantially greater than that in WT mice. In addition to
their potential as CD8+ T cell effector
mechanisms, most, if not all, of these molecules play regulatory roles
ranging from T cell homeostasis to enhancement of Ag presentation. The
expansion of LM-specific CD8+ T cells depends on
the infecting dose (our unpublished observation) (46).
PKO/GKO mice show a moderate delay in the clearance of LM in both
spleen and liver compared with WT mice, and the increased duration of
the infection may account for the increased Ag-specific
CD8+ T cell response seen in double-deficient
mice. Also, recent studies have shown a role for perforin in
down-regulating Ag-specific T cell responses during chronic viral
infection (47) and in response to superantigen stimulation
(48). In addition, the antiproliferative and cytotoxic
effects of IFN- could participate in the regulation of T cell
expansion (49, 50, 51). Further analysis of single- and
double-deficient mice should clarify these issues.
Finally, the ability of PKO/GKO mice to develop CD8+ T cell-mediated resistance to virulent LM after infection with an attenuated LM strain demonstrates that vaccination-induced immunity can overcome the lack of a major cytolytic pathway and a cytokine that is critical for resistance to acute infection. These studies suggest that specific gene knockout mice can be used as models of immune deficiency to learn whether an immune response can be generated that overcomes the specific dysfunction. Such studies may impact vaccine strategies for immunocompromised individuals.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, University of Iowa, 3-512 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242.
3 Abbrevations used in this paper: LM, Listeria monocytogenes; PKO, perforin knockout mice; GKO, IFN- knockout mice; WT, wild type; PKO/GKO, perforin/IFN- double-deficient mice; LLO, listeriolysin O; LD50, lethal dose for 50%; CRA, 51Cr release assay; MIP-1, macrophage inhibitory protein-1.
4 D. W. White, V. P. Badovinac, X. Fan, and J. T. Harty. Adaptive immunity against Listeria monocytogenes in the absence of the p55 TNF receptor type 1. Submitted for publication.
Received for publication December 30, 1999. Accepted for publication March 23, 2000.
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, 2 x 106; and E,
•, 7 x 106.
, average value ± SD. Data
are pooled from two independent experiments with three mice per group
in each experiment. CFU analyses were performed on livers with a limit
of detection of 500 LM/g of tissue. PKO/GKO mice with detectable
infection had <2000 LM/g of liver.