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*
Immunology Graduate Program and
Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235; and Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology, and
Department of Pathology, University of Chicago, Chicago IL 60637
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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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LM-specific CD8+ T cells respond to Ag presented by both classical (class Ia) and nonclassical (class Ib) MHC. Spleen cells isolated from mice 6 days following a primary infection contain CTL that recognize LM Ags in the context of the class Ib molecules, M3 and Qa-1b (12, 13). These class Ib molecules differ from conventional class Ia MHC in that they exhibit limited polymorphism, are expressed at low levels on the cell surface, and bind a limited repertoire of peptide ligands (14, 15). M3 has a short hydrophobic peptide binding groove that preferentially binds peptides beginning with N-formyl methionine. Although mitochondria are a limited source for such epitopes, prokaryotic organisms initiate protein synthesis with formylated methionine, which has implicated M3 as a specialized class I molecule for generating antimicrobial immunity. In support of this theory, three LM-derived N-formylated peptides have been identified that are recognized by antilisterial CTL (16, 17, 18). While Qa-1b has been shown to function in the regulation of NK cells (19, 20), CD8+ T cells isolated from mice infected with LM and the intracellular pathogen Salmonella typhimurium recognize Qa-1b-presented Ag (21, 22). The LM-derived peptide epitope(s) presented by Qa-1b has yet to be identified.
We have previously demonstrated that presentation of LM Ags by MHC class Ib molecules alone is sufficient for generating protective CD8+ T cell immunity (23). MHC class Ia-deficient mice (B6.Kb-/-Db-/-) receiving a sublethal LM infection exhibit a 3- to 4-fold expansion of class Ib-restricted CD8+ T cells that are capable of lysing LM-infected target cells in vitro. These animals also clear an in vivo infection with similar kinetics as wild-type B6 mice, which generate both class Ia- and class Ib-restricted antilisterial CTL. While these observations demonstrate that class Ib-restricted CTL provide significant protection following a primary LM infection, their ability to elicit an enhanced memory response to a secondary infection has not yet been shown. It is also not fully understood whether class Ib-restricted T cells exhibit unique or overlapping effector functions with class Ia-restricted CTL. Data from a recent study suggest that M3-restricted CD8+ T cells provide rapid early protection following a primary LM infection, but contribute little to the memory response (24). Because these experiments analyzed the response to only a single class Ib-peptide complex, the protection generated from a polyclonal population of class Ib-restricted CTL during a secondary immune response remains unclear. In this study we have analyzed the responses of class Ia- and class Ib-restricted CD8+ T cells during primary and secondary LM infections. Furthermore, we have examined the relative contribution of M3 in the generation of a class Ib-restricted CTL response to LM in both wild-type as well as class Ia knockout animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, B6.Kb-/-Db-/-, and TAP-1-/- mice were bred and maintained in animal colonies at the University of Texas Southwestern Medical Center (Dallas, TX). B6.Kb-/-Db-/- animals were generated as previously described (25) and have been backcrossed onto the C57BL/6 background six times.
Bacteria
LM 10403 serotype 1 was originally provided by H. G.
A. Bouwer (Veterans Affairs Medical Center, Portland, OR). Bacteria
were grown on brain-heart infusion (BHI) agar plates (Difco, Detroit,
MI), and virulent stocks were maintained by repeated passage through
C57BL/6 mice. The LD50 for B6 mice is 
2
x 104 bacteria. For infection of cell lines and
animals, log phase cultures of LM grown in BHI broth were washed twice
and diluted in PBS.
Cell lines and reagents
The J774 macrophage line (H2d), and the J774 transfectant expressing H2- Kb (J774:Kb) were grown in DMEM (Life Technologies, Gaithersburg, MD). TAP-2-deficient RMA-S cells were maintained in RPMI 1640 medium (Life Technologies). All medium was supplemented with 10% FCS, 25 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 20 µM 2-ME. Cells were grown at 37°C in humidified air containing 7% CO2. For experiments in which cells would be infected with LM, antibiotic-free medium was used for passage.
Bone marrow macrophage cultures
Bone marrow was harvested from femurs of mice and cultured in T75 flasks at 2 x 105/ml (6 x 106 cells/flask) in DMEM supplemented with 15% FCS, 30% L cell supernatant (as a source of CSF-1), and 20 µg/ml gentamicin (Life Technologies). Cell cultures were incubated for 6 days at 37°C and used as macrophage targets for in vitro proliferation assays.
Antibodies
For flow cytometric analysis, anti-CD4 PE (RM4.5), anti-CD4 FITC (GK1.5), anti-CD8 FITC (53-6.7), and anti-CD44 PE (IM7) were purchased from PharMingen (San Diego, CA). Anti-CD8-PE (CT-CD8a) was purchased from Caltag (Burlingame, CA). Spleen cells (1 x 106) were stained for 20 min on ice with the appropriate concentrations of mAbs and subsequently washed with PBS containing 1% FCS and 0.1% NaN3. Cells were acquired using a FACScan flow cytometer and analyzed using CellQuest software (Becton Dickinson, Mountain View, CA).
For T cell depletions, anti-CD4 mAbs 2B6 (rat IgM) and GK1.5 (rat IgG2b), and anti-CD8 mAbs 3.155 (rat IgM) and 53-6.72 (rat IgG2a) were semipurified from hybridoma-cultured medium by 50% ammonium sulfate precipitation and dialyzed three times against PBS. For CTL blocking experiments, mAb 130 (anti-M3) and 34-5-8 (anti-Dd) hybridoma-conditioned medium was used at a one-fourth final dilution.
Peptides
Peptide synthesis was performed by automated solid phase techniques using standard F-moc chemistry on a Rainin Symphony peptide synthesizer (Rainin, Woburn, MA). The homogeneity of each peptide was determined by reverse phase HPLC and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Stock solutions (2 mM) were prepared by dissolving lyophilized peptide in DMSO (Sigma, St. Louis, MO).
Generation of LM-specific T cells
For primary LM infection, mice were injected with 2 x
103 bacteria (0.1 LD50) in
200 µl of PBS via the tail vein, and spleen cells were harvested on
the indicated day following infection. For secondary infection, LM
immune animals that had received a primary infection 3–4 wk previously
were injected with 2 x 105 bacteria (10
LD50). Spleen cells were subsequently isolated on
the indicated day for assay. Effector cells for
51Cr release CTL assays were generated by
culturing spleen cells (2 x 106/ml) from
day 6 primary infected mice in RPMI medium containing 1 µg/ml Con A
(Sigma) for 72 h at 37°C.
51Cr release CTL assays
Monolayers of J774 target cells grown in antibiotic-free medium were infected with LM for 1 h at a multiplicity of infection of 5:1. Cells were then washed three times with warm PBS, and DMEM containing 40 µg/ml gentamicin was added. Three hours after infection, LM-infected and noninfected J774 cells were harvested and labeled with 51Cr for 1 h at 37°C. Target cells were washed twice and added to a 96-well round-bottom plate at 5 x 103/well in 50 µl of DMEM containing gentamicin. Control medium, mAb 130 (anti-M3), or 34-5-8 (anti-Dd) hybridoma supernatant (50 µl) was added to target cells and allowed to incubate for 30 min at 37°C. Titrations of effector cells were then added in 100 µl of medium. Spontaneous and maximum release wells received 100 µl of medium or 1% SDS, respectively. The assays were terminated 4 h later when 100 µl of supernatant was harvested from each well and counted on a Micromedic ME Plus gamma counter (Micromedic, Horsham, PA). The percent specific lysis was calculated as 100 x (experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm). Data presented are the means of triplicate wells. Spontaneous release for LM-infected J774 target cells did not exceed 25%.
IFN-
enzyme-linked immunospot (ELISPOT) assays
Ninety-six-well Multiscreen HA plates (Millipore, Bedford, MA)
were coated overnight (50 µl/well) at 4°C with rat anti-mouse
IFN- mAb (R4-6A2, PharMingen) at 10 µg/ml in PBS. RMA-S stimulator
cells were incubated for 14 h at 25°C with 10 µM peptide,
washed twice, and irradiated (7500 rad) before assay. J774 macrophage
stimulator cells were infected with LM for 4 h before assay as
described above. In certain experiments, LM-infected J774 cells were
preincubated for 30 min at 25°C with the indicated mAb before
addition to the assay. Stimulator cells (1 x
105/well) were mixed with the appropriate
concentration of effector spleen cells in RPMI medium containing 30
U/ml IL-2 and plated in triplicate wells. For T cell-depleted effector
groups, 5 x 106 spleen cells were incubated
for 30 min on ice with 1 ml of 2B6 mAb (anti-CD4) or 3.155 mAb
(anti-CD8) diluted 1/10 in RPMI medium. Cells were then washed
twice and incubated with 10% rabbit complement (Cedarlane, Ontario,
Canada) in a 37oC water bath for 30 min. Effector
cells were washed twice, diluted, and added to the assay. After 24-h
incubation at 37°C, the plates were washed free of cells with
PBS/0.1% Tween-20 and incubated overnight at 4°C with biotinylated
rat anti-mouse IFN- mAb (XMG1.2, PharMingen) at 5 µg/ml.
Plates were washed four times, and 75 µl of streptavidin-peroxidase
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. After
2-h incubation at 25°C, plates were washed four times and developed
with a substrate solution of 300 µg/ml 3-amino-9-ethylcarbozole
(Sigma), 0.1 M sodium acetate buffer, and 0.5 µl/ml 30% hydrogen
peroxide. Spots were counted using a dissecting microscope. The mean
number of spots from triplicate wells was calculated for each responder
animal. Data are presented as the mean number of spots for two animals
per group. In certain experiments, ELISPOT data were used to calculate
the number of CD8+ T cells responding to class
Ia- or class Ib-presented Ag. Effector spleen cells were analyzed by
flow cytometry to determine the percentage of
CD8+ T cells, and thus the total number of
CD8+ T cells per well. The number of spots per
1 x 103 CD8+ T cells
was calculated as (total number of spots - number of spots from
CD8+ T cell depleted groups) x (1000/number
of CD8+ T cells/well). Data calculated for J774
stimulators are described as the class Ib response. The class Ia
response is calculated by subtracting J774 response numbers from
J774:Kb response numbers.
In vitro proliferation assays
Bone marrow-derived macrophages were cultured for 6 days as previously described. Cells were harvested with trypsin-EDTA (Life Technologies), washed twice, and added to a 48-well plate (2 x 105/well) in 500 µl of antibiotic-free DMEM. Macrophages were cultured for an additional 18 h at 37°C, then infected for 90 min with LM at a multiplicity of infection of 10:1. Monolayers were washed three times with warm PBS and covered with 500 µl of RPMI medium containing 20 µg/ml gentamicin. Four hours following initiation of infection, 2 x 106 effector cells were added to each well. T cell-enriched effector spleen cells were generated as follows. Day 6 primary immune and day 3 secondary immune animals were generated as described above. Splenocytes from three animals per group were pooled and depleted of RBC using erythrocyte lysing buffer (R&D Systems, Minneapolis, MN). As a primary step for autologous macrophage removal, spleen cells were cultured (1 x 107/ml in RPMI) on 100- x 15-mm petri dishes for 90 min at 37°C. Nonadherent cells were harvested, washed once, and run over mouse T cell enrichment columns (R&D Systems). Recovered cell populations, which contained 80–90% T cells, were washed twice in HBSS and incubated (1 x 107/ml) for 15 min in a 37°C water bath with HBSS containing 1 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR). Effector cells were washed twice, resuspended in RPMI medium, and added to noninfected or LM-infected macrophage cultures. Cell samples were harvested at 24, 36, and 48 h after effector addition, and the CFSE levels of CD8+ T cells were analyzed by flow cytometry.
In vivo CFU analysis of the LM memory response
C57BL/6 and B6.Kb-/-Db-/- mice received a primary sublethal i.v. infection of 2 x 103 LM. Four weeks later, LM immune animals were divided into three groups, which received no treatment, were depleted of CD4+ T cells, or were depleted of CD8+ T cells. For in vivo T cell depletions, animals received i.p. injections of GK1.5 (anti-CD4) or 53-6.72 (anti-CD8) mAbs on days -3, -2, and -1. Lymph node cells were harvested at the time of sacrifice and were analyzed by flow cytometry to ensure that depletion of the proper subset had occurred. On day 0, naive control animals and LM-immune animals were infected with 2 x 105 LM. Two days following infection, spleens and livers were removed and homogenized in sterile water using a glass Dounce tissue grinder (Kontes, Vineland, NJ). Serial 10-fold dilutions of homogenate were plated in triplicate on BHI agar plates and were incubated for 24 h at 37°C. Colony counts were averaged and corrected for dilution to yield the LM CFU per organ. The CFU limit of detection is 50 for spleen and 100 for liver. Data are presented as the mean CFU from three animals per group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To better understand the role of M3 Ag presentation in the class
Ib response to LM infection, an M3-specific mAb (mAb 130) was tested
for its ability to block CTL recognition of LM-infected J774 macrophage
targets. Spleen cells from B6 and B6.Kb-/-
Db-/- mice infected for 6 days with LM were
stimulated in vitro with Con A for 3 days and used as effectors in a
51Cr release assay. Both effector groups
specifically lysed LM-infected J774 cells, whereas noninfected targets
were not recognized (Fig. 1
). No lytic
activity was observed using effector cells from naive B6 or
B6.Kb-/-Db-/-
animals (data not shown). J774 (H2d)
expresses allogeneic MHC class Ia molecules, which suggests that the
observed LM-specific CTL activity is MHC class Ib restricted. The
preincubation of LM-infected J774 cells with mAb 130 drastically
reduced the level of target cell killing by B6 and
B6.Kb-/-Db-/-
antilisterial CTL, whereas an isotype control mAb had no effect. These
data demonstrate that M3 plays a major role in the cytotoxic T cell
response restricted by nonclassical class I molecules.
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Three LM-derived peptide epitopes presented by M3 to antilisterial
CTL have previously been identified (16, 17, 18). We examined
the CD8+ T cell response to these Ags following a
primary or secondary LM infection in B6 and
B6.Kb-/-Db-/- mice to
assess whether there is an enhanced M3-restricted memory response.
TAP-deficient RMA-S cells were incubated in the presence or the absence
of 10 µM peptide for 14 h at 25°C, irradiated, and used as
stimulator cells in an IFN- ELISPOT assay. Primary immune effectors
were generated from B6 and
B6.Kb-/-Db-/- mice
infected for 6 days with 2 x 103 LM.
Splenocytes from these animals specifically recognized RMA-S cells
pulsed with the three LM-derived epitopes, whereas no response was
observed from naive
B6.Kb-/-Db-/- or B6
spleen cells (Fig. 2). Depletion of
CD8+ T cells from these effector populations
before assay resulted in the loss of Ag-specific spot formation (data
not shown). RMA-S cells pulsed with f-MIGWII generated the strongest
response from both B6 and
B6.Kb-/-Db-/- CTL,
while f-MIVIL and f-MIVTLF were recognized at approximately equal, yet
lower, levels. These data are in accordance with our previous
observations using 51Cr release CTL assays
(23). It has previously been shown that memory
CD8+ T cells are rapidly activated following a
secondary LM infection (9, 10). To analyze the memory
response to these M3-presented epitopes, LM-immune mice (infected 4 wk
previously) were given a secondary challenge of 2 x
105 LM, and their spleen cells were assayed 3
days later. Secondary immune splenocytes from both B6 and
B6.Kb-/-Db-/- mice
contained less M3 Ag-specific CD8+ T cells than
primary immune effectors, even when assayed 5 days postinfection (data
not shown). Although RMA-S cells pulsed with f-MIGWII stimulated the
strongest response from memory CD8+ T cells,
recognition of all three Ags was significantly diminished.
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The use of peptide-pulsed RMA-S cells to detect LM-specific CTL is
limiting in that only the response against the three known peptide
epitopes presented by M3 is measured. Therefore, to detect all LM
epitopes presented by class Ib molecules to CD8+
T cells, we used LM-infected J774 cells as stimulators in an ELISPOT
assay. We assume that J774 cells present known as well as unknown
epitopes derived from LM infection. Spleen cells from primary infected
B6 and B6.Kb-/-Db-/-
mice contained effectors that specifically recognized LM-infected J774
cells (Fig. 3), but not uninfected
stimulators (data not shown). There was an 10-fold increase in the
number of spots detected using LM-infected stimulators compared with
peptide-pulsed RMA-S cells (compare Fig. 2
with Fig. 3), demonstrating
that these target cells are more efficient for observing an LM-specific
CTL response. Although variations were observed in the LM-specific
responses between individual infected animals, ELISPOT results most
often ranged from 75–120 spots/1 x 104
spleen cells when assayed against J774 stimulators. The depletion of
CD8+ T cells from these responder populations
almost completely abolished LM-specific spot formation, whereas the
depletion of CD4+ T cells had little effect. The
CTL response to LM-infected J774 cells was substantially reduced with
the addition of anti-M3 mAb, further demonstrating that M3 is the
dominant restriction element for class Ib-restricted antilisterial
CTL.
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H2-Kb is the dominant class Ia restriction
element for B6 antilisterial CTL (26, 27). We transfected
J774 cells with Kb
(J774:Kb) to allow for the comparison of class
Ia- and class Ib-restricted responses following a primary or secondary
LM infection. Spleen cells isolated from B6 mice 6 days following a
primary LM infection responded to LM-infected J774 and
J774:Kb cells equally well in an ELISPOT assay
(Fig. 4A). In contrast, we
observed a greatly enhanced response to J774:Kb
stimulators using B6 memory effectors isolated 5 days following a
secondary LM infection. The memory response to J774, and presumably
class Ib-presented Ag, was substantially lower, almost equal to the
response observed for primary immune effectors.
B6.Kb-/-Db-/- memory
responders also failed to demonstrate an enhanced class Ib-restricted
CTL response to LM-infected target cells (Fig. 4B). J774 and
J774:Kb stimulator cells elicited similar
responses from
B6.Kb-/-Db-/-
responders, as would be expected from the lack of class Ia-restricted
CTL in these animals.
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We examined the rate at which class Ia- and class Ib-restricted
CD8+ T cells expand and contract in B6 and
B6.Kb-/-Db-/- mice
following a primary LM infection. Spleen cells were harvested at
various times following a sublethal infection and analyzed by ELISPOT
using LM-infected J774 and J774:Kb stimulator
cells. The LM CFU per spleen was also measured to monitor the rate
of bacterial clearance. In wild-type B6 mice, LM-specific
CD8+ T cells were first detected between days 2
and 4 postinfection and dramatically expanded between days 4 and 6
(Fig. 5A). There was almost
equivalent recognition of J774 and J774:Kb
stimulators throughout these time points, suggesting that the majority
of CD8+ T cells were responding to class
Ib-presented Ag. However, after day 6 LM-infected
J774:Kb cells were still capable of stimulating a
strong CTL response, while the recognition of J774 cells was
down-regulated to almost undetectable levels. These observations are
further illustrated in Fig. 5C, in which the number of
CD8+ T cells responding to class Ia- or class
Ib-presented Ag is shown. These data indicate that in wild-type B6
animals, class Ia- and class Ib-restricted CD8+ T
cells are distinct populations that expand and contract at different
times following a primary LM infection. At their peak response,
class Ia and class Ib CTL represent 3 and 5% of
CD8+ T cells, respectively.
B6.Kb-/-Db-/- animals
demonstrated a similar kinetic CD8+ T cell
response to class Ib-presented Ags (Fig. 5, B and
D). Although a vigorous expansion was observed within the
first 6 days of infection, recognition of LM-infected target cells was
significantly diminished by day 8 and was almost undetectable by day
10. At the peak response on day 6, 10% of
B6.Kb-/-Db-/-
CD8+ T cells responded to infected targets. As we
have previously demonstrated (23), B6 and
B6.Kb-/-Db-/- animals
cleared LM from the spleen with similar kinetics, reaching undetectable
levels by day 8 postinfection (Fig. 5, A and B,
dashed line).
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A similar kinetic analysis was performed to examine the
CD8+ T cell memory response to secondary LM
infection. B6 and B6.Kb-/-Db-/- animals
received a primary sublethal infection, and 4 wk later were
rechallenged with 2 x 105 LM. Spleen cells
were isolated from mice infected for various times and were analyzed in
an ELISPOT assay. As shown in Fig. 6, A and C, the dominant memory
CD8+ T cell response in wild-type B6 animals was
class Ia restricted. Recognition of LM-infected
J774:Kb cells dramatically expanded between days
2 and 6 postinfection, whereas the class Ib response against wild-type
J774 cells remained significantly lower. Both responses peaked on day 6
postinfection, with class Ia- and class Ib-restricted CTL representing
10 and 3% of CD8+ T cells, respectively. A
limited expansion of class Ib-restricted CTL was also observed in
B6.Kb-/-Db-/- animals
(Fig. 6, B and D). The kinetics and magnitude of
the memory CD8+ T cell response in these animals
closely resembled those following a primary infection. Despite this
lack of an enhanced memory CTL response,
B6.Kb-/-Db-/- mice
clear a lethal LM infection as efficiently as wild-type B6 mice, with
no detectable CFU observed by day 2 postinfection (Fig. 6, A
and B, dashed line).
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The previous results indicate that class Ib-restricted memory
CD8+ T cells fail to undergo significant
expansion in vivo. We examined whether these memory CTL retain the
ability to proliferate and expand when provided with Ag in vitro.
Spleen cells were harvested from B6 and
B6.Kb-/-Db-/- mice 6
days following a primary infection or 3 days following a secondary
infection. T cells were enriched from these splenic populations and
labeled with the cytosolic fluorescent dye CFSE. There is an 2-fold
decrease in CFSE fluorescence with every cell division, allowing us to
measure the proliferative response of CD8+ T
cells when cultured with LM-infected macrophages. As shown in Fig. 7, when B6 and
B6.Kb-/-Db-/- memory T
cells are cultured with syngeneic macrophages infected with LM, a
significant loss of CFSE can be detected in a population of
CD8+ T cells over the course of 48 h. This
expansion is LM specific, as little CFSE loss is observed when these
effectors are cultured with noninfected macrophages. Bone
marrow-derived macrophages from B6,
B6.Kb-/-Db-/-, and
TAP-1-/- mice were used to further analyze the
ability of class Ia and class Ib Ag presentation to stimulate expansion
of LM-specific CTL. The data presented in Table I show the percentage of
CD8+ T cells with diminished levels of CFSE after
48 h of culture. Primary and secondary immune
CD8+ T cells from both B6 and
B6.Kb-/-Db-/- animals
demonstrated significant expansion in the presence of LM-infected B6
macrophages, whereas naive B6 and
B6.Kb-/-Db-/- (data not
shown) CD8+ T cells did not.
B6.Kb-/-Db-/-
macrophages were also capable of stimulating CTL proliferation,
presumably through class Ib presentation of LM Ags. The response
against LM-infected TAP.1-/- macrophages, which
express very low levels of class I molecules, was diminished for all
effector T cell groups. These data demonstrate that memory class
Ib-restricted CTL have the capacity to proliferate and expand against
LM-infected target cells in vitro.
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We wished to determine whether significant protection is provided
by class Ib-restricted memory CD8+ T cells in
response to a secondary lethal LM infection. B6 and
B6.Kb-/-Db-/- mice
received a primary sublethal dose of LM, and 4 wk later were left
untreated or were depleted of CD4+ T cells or
CD8+ T cells in vivo. LM-immune and naive control
mice were infected with 2 x 105 LM, and 2
days later the spleens were harvested, and the LM CFU were measured.
Both B6 (Fig. 8A) and
B6.Kb-/-Db-/- (Fig. 8B) intact LM immune animals demonstrated significant
protection compared with naive control animals (4.8 and 4.3
log10 protection, respectively). Although this
protective memory response was partially reversed with
CD4+ T cell depletion, almost all protection was
lost in both B6 and
B6.Kb-/-Db-/- LM-immune
animals with the depletion of CD8+ T cells.
Similar results were observed in the livers of these animals (data not
shown). These data demonstrate that
B6.Kb-/-Db-/- mice show
protection equivalent to that observed in wild-type B6 mice, and that a
significant part of this response is mediated by class
Ib-restricted CTL.
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The rapid expansion of class Ib-restricted CTL that we observed
following a primary LM infection has similar kinetics as the memory
response of class Ia-restricted CTL. It has been suggested that M3 may
prime CD8+ T cells through the presentation of Ag
derived from normal bacterial flora, thus eliciting a "memory"
response when these T cells encounter Ag derived from foreign
pathogenic bacteria (28). High expression levels of CD44
on murine T cells has been described as a phenotype for activated or
memory cells (29). We analyzed the level of CD44
expression on CD8+ and CD4+
T cells in naive B6 and
B6.Kb-/-Db-/- animals.
While 10% of splenic CD8+ T cells in B6 mice
were CD44 high, this percentage increased to 68% in
B6.Kb-/-Db-/- animals.
CD44 expression on CD4+ T cells, in contrast, was
identical in these two mouse strains (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The class Ib molecules M3 and Qa-1b have
previously been shown to function as restriction elements for
LM-specific CTL (12, 13). However, the relative
contribution of each in the stimulation of CD8+ T
cell responses has not been defined. Here, we show that an M3-specific
mAb significantly inhibits class Ib-restricted CTL recognition of
LM-infected target cells in both 51Cr release and
IFN- ELISPOT assays. These data demonstrate that M3 is the dominant
restriction element for class Ib-restricted antilisterial CTL, and that
the contributions of other nonclassical molecules, including
Qa-1b, are minimal. A strong M3-restricted CTL
response can most likely be attributed to the unique ability of this
molecule to bind and present N-formylated peptides. Recent
studies using M3-restricted TCR transgenic animals have demonstrated
that formylated mitochondrial peptides in the context of M3 are capable
of positively selecting CD8+ thymocytes
(30, 31). The transgenic TCR were specific for LM Ags,
which supports the hypothesis that a pool of peripheral
CD8+ T cells selected on M3 responds to the
presentation of bacteria-derived epitopes. Although three such
LM-derived N-formylated peptides have been identified
(16, 17, 18), our data suggest that M3 presents additional
epitopes. Thus, the class Ib-restricted CTL response against
LM-infected J774 cells is 10-fold higher than the response against
RMA-S cells pulsed with the known M3-restricted peptides. Although it
may be argued that infected macrophages are more efficient at
presenting these epitopes than peptide-pulsed tumor cells, we have been
able to generate an LM-specific CTL line that is M3
restricted, but does not respond to the three
known peptides (M. S. Seaman and J. Forman, unpublished
observations). These data further indicate that more M3-presented LM
Ags have yet to be identified. Having demonstrated that M3 plays a
significant role in activating LM-specific CTL, it will be of interest
to determine whether similar responses are elicited against other
bacterial pathogens. Recently, it has been shown that M3 is capable of
presenting N-formylated peptides derived from
Mycobacterium tuberculosis to CD8+ T
cells.4
Our results demonstrate that following a primary LM infection, B6 and B6.Kb-/- Db-/- animals both exhibit a similar rapid expansion of class Ib-restricted antilisterial CTL between days 4 and 6 postinfection, which correlates with clearance of LM CFU in the spleens of these animals. In comparison, we observed a smaller and delayed expansion of class Ia-restricted CTL in B6 mice that occurs after most of the infection has already been cleared. It remains unclear why the kinetic responses of these effector populations differ. The accelerated expansion of class Ib-restricted CTL in response to a primary LM infection is comparable to that observed later for class Ia-restricted memory CTL. Although NK T cells have been shown to exhibit rapid activation responses (32), phenotypic analysis of CD8+ T cells in B6.Kb-/-Db-/- animals provides no evidence that they belong to this unique cell population (data not shown). It has previously been hypothesized that M3 may be capable of presenting Ags derived from normal bacterial flora to CD8+ T cells, thus priming these effectors for later encounters with pathogenic microbes (28). This report demonstrated that animals housed in a conventional colony could mount CTL responses to M3-restricted LM Ags without prior sensitization. It has also been shown that M3-restricted CTL clones specific for LM Ags can exhibit cross-reactive responses to multiple other N-formylated peptides, including Ags derived from a variety of Gram-positive and Gram-negative bacteria (33). We have demonstrated that naive class Ib-restricted CD8+ T cells, as observed in B6.Kb-/-Db-/- animals, express high levels of CD44, a phenotypic marker for memory cells. Although these data are not conclusive, it does support the hypothesis that these cells have been previously primed and may further indicate that a portion of these CD8+ T cells in class Ia-deficient mice are M3 restricted. However, it should be noted that if the rapid kinetics of the M3-restricted primary response represents reactivation of memory cells, it does not lead to rapid clearance of LM. We observed that B6.Kb-/-Db-/- mice previously infected with LM cleared a secondary infection much more rapidly than naive animals. Our animals were housed in a specific pathogen-free colony, which suggests that the natural microbial environment, rather than previous pathogen exposure, would be responsible for the priming of M3-restricted CD8+ T cells.
Other factors that may contribute to the distinct kinetic CTL responses we observed are the nature of LM Ags presented by class Ia and class Ib molecules and their persistence in vivo. The generation of known class Ia-restricted epitopes has been shown to require proteosome-dependent processing of LM proteins released in the host cell cytosol (34, 35, 36). The three known M3-restricted peptides, in contrast, are secreted by LM or clipped from the bacterial cell surface (16, 17, 18), which may result in rapid binding and cell surface presentation by M3. Although one report has demonstrated that presentation of the M3-restricted f-MIVTLF epitope is proteosome dependent (37), it remains unclear whether further processing of all M3 Ags is required. The life span of LM Ags in vivo may regulate the rate at which antilisterial CTL responses are down-regulated. Following a sublethal primary LM infection, we observed a rapid loss of class Ib-restricted CTL activity that closely followed bacterial clearance. These results could indicate that M3 Ags have a relatively short half-life on the cell surface, and that continual bacterial secretion of these epitopes is required for sustaining a class Ib-restricted CTL response. It has been demonstrated that splenocytes pulsed with the LM f-MIGWII epitope exhibit stable M3/peptide complexes on the cell surface (38), which may indicate that the LM N-formylated peptides themselves are less stable in the intracellular environment. Class Ia-restricted Ags, in contrast, may be more efficiently harbored by professional APCs or may remain bound to class I molecules longer, thus allowing for expansion of these CTL populations after LM CFU can no longer be detected.
Such a difference in Ag persistence could also explain our observations that class Ia-restricted CD8+ T cells exhibit a greatly enhanced response following secondary infection, whereas the expansion of class Ib-restricted memory CTL is minimal. Competition between these CTL populations cannot explain this difference, as limited expansion of the class Ib-restricted population is also observed in MHC class Ia-deficient animals. Both B6 and B6.Kb-/-Db-/- LM-immune animals are capable of eradicating a lethal dose of LM within 2 days postinfection. Rapid clearance of LM may limit the amount and duration of Ag presented to M3-restricted CD8+ T cells, thus failing to stimulate a robust proliferative response as observed with class Ia-restricted CTL. Our CFSE proliferation assays further support this hypothesis. When class Ib-restricted memory CTL are provided with Ag in vitro, their proliferative expansion is comparable to that of class Ia-restricted memory CTL. It is also possible that with LM clearance in vivo, class Ib-restricted CTL become more susceptible to apoptosis, resulting in a faster down-regulation of this response. Whether class Ia and class Ib CD8+ T cells exhibit differences in their ability to survive in an inflammatory environment after LM clearance remains to be examined.
Despite the fact that class Ib-restricted CTL exhibit limited in vivo expansion following secondary infection, we have demonstrated that these memory cells play a significant role in protection. Thus, in vivo depletion of CD8+ T cells in LM immune B6.Kb-/-Db-/- animals severely impaired the ability to protect against a lethal LM challenge. These studies along with our kinetic analysis of the LM memory response indicate that CD8+ T cell protection is important within the first few days of infection. These data are important in demonstrating that limited in vivo expansion of class Ib-restricted CTL does not necessarily reflect on the contribution of these effector cells in protection.
In summary, we have demonstrated that M3-restricted CTL play an important role in the adaptive immune response to LM. These effector cells probably account for the rapid bacterial clearance observed after a primary infection. Future experiments to test CD8+ T cell immunity in M3-deficient animals will help to further understand the role of this nonclassical MHC molecule. A better understanding of LM Ag processing and presentation by MHC class Ia and class Ib molecules will also provide important insights into how these antilisterial CTL populations are activated and maintained in vivo.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James Forman, Center for Immunology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235-9093.
3 Abbreviations used in this paper: LM, Listeria monocytogenes; BHI, brain-heart infusion; CFSE, carboxyfluorescein diacetate succinimidyl ester; ELISPOT, enzyme-linked immunospot.
4 T. Chun, N. V. Serbina, B. Wang, N. M. Chiu, J. L. Flynn, and C. R. Wang. 2000. Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis. Submitted for publication.
Received for publication May 22, 2000. Accepted for publication August 4, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon production by natural killer cells is important in defense against murine listeriosis. Infect. Immun. 59:2892.This article has been cited by other articles:
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) or were infected for 4 h
with LM (
) and incubated in the presence of mAb 130 (•) or an
isotype-matched control mAb (
). Results are expressed as the percent
specific 51Cr release and are the means of triplicate
wells.
), f-MIGWII (
), or f-MIVTLF
(
) were
also assayed against LM-infected stimulators. Results are presented as
the mean number of spots detected from two animals per group ±
SEM and are representative of three independent experiments performed.
*, p < 0.05 compared with untreated effector
cells anti-LM-infected J774 stimulators (by Student’s
t test).
). The horizontal dashed line represents the CFU
limit of detection. C and D, Data from
graphs A and B were used to calculate the
number of CD8+ T cells from B6 (C) and
B6.Kb-/-Db-/- (D) mice
responding to class Ia (open symbols) or class Ib (filled symbols)
presented Ag. Calculations were performed as described in
Materials and Methods.
