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*
Interdisciplinary Graduate Program in Immunology and
Department of Microbiology, University of Iowa, Iowa City, IA 52242; and
Sections of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 cytoplasm, LM multiplies and polymerizes host-derived F-actin, thereby initiating movement and direct spread to neighboring host cells (6, 7, 8, 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 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 response to LM (3, 11, 12, 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 cells.
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 results have demonstrated that IFN- is not required for
the development 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 infection in SCID mice (18).
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 can mediate cytolysis in vitro (19, 20). 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 (21, 22, 23). The absolute requirement for this pathway 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 pathway 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 undemonstrated.
Two studies on the role of perforin in secondary resistance and CD8+ T cell-mediated immunity to LM have been reported previously (29, 30). The first report demonstrated that CD8+ splenocytes 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-2b PO 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 immunity (30). However, this report concentrated on CD8+ T cells specific 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-2b haplotype 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-2b PO mice as well as the analysis of CD8+ T cells specific for more than one LM Ag. To address these issues, we generated H-2d MHC PO mice by backcross of H-2b PO mice with BALB/c. The CD8+ T cell response to LM is well characterized in the H-2d haplotype (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 perforin-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.
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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) and crossed with PO (H-2b MHC) mice kindly provided by Dr. W. R. Clark (25). F1 mice were backcrossed to BALB/c and H-2d/d perforin+/- 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/d perforin+/- mice were backcrossed to BALB/c three additional times and then intercrossed to generate H-2d/d perforin-/- (H-2d PO) mice. H-2d PO 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.
Bacteria
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, 34, 35, 36). 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 ml per animal. Aliquots were plated onto tryptic soy agar containing 50 µg/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-2d MHC) (American Type Culture 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 Island, 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 µg/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 described (17) using FITC-conjugated
anti-CD8 (53.6-7; Sigma), PE-conjugated anti-CD4 (H129.19;
Sigma), mouse anti-mouse H-2Kb IgG2b (Y-3; ATCC), and
mouse anti-mouse H-2Kd IgG2a (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-2d
PO mice using methods previously described (37). Briefly, 2–4 x
107 splenocytes from mice injected 7–10 days previously
with 103–4 CFU of virulent LM strain 10403s or XFL303 were
incubated in RP10 with 3 x 106 irradiated (150 Gy)
stimulator cells (P815-LLO cells or P815-p60 cells). Subsequent weekly
restimulations were conducted by combining 1–3 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
the case of T cell lines specific for NP 118–126, P815-derived
stimulator cells were left out and 3 x 107 irradiated
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 indicated 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 51Cr was calculated by the formula: 100 x (experimental cpm - spontaneous cpm)/(total cpm - spontaneous cpm). Spontaneous release was less than 20% of total in all experiments.
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 phosphatase-conjugated goat
anti-rabbit Ig (Sigma), and alkaline phosphatase substrate
(Sigma) were added sequentially according to the manufacturer’s
protocol. OD was measured at 405 nm. Limit of detection was less than
50 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 determined using adoptive transfer assays, as described previously (44, 17, 30). Briefly, RBC-depleted splenocytes from donor mice immunized 7–10 days previously with 103–4 virulent LM strain 10403s or XFL303 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 0.5-ml into naive BALB/c host mice. Within 2 h, host mice, including uninjected controls, were challenged i.v. with the indicated dose of virulent LM 10403s or XFL303. 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 streptomycin, and calculating colony count averages after overnight incubation at 37°C.
ELISPOT
The prevalence of activated Ag-specific CD8+ T cells
in the spleens of BALB/c and PO mice was determined by ELISPOT
analysis, as previously described (46, 47, 48, 49). Briefly, splenocytes
(5 x 103-105/well) were cocultured with
P815-LLO target cells (105/well) for 24–48 h in
flat-bottom 96-well plates that had been previously coated with rat
anti-mouse IFN- mAb (R4-6A2; PharMingen). Following lysis of the
cells with distilled water and rinses with PBS containing 0.2%
Tween-20, the plates were incubated with rabbit anti-IFN-
polysera. After rinsing, the plates were incubated with donkey
anti-rabbit Ig conjugated to alkaline phosphatase (Jackson).
Further rinsing was followed by the addition of 5-bromo-4
chloro-3-indolyl phosphate (BCIP) substrate (Sigma) in AMP
buffer impregnated with 0.75% agarose. The reaction was developed at
37°C and spots were counted using a dissection microscope. The
average frequency of responders from triplicate determinations was
multiplied by the total number of splenocytes to calculate responders
per spleen.
Analysis of CD8+ T cells using Kd-peptide tetramer complexes
Kd-peptide tetramer complexes were generated and used as previously described (50), with minor modifications. For staining, approximately 5 x 106 lympholyte-M (Cedarlane) separated splenocytes were blocked with anti-FcR mAb (2.4G2, gift of T. Waldschmidt at the University of Iowa) before staining with FITC-conjugated anti-CD8 (53.6-7; Sigma) and PE-conjugated Kd-peptide tetramer complexes for 1 h at 4°C. After washing, the cells were resuspended in PBS containing 0.01% sodium azide, 1% BSA, and 1 µg/ml propidium iodide, and then analyzed on a FACScan using CyCLOPS software (Cytomation, Fort Collins, CO). Lymphocytes (determined by forward scatter and side scatter) that excluded propidium iodide were analyzed for CD8 and Kd-peptide tetramer-specific staining. The frequency of CD8+ cells and CD8+, tetramer+ cells was used with the total splenocyte count to calculate the responders per spleen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We bred the PO mutation onto the BALB/c (H-2d) background in which multiple LM Ags have been described (31). Experiments in which we compared naive H-2d PO mice with BALB/c mice did not reveal a significant difference in the LD50 of virulent LM (data not shown). This result is consistent with studies that compared H-2b PO mice with wild-type C57BL/6 mice (29, and data not shown) and verifies that perforin is not required for resistance to primary LM infection.
We next compared the antilisterial activity of immune splenocytes
derived from H-2d PO mice with those from BALB/c mice.
Since the resistance of naive PO and BALB/c mice to primary LM
infection was similar, we used standard sublethal doses of virulent LM
(
0.1 LD50) to immunize H-2d PO and BALB/c
donor mice. Seven days postimmunization, equivalent numbers of BALB/c
or PO donor splenocytes were transferred into naive BALB/c host mice
that were subsequently challenged with a high dose of virulent LM.
Bacterial counts in the spleens and livers of splenocyte recipient and
control mice 3 days postchallenge demonstrated that BALB/c-derived
splenocytes provided dramatic antilisterial protection in both the
spleen (Fig. 1
A) and the liver
(Fig. 1B), reducing bacterial recovery >100,000-fold and
>10,000-fold, respectively, compared with mice that did not receive
splenocytes. This activity was primarily mediated by CD8+
cells since depletion of the CD8+ compartment with Ab and
complement before transfer eliminated the majority of the protection
(Fig. 1, A and B). Immunity mediated by
splenocytes derived from H-2d PO mice was comparable with
that provided by wild-type splenocytes in the liver (8,000-fold
reduction), but somewhat less than that provided by wild-type
splenocytes in the spleen (1,000-fold reduction). Depletion of
CD8+ cells from H-2d PO-derived splenocytes had
a modest effect on the level of immunity transferred, but did not
eliminate immunity mediated by these cells (Fig. 1, A and
B). These results recapitulate those from H-2b
mice (29, 30) and further support the model that perforin plays a role
in the normal immune response to LM, especially in the spleen, and that
splenocytes other than CD8+ T cells may play a significant
role in adaptive immunity to LM in the PO mouse.
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Generation and characterization in vitro of CD8+ T cell lines specific for three known antigenic peptides expressed by recombinant LM XFL303
Experiments with LLO-specific CD8+ T cell lines from H-2b PO mice previously identified a perforin-independent pathway by which CD8+ T cells mediate antilisterial immunity (30). To determine whether this result was restricted to LLO-specific CD8+ T cells, we generated and characterized CD8+ T cell lines specific for multiple Ags from H-2d PO and syngeneic control (BALB/c as well as H-2d perforin+/-) mice that had been immunized with virulent LM XFL303. XFL303 is a recombinant strain of virulent LM that secretes the LCMV-derived NP 118–126 epitope in the context of a dihydrofolate reductase (DHFR) fusion protein and strongly activates CD8+ T cells that recognize NP 118–126 bound to H-2Ld in the H-2d mouse (33). Splenocytes from H-2d PO mice immunized 7 days previously with a sublethal dose of virulent LM XFL303 were restimulated in vitro with H-2d target cells expressing LLO 91–99, p60 217–225, or NP 118–126 peptide. CD8+ T cell lines specific for NP 118–126 were also generated from control (BALB/c and perforin+/-) mice. BALB/c-derived CD8+ T cell lines specific for LLO 91–99 and p60 217–225 have been described previously (44, 34).
After several in vitro restimulations, all lines were >95%
CD8+CD4-, as measured by flow cytometry (data
not shown). To verify Ag specificity and cytolytic activity, we
performed 51Cr release assays. All CD8+ T cell
lines mediated cytolysis of H-2d MHC target cells in the
presence, but not in the absence, of the appropriate Ag (Fig. 2, A–D). As expected,
CD8+ T cells derived from control mice (Fig. 2D)
mediated higher levels of specific lysis in shorter time period than
did CD8+ T cells derived from PO mice (Fig. 2, A–C). Delayed cytolysis in the absence of perforin is
consistent with previous studies that have documented the importance of
perforin in cytolytic assays in vitro (24, 25, 51, 52).
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). Whereas peptide-coated,
CD95-expressing (P815-Fas (38)) target cells were lysed at high levels
in a 51Cr release assay by PO-derived CD8+ T
cells, peptide-coated P815 cells that express very low levels of CD95
(P815) were not lysed (Fig. 3A). PO CD8+ T cell
lines specific for p60 and NP also exhibited increased lysis of target
cells expressing CD95 compared with target cells that express minimal
CD95 (data not shown). Similar results were obtained with another pair
of target cells that differ in expression of CD95, namely
L1210F+ and L1210F- (39, 25). We observed
specific lysis of CD95-expressing targets (Fig. 3B) and
background levels of lysis of targets that do not express CD95 (data
not shown). Finally, Ag-specific lysis of L1210F+ cells by
PO CD8+ T cells was inhibited by a mAb specific for CD95,
but not by control rat IgG (Fig. 3B). Complete inhibition
was observed at most E:T ratios using 5 µg/ml of anti-CD95 mAb, a
concentration that did not kill target cells nor the CD8+ T
cells in the time frame of the 51Cr release assay (data not
shown). While these results do not rigorously rule out
perforin-independent, CD95-independent mechanisms of lysis by
PO-derived CD8+ T cells, they do establish the presence of
a CD95-dependent pathway.
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(Fig. 2
, E–H) and TNF (Fig. 2, I–L) in
an Ag-specific fashion. IFN- production by H-2d PO
CD8+ T cells specific for LLO and p60 was also verified by
ELISPOT analysis (data not shown).
In general, we observed higher levels of IFN- production by
PO-derived CD8+ T cells compared with wild-type cells (Fig. 2), a finding that is consistent with data from Sad and colleagues
(53).
In vivo immunity can be mediated by PO CD8+ T cells specific for a range of LM-derived peptides
Our previous studies demonstrated that LLO-specific
CD8+ T cells from H-2b PO mice can provide
significant immunity to LM. To test whether perforin-independent
CD8+ T cell-mediated immunity is generalizable to other
Ags, we tested the ability of H-2d PO CD8+ T
cells specific for LLO 91–99, p60 217–225, and NP 118–126 to mediate
immunity against rLM XFL303 in vivo. CD8+ T cells were
transferred into naive BALB/c host mice that were subsequently
challenged with approximately 10 LD50 of virulent LM
XFL303. PO-derived CD8+ T cells specific for LLO 91–99,
p60 217–225, and NP 118–126 all provided antilisterial immunity in
the liver (Fig. 4, E–F).
PO-derived CD8+ T cells also reduced bacterial counts in
the spleen (Fig. 4, A–C), albeit to a lesser degree.
BALB/c-derived CD8+ T cells specific for NP 118–126
provided high levels of antilisterial immunity in both the spleen (Fig. 4D) and the liver (Fig. 4H). Immunity mediated by
BALB/c-derived CD8+ T cells specific for LLO 91–99 and p60
217–225 has been described previously (44, 34). Consistent with our
previous studies, the degree of immunity in the spleen mediated by
PO-derived CD8+ T cells was typically less compared with
that which is usually observed with BALB/c-derived CD8+ T
cells (Fig. 4, A–C versus D).
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). Similarly, all mice that received PO
CD8+ T cells specific for LLO survived a lethal challenge
with virulent LM 10403s (Table I).
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). These results demonstrate that perforin-independent mechanisms of antilisterial immunity mediated by CD8+ T cells are not restricted to CD8+ T cells that recognize LLO.
Analysis of priming and expansion of the CD8+ T cell compartment in PO versus syngeneic BALB/c mice
We previously hypothesized that splenocytes from PO mice might be deficient in mediating antilisterial immunity due to inefficient priming of LM-specific CD8+ T cells in these mice (30). To address this issue, we quantitated LLO-specific CD8+ T cells in PO and BALB/c mice following primary and secondary challenge with LM.
Phenotypic as well as functional assays are now available to identify
and quantitate Ag-specific CD8+ T cells ex vivo (54). The
utility of Kd-peptide tetramer complexes in the phenotypic
analysis of Ag-specific CD8+ T cells in wild-type mice
undergoing a response to LM has been demonstrated previously (50). We
applied this technique to address the issue of priming in the PO mouse.
The specificity of these reagents in the PO system was verified using
H-2d PO CD8+ T cell lines. Kd
tetramers folded with the peptide LLO 91–99
(KdLLO) stained LLO 91–99-specific
CD8+ T cells from the H-2d PO mouse, but failed
to stain H-2d PO CD8+ T cells specific for p60
217–225 (Fig. 5A). A control
reagent generated by folding Kd tetramers with a self
peptide ((50), KdJAK1) did not stain PO-derived
CD8+ T cells specific for LLO 91–99 (Fig. 5A).
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, B and C).
This value for BALB/c mice is slightly higher than that previously
reported (50), due to higher background staining in naive mice
(0.53 ± 0.24%
CD8+KdLLO+ cells in the
BALB/c (n = 9) and 0.49 ± 0.28% in the
H-2d PO (n = 7)) and the fact that, in this
study, we did not differentiate between CD62Lhigh (naive)
and CD62Llow (activated) CD8+ T cells.
Expansion of LLO 91–99-specific CD8+ T cells could clearly
be observed compared with background staining of naive splenocytes
(Fig. 5B). Reinfection of immune BALB/c and H-2d
PO mice resulted in further expansion of the LLO 91–99-specific T cell
population (Fig. 5, B and C). As was the case
with the primary response, the secondary response to LLO 91–99 in
BALB/c and H-2d PO mice was similar (Fig. 5C),
accounting for 11 and 14%, respectively, of all CD8+ T
cells in the representative mice shown in Fig. 5B. Every analysis using KdLLO was accompanied by an analysis on the same cells with KdJAK1. We did not observe a significant increase in the percentage of CD8+KdJAK1+ cells in animals undergoing a response to LM compared with naive animals (data not shown). The percentage of cells that stained positive using the control reagent was similar to the percentage of cells that stained positive with KdLLO from naive animals (data not shown).
ELISPOT analysis was also performed on splenocytes from
H-2d PO and control BALB/c animals undergoing a primary and
secondary response to LM (48). The number of cells that specifically
produce IFN- in response to LLO 91–99 was similar to the number of
CD8+ cells specific for LLO 91–99, as determined by
tetramer analysis (data not shown). In sum, we do not find any
evidence, using functional as well as phenotypic analyses, that the
CD8+ T cell compartment in PO mice is deficient in
Ag-specific expansion in response to LM. Thus, deficiencies in
CD8+ T cell immunity in PO mice are a consequence of the
efferent and not the afferent immune response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene knockout mouse-derived CD8+ T cells
specific for either LLO 91–99 (44) or p60 217–225 (34) provide
immunity to LM infection. In this study, we extend this finding to
BALB/c-derived CD8+ T cells specific for the NP epitope
expressed by rLM XFL303, further demonstrating that the recombinant Ag
functions as a target for protective CD8+ T cells in a
similar fashion to endogenous LM Ags.
Activated CD8+ T cells elaborate a number of effector
functions following Ag-specific stimulation in vitro. A current
challenge is to understand the relative importance of these effector
mechanisms in CD8+ T cell-mediated immunity against
different pathogens in vivo. Two previous reports addressed the role of
perforin in CD8+ T cell-mediated immunity to LM (29, 30).
The first by Kagi and colleagues showed that the CD8+
compartment in the spleens of PO mice is deficient, compared with
wild-type mice, in antilisterial activity. This result, which has since
been independently corroborated (30, and Fig. 1), suggests that
perforin plays an important role in the normal antilisterial response.
The second report, which used Ag-specific CD8+ T cell
lines, revealed a perforin-independent pathway that results in
significant CD8+ T cell-mediated immunity against LM. The
relative importance of perforin-dependent versus perforin-independent
pathways of antilisterial resistance in immunocompetent hosts is not
known. Furthermore, the precise mechanisms involved in
perforin-independent resistance to LM mediated by CD8+ T
cells remain undefined. Our previous studies demonstrated that both PO
and wild-type Ag-specific CD8+ T cells provide
antilisterial immunity in vivo in MRL lpr/lpr
host mice (30). Thus, although PO CD8+ T cells conducted
Ag-specific cytolysis in vitro, which was dependent on CD95 (30), we
found no evidence for a CD95-dependent pathway of antilisterial
resistance in vivo. The role of CD95 in immunity to LM in vivo has been
further addressed in a recent report by Jensen and colleagues (55).
They have shown that mice deficient in both perforin and CD95 are more
susceptible than mice deficient in perforin alone to primary and
secondary listeriosis. While this study did not specifically examine
CD8+ T cells in vivo, these results suggest a role for CD95
in the development or expression of antilisterial resistance in the
absence of perforin. In the present study, we confirm the presence of a
CD95-dependent pathway of Ag-specific cytolysis in vitro mediated by PO
CD8+ T cells of the H-2d haplotype (Fig. 3).
The ability of H-2d CD8+ T cells to mediate
antilisterial immunity in H-2dlpr/lpr host mice has not been tested.
By performing the present experiments with PO mice of the H-2d haplotype, we were able to address two previously unresolved issues. First, we show that PO CD8+ T cells specific for three epitopes, LLO 91–99, NP 118–126, and p60 217–225, can provide immunity to LM infection. This result demonstrates that perforin-independent mechanisms are not limited to CD8+ T cells specific for a single Ag and suggests the possibility that CD8+ T cells, regardless of Ag specificity, may mediate antilisterial resistance in a perforin-independent fashion in the normal mouse. This result argues against the previously untested possibility that CD8+ T cell lines from PO mice mediate efficient antilisterial immunity based on their oligoclonal specificity for LLO (30).
While our data demonstrate potent, perforin-independent, Ag-specific
resistance to LM mediated by CD8+ T cells (Table I), our
data also confirm a deficiency due to the absence of perforin, which is
especially notable in the spleen (29, 30, 55, and Fig. 4). These
results suggest that optimal antilisterial immunity mediated by
CD8+ T cells may involve distinct effector functions in the
liver and spleen. A model by which CD8+ T cells mediate
resistance to LM infection in the liver in the absence of cytolysis is
consistent with data from a model of hepatitis (56, 16), and may imply
a greater role for cytokines versus cytolytic activity in this target
organ. Elucidating the mechanisms by which distinct effector functions
are triggered in vivo, or the factors that determine the susceptibility
of an infected cell to perforin-dependent cytolysis, remains a topic of
current interest.
The second issue, whether priming of CD8+ T cells is
deficient in PO mice, was addressed by functional as well as phenotypic
analysis. Three current methods have replaced limiting dilution assays
and subsequent CTL precursor frequency analysis as techniques to
quantitate the expansion of Ag-specific CD8+ T cells in
response to pathogens. ELISPOT analysis, intracellular staining for
cytokines, and flow-cytometric analysis using MHC class I tetramers
have all provided equivalent estimates of the degree of expansion of
CD8+ T cells in response to individual pathogens (48, 50, 57, 58). Neither ELISPOT analysis for IFN--producing
CD8+ T cells, nor flow-cytometric analysis for
CD8+ T cells specific for H-2Kd bound to LLO
91–99 (Fig. 5), showed a defect in priming of LM-specific
CD8+ T cells in PO mice relative to BALB/c mice. This
result indicates that the deficiency of PO-derived CD8+
splenocytes in mediating antilisterial immunity cannot be attributed to
a failure in priming after immunization, as was previously suggested
(30).
Our findings to date do not account for the relative deficiency of polyclonal CD8+ splenocytes versus in vitro restimulated, oligoclonal CD8+ T cell lines to mediate antilisterial immunity. Possible explanations that remain to be tested include differences in homing following i.v. injection between splenocytes and restimulated lines, and the impact of cells other than CD8+ T cells, which are present in a dose of splenocytes, but missing from a dose of restimulated CD8+ T cells. In addition, while we now have the tools to estimate the number of Ag-specific CD8+ T cells typically injected into a recipient mouse during an adoptive transfer experiment, we do not know the proportion of those Ag-specific CD8+ T cells that actually mediates antilisterial immunity in vivo following transfer. In vitro manipulations might increase or decrease the fraction of cells that are capable of mediating antilisterial immunity in vivo. The possibility that in vitro restimulation might enhance alternative pathways of antimicrobial resistance by CD8+ T cells could have important implications for adoptive immunotherapy against pathogens as well as tumors (59).
|
Acknowledgments |
|---|
, and S. Xue and S.
Perlman for reagents and assistance with ELISPOT analyses. The expert
technical assistance of Andrea Vivado and Gail Mayfield is greatly
appreciated. We also thank J. Fishbaugh and T. Waldschmidt
for reagents and assistance with flow-cytometric analysis. |
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: LM, Listeria monocytogenes; ELISPOT, enzyme-linked immunospot assay; LCMV, lymphocytic choriomeningitis virus; LLO, listeriolysin O; NP, nucleoprotein; PE, phycoerythrin; PO, perforin deficient.
Received for publication August 11, 1998. Accepted for publication October 7, 1998.
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