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*
Interdisciplinary Program in Immunology and
Department of Microbiology, University of Iowa, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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was neutralized in L. monocytogenes-infected
animals, demonstrating that multiple signals exist to overcome CD40L
blockade of CD8+ T cell cross-priming during bacterial
infection. These data support a model in which DCs can be stimulated in
vivo through signals other than CD40, becoming APC that can effectively
stimulate CD8+ T cell responses against exogenous Ags
during infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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can cause the up-regulation of B7.1,
B7.2, and MHC class II as well as the secretion of IL-12 by DCs
(6, 7). Several studies have examined CD8+ T cell responses in CD40L-/- mice after viral infection. CD8+ T cell responses appear to be normal in response to many viruses including lymphocytic choriomeningitis virus (LCMV), suggesting that viral infection may in some instances bypass the requirement for CD40L in DC activation (8). Some deficiencies have been observed in primary CD8+ T cell responses after vesicular stomatitis virus (VSV) infection and memory CD8+ T cell responses after LCMV infection (9, 10). Early T cell independent inflammatory responses to infection with the intracellular bacterium L. monocytogenes (LM) and T cell-mediated protective immunity appear to be unaffected by the absence of CD40L (11). Recent evidence has also shown a minimal impact on the splenic CD8+ T cell response to secreted LM Ags in CD40-/- mice (12). However, these studies have measured responses generated through the endogenous MHC class I presentation pathway. Thus, it is not known whether CD40L-CD40 interactions are required for naive CD8+ T cell priming against exogenously processed Ags expressed by pathogens.
This study examines the role of CD40L-CD40 interactions in CD8+ T cell priming against LM Ags presented by either the endogenous or exogenous MHC class I presentation pathway. LM is a Gram-positive facultative intracellular bacterial pathogen that can enter and multiply within phagocytic and nonphagocytic cells. LM has the ability to escape from the membrane-bound phagosome after infection and enter the host cell cytoplasm (13). Secreted bacterial Ags can then be processed through the endogenous MHC class I presentation pathway, which involves proteolytic degradation by the proteasome and transport into the endoplasmic reticulum by the TAP complex. Nonsecreted bacterial Ags, although not accessible to the endogenous pathway, may become accessible to exogenous MHC class I presentation pathways after destruction of the organism (14). Consistent with this idea, infection of mice with recombinant LM strains expressing an H-2Ld-restricted epitope from the nucleoprotein of LCMV (NP118–126) as either a secreted or a nonsecreted fusion protein prime NP118–126-specific CD8+ T cell responses that differ in magnitude by only 3- to 4-fold (14).
To examine the role of CD40L-CD40 interactions in CD8+ T cell priming against exogenously processed bacterial Ags, CD8+ T cell responses were measured in mice treated with CD40L-blocking Abs and infected with recombinant LM. Our data demonstrate that although CD40L blocking Ab treatment prevented cross-priming of CD8+ T cells against a model Ag, it does not inhibit priming against LM Ags. In addition, we show that LM infection overcomes CD40L blockade to allow CD8+ T cell cross-priming against a model exogenous Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old female BALB/c (H-2d MHC) and CB6F1 (H-2bxd MHC) mice were purchased from National Cancer Institute (Frederick, MD). Virulent LM strains used in this study were recombinant strain XFL304 (LM-NPns) expressing a nonsecreted fusion protein containing the LCMV NP118–126 epitope and strain XFL303 (LM-NPs) expressing the same fusion protein in secreted form (14). Attenuated LM strain DP-L1942 (ActA- LM), which carries an in-frame deletion in the actA gene (15), and attenuated LM strain DP-L2161 (listeriolysin O (LLO)- LM), which carries an in-frame deletion in the hly gene (16), were also used. Growth and maintenance of all LM strains were described previously (17).
Infection with LM
Age- and sex-matched adult BALB/c or CB6F1 mice were infected by i.v. injection with 2 x 103 LM-NPns or LM-NPs, 1 x 106 ActA- LM, and 1 x 108 LLO- LM. Actual numbers of CFU injected were determined for each experiment by plate count.
Cross-priming with OVA-loaded splenocytes
Age- and sex-matched CB6F1 mice were immunized with OVA-loaded BALB/c splenocytes as previously described (18, 19). Briefly, RBC-depleted BALB/c splenocytes were incubated with freshly prepared OVA (10 mg/ml) in hypertonic medium (RPMI 1640, 0.5 M sucrose, 10% polyethylene glycol 1000, and 10 mM HEPES, pH 7.2) for 10 min at 37°C. Splenocytes were then diluted in warm hypotonic medium (60% HBSS and 40% H2O) and incubated for 2 min at 37°C. Cells were washed twice in HBSS and resuspended at 60–70 x 106 splenocytes/ml. CB6F1 mice received 0.5 ml/animal i.v. For coimmunization with LM and OVA-loaded splenocytes, i.v. infection with the indicated number of organisms was performed first followed 2–4 h later by injection of OVA-loaded splenocytes.
Cells lines and Abs
Hybridomas producing anti-CD40L mAb MR-1 (20)
(a gift from Dr. T. Waldschmidt, University of Iowa, Iowa City, IA) and
anti-TNF (XT22 and XT3 used in combination at a mass ratio of 1:1)
(21) were grown in DMEM supplemented with 10% FCS,
L-glutamine, and antibiotics. Hybridomas producing
anti-IFN- (XMG1.2) (22) were grown in RPMI 1640
supplemented with 10% FCS, L-glutamine, and antibiotics
(RP10) (23). Ab was purified from culture supernatant by
affinity chromatography with protein G (Amersham Pharmacia, Piscataway,
NJ). Each batch of Ab was concentrated to 2 mg/ml and assessed for
endotoxin contamination by Limulus amebocyte lysate assay
(Associates of Cape Cod, Falmouth, MA). All preparations contained <15
ng/ml endotoxin.
Anti-CD40L Ab MR-1 has been used in multiple studies to block CD40L in vivo (24). To determine whether our purification procedure was sufficient in yielding MR-1 that could effectively block CD40L in vivo, MR-1- or hamster Ig (hIg)-treated BALB/c mice were immunized with SRBC. Immunization results in CD40L-dependent germinal center formation within 4 days in control hIgG (Jackson ImmunoResearch, West Grove, PA)-treated animals or animals receiving no Ab treatment. In animals that received 1 mg MR-1 in total, given on days -2, 0, 2, and 4, surface staining revealed a 70–85% decrease in germinal center B cells (data not shown), a level below that of germinal center B cells found in naive animals housed in our animal facility. Similar data were obtained 7 days after SRBC immunization (data not shown).
Intracellular cytokine staining of splenocytes
The number of CD8+ T cells specific for
OVA257–264 in the context of
H-2Kb, NP118–126 in the
context of H-2Ld, LLO91–99
in the context of H-2Kd, or
p60217–225 in the context of
H-2Kd was determined by intracellular cytokine
staining for IFN- as previously described (25).
RBC-depleted splenocytes from immunized mice were incubated for 5–6 h
in 2 µl/ml brefeldin A at 37°C with or without synthetic peptides
at 200 nM. For staining, cells were placed on ice, washed, and
incubated with Ab directed against the FcII/III receptors (2.4G2)
and FITC-conjugated anti-CD8 mAb (BD PharMingen, San Diego, CA),
fixed, permeabilized, and stained with PE-conjugated anti-IFN-
mAb according to manufacturer’s instructions (BD PharMingen).
Data were acquired on FACScan flow cytometer (Becton Dickinson,
Mountain View, CA) using Summit software. One hundred thousand events
were collected for analysis with FlowJo software (Tree Star, San
Carlos, CA). The gate for IFN-+ cells
was selected so that the percentage of IFN-+
cells in the unstimulated sample for each mouse was 0.2% or less of
CD8+ splenocytes. This value was subtracted from
the peptide-stimulated values to determine the frequency of Ag-specific
CD8+ T cells. Total numbers of epitope-specific
CD8+ T cells/spleen were calculated using this
frequency, the percentage of CD8+ T cells in each
sample, and the total number of splenocytes per animal.
IFN- ELISPOT
Ag-specific CD8+ T cell responses were
measured by IFN- ELISPOT analysis as previously described
(26). Briefly, splenocytes
(104–105/well) were
cultured with medium alone or with 100 nM synthetic peptide
(LLO91–99 or NP118–126)
for 36–48 h in flat-bottom 96-well plates that had been previously
coated with rat anti-mouse IFN- mAb (RA-6A2, BD PharMingen).
Following washes with PBS-0.05% Tween, the plates were incubated for
3 h at room temperature with rabbit anti-IFN- antiserum (a
gift from Dr. J. Cowdery, University of Iowa). After washing, the
plates were incubated for 4–5 h with donkey anti-rabbit Ig
conjugated to alkaline phosphatase (Jackson ImmunoResearch). Further
washing was followed by addition of 1 mg/ml
5-bromo-4-chloro-3-indolylphosphate (Sigma, St. Louis, MO) buffer with
0.75% agarose. The reaction was developed for 0.5–1 h at 37°C and
was stopped by storage at 4°C. Spots were counted using a dissecting
microscope. The average frequency of responders from triplicate wells
was multiplied by the total number of splenocytes in calculating the
number of responders per spleen. Background (no peptide stimulation)
was subtracted from this value to determine the total number of
Ag-specific cells per spleen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disruption of CD40L-CD40 interactions has been shown to prevent CD8+ T cell cross-priming against model Ags (1, 2). We first determined whether our preparation of MR-1 (CD40L blocking Ab) was capable of disrupting CD40L-CD40 interactions in vivo by measuring CD8+ T cell cross-priming against OVA-loaded splenocytes in animals treated with MR-1.
CB6F1 (H-2bxd) animals were treated with MR-1 or
hIg and injected i.v. with OVA-loaded BALB/c
(H-2d) splenocytes. Priming of an
H-2Kb-restricted CD8+ T
cell response to the OVA257–264 epitope in
this experiment requires cross-presentation, since the Ag is loaded
into BALB/c splenocytes, which lack the necessary MHC class
I-presenting molecule. OVA257–264-specific
CD8+ T cell responses were quantitated 7 days
after injection using intracellular IFN- staining of splenocytes to
detect Ag-specific cells (25). Representative mice treated
with hIg control Abs or MR-1 are shown in Fig. 1
A. In the absence of peptide
stimulation, few CD8+ T cells make IFN-.
However, when splenocytes from hIg-treated, OVA-immunized animals were
incubated with the OVA257–264 peptide,
approximately 0.75% of CD8+ T cells produced
IFN- after subtraction of background. The total number of
Ag-specific cells ranged from 0.4–1 x
105/spleen in this experiment and is
representative of 12 independent experiments (Fig. 1B). In
contrast, CD8+ splenocytes from MR-1-treated,
OVA-immunized animals failed to make a detectable IFN- response over
background after stimulation with synthetic
OVA257–264 (Fig. 1, A and
B). In total, 4 of 36 MR-1-treated mice and 33 of 36
hIg-treated mice had responses above the limit of detection. Similar
results were reported with CD40L-/- and
CD40-/- mice (2). Thus, MR-1
treatment effectively prevents detectable cross-priming of
CD8+ T cells against a model exogenous
Ag.
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We investigated the role of CD40L-CD40 interactions in
CD8+ T cell priming against secreted
(endogenously processed) and nonsecreted (exogenously processed) LM
Ags. Mice treated with MR-1 or hIg control Abs were infected with
2 x 103 LM-NPns (
0.1
LD50) that expresses a well-characterized
CD8+ T cell epitope from the nucleoprotein of
LCMV (NP118–126) as a nonsecreted fusion
protein. As an internal control, CD8+ T cell
priming against a secreted, endogenously processed epitope from the LM
molecule LLO91–99 was also measured. Ag-specific
CD8+ T cells were detected by IFN-
intracellular staining 7 days after infection. Responses of
approximately 3.3–3.9 x 105
LLO91–99-specific cells/spleen were detected
from both groups of animals (Fig. 2A). Similarly, the total
number of NP118–126-specific
CD8+ splenocytes from both MR-1- and hIg-treated
animals ranged from 6 to 8 x 104
cells/spleen (Fig. 2B). Splenocytes from uninfected mice did
not produce IFN- after stimulation with either peptide (data not
shown). As a control for the course of infection, liver CFU analysis
was performed on days 3 and 7, as infections in the spleen and liver
are generally parallel (J. Harty, unpublished observations). MR-1- and
hIg control Ab-treated animals showed no difference in infection at
these time points and did not have CFU in the liver above the limit of
detection (150 organisms/g liver) on day 7 (data not shown). This
demonstrates that in contrast to cross-priming against OVA, naive
CD8+ T cell priming against nonsecreted or
secreted Ags expressed by LM is not inhibited by MR-1 treatment.
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ELISPOT
(Fig. 2
, C and D). In a preliminary experiment
memory CD8+ T cell responses to
NP118–126 were at or below the level of
detection using intracellular cytokine staining for IFN-. Therefore,
the data shown in Fig. 2, C and D, were
determined using the ELISPOT assay, which allows detection of low
frequencies of Ag-specific CD8+ T cells. A 2- to
3-fold decrease in the number of Ag-specific cells was observed in
MR-1-treated mice compared with control animals, but this revealed
statistical significance only for
NP118–126-specific cells. However, since both
LLO91–99- and
NP118–126-specific cells were reduced in number,
and a slightly larger spleen size was seen in the hIg control group, it
is unlikely that this difference is biologically significant.
CD8+ T cell recall responses were also examined
in animals that received transient CD40L blockade during primary
infection with LM-NPns. For challenge, the mice were infected with
LM-NPs, which is isogenic with LM-NPns except that LM-NPs secretes the
NP118–126 fusion protein and primes a 3- to
4-fold higher NP118–126-specific response
(14). On day 30 after primary infection, mice were
challenged with 2 x 105 LM-NPs
(10LD50 for naive mice; Fig. 2, E
and F). Both MR-1-treated and hIg-treated animals contained
3–5 x 106
LLO91–99-specific CD8+ T
cells (Fig. 2E) and 2.4–2.7 x
106 NP118–126-specific
CD8+ T cells (Fig. 2F) in the spleen
as determined by IFN- intracellular staining five days after
infection. Challenge of immune mice with LM-NPns also resulted in
comparable levels of secondary Ag-specific CD8+ T
cell responses between MR-1- and hIg-treated animals (data not shown).
These experiments illustrate that MR-1 treatment during the primary
exposure to Ag does not impact the ability of
CD8+ T cells to respond to either secreted or
nonsecreted Ags (Fig. 2). Memory responses to
NP118–126 were significantly decreased 2- to
3-fold compared with control animals, but this did not result in any
deficiency in CD8+ T cell responses to a
secondary challenge.
Infection with rLM overcomes CD40L blockade in cross-priming
We next asked whether LM infection could overcome the capacity of
MR-1 to block CD8+ T cell cross-priming against
OVA-loaded splenocytes. MR-1 treatment blocked the
OVA257–264-specific CD8+ T
cell response in CB6F1 animals immunized with OVA-loaded splenocytes
(Figs. 1 and 3). In contrast, animals
treated with MR-1, infected with 2 x 103
LM-NPns that lacks the OVA257–264 epitope, and
immunized with OVA-loaded splenocytes, made a substantial
OVA257–264 response of 3.3 x
104 CD8+ T cells/spleen
(Fig. 3). This response was not significantly different from
OVA-immunized, uninfected animals treated with hIg control Abs. There
was a significant increase in the number of
OVA257–264-specific CD8+ T
cells (6-fold) under conditions of infection and hIg treatment,
suggesting that infection results in an enhanced environment for
CD8+ T cell cross-priming. The experiment in Fig. 3 demonstrates that LM infection is able to overcome CD40L blockade in
cross-priming of naive CD8+ T cells against
OVA-loaded splenocytes.
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The mechanism by which LM infection overcomes CD40L blockade of
cross-priming is unknown, but may relate to specific properties of the
organism or the host response to LM. To address this issue, we asked
whether infection with the attenuated LM strain DP-L1942
(ActA- LM) would also be able to overcome MR-1
treatment. ActA- LM has an in-frame deletion in
the actA gene, which is required for intracellular spread of
LM to neighboring host cells (15). Since this strain is
highly attenuated, mice in this experiment were infected with 1 x
106 ActA- LM (0.1
LD50). CB6F1 animals were treated with MR-1 or
hIg as described, infected with ActA- LM, and
immunized with OVA-loaded splenocytes. Similar to the experiments with
virulent LM, OVA257–264-specific
CD8+ T cell responses were only found in
MR-1-treated animals when they were also infected with LM (Figs. 3 and 4). The magnitude of the response in
MR-1-treated, ActA- LM-infected animals was
approximately 105 Ag specific cells/spleen and
was equivalent to that seen in uninfected hIg-treated animals. These
data show that infection with either virulent or attenuated LM enhances
CD8+ T cell cross-priming against OVA-loaded
splenocytes, overcoming CD40L blockade in MR-1-treated animals and
increasing the Ag-specific response in hIg-treated animals.
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B). A lower dose of 1 x
103 ActA- LM resulted in
measurable priming against an endogenous, secreted LM epitope
(p60217–225) that primes similar numbers of
Ag-specific cells to OVA during LM infection (27).
However, this dose of infection failed to overcome CD40L blockade of
OVA257–264-specific CD8+ T
cell priming. Immunization with 1 x 106
heat-killed ActA- LM also did not result in
measurable CD8+ T cell priming against OVA or LM
Ags. This result demonstrates that bacterial components alone, at least
at the level tested here, are not sufficient to overcome CD40L blockade
of CD8+ T cell cross-priming and that active
infection must take place in the host.
A second attenuated strain, DP-L2161 (LLO- LM),
was also used to infect CB6F1 animals treated with MR-1 or hIg and
immunized with OVA-loaded splenocytes (Fig. 4C).
LLO- LM has an in-frame deletion in the
hly gene, which encodes the LLO virulence factor
(16). Loss of functional LLO results in organisms that are
highly attenuated due to an inability to escape from the host cell
phagosome (28, 29). A lower number of
OVA257–264-specific CD8+ T
cells was measured in mice infected with 1 x
108 (LD50
>109) LLO- LM and treated
with hIg compared with hIg-treated animals infected with
ActA- LM (Fig. 4, compare A and
C). Many MR-1 treated, LLO-
LM-infected animals had no OVA257–264-specific
response above the level of detection (7 of 12 animals). However,
some animals did have measurable
OVA257–264-specific responses ranging from
approximately 1 x 104 to 1 x
105 CD8+ T cells/spleen.
This indicates that infection with LLO- LM can
overcome CD40L blockade of cross-priming, but does so less efficiently
than virulent LM or ActA- LM.
Neutralization of TNF or IFN- does not prevent a response to
OVA-loaded splenocytes during infection
During LM infection, various proinflammatory cytokines, such as
IFN- and TNF, are generated that could serve as activation signals
for DCs. TNF in particular stimulates the maturation of DCs and
promotes cross-presentation of model Ags in vivo in the absence of
CD40L (30, 31). To determine whether TNF or IFN- is
important for the ability of LM infection to overcome CD40L blockade of
cross-priming, CB6F1 animals were treated with MR-1 and TNF- or
IFN--neutralizing Abs. Cytokine-neutralizing Abs were used at a
dosage previously determined to be effective for neutralization in vivo
(32). Animals were then infected with 1 x
106 ActA- LM and immunized
with OVA-loaded splenocytes. Animals treated with TNF-neutralizing Abs
had enlarged spleens (3–4 x 108
splenocytes), and six of eight animals had organisms remaining in the
liver 7 days after infection (data not shown). All other treatment
groups had similar spleen sizes (2 x 108
splenocytes) and had cleared the infection by day 7. The total number
of OVA257–264-specific
CD8+ T cell was not significantly different in
TNF-- or IFN--treated animals compared with control
rat Ig-treated animals (Fig. 5). However, the lack of
statistical significance in TNF-depleted mice may be impacted by the
increased level of infection in these animals. This experiment
demonstrates that neutralization of these two cytokines under
conditions of CD40L blockade and bacterial infection does not prevent
naive CD8+ T cell priming against OVA-loaded
splenocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have all
been shown to result in the maturation of DCs in vitro, and
anti-CD40 Abs have been shown to promote CD8+
T cell priming against exogenous model Ags in vivo in the absence of
CD4+ T cells (3, 7). Although MR-1 treatment prevented CD8+ T cell cross-priming against OVA-loaded splenocytes, it did not affect CD8+ T cell priming against nonsecreted or secreted LM Ags. It has been shown that CD4+ T cells or CD40L are not required for CD8+ T cell priming under conditions of viral infection and that there is minimal impact on the splenic CD8+ T cell response to secreted LM Ags in CD40-/- mice (8, 12). However, these studies did not address the issue of priming against Ags that do not have access to endogenous MHC class I processing pathways. It has additionally been demonstrated that memory CD8+ T cell responses decline in the absence of CD40L. Although we saw a significant decline in the CD8+ T cell memory response to NP118–126, secondary responses to both NP118–126 and LLO91–99 were unaffected in MR-1-treated mice in our system. Since our MR-1 treatment was only performed transiently during the initial exposure to Ag, it is unknown whether constant blockade of CD40L in these animals would affect primary or memory CD8+ T cell responses after LM infection.
Consistent with the idea that immunization with model Ags may not
reflect the host response to infection, we found that both virulent and
attenuated LM could overcome CD40L blockade of
CD8+ T cell cross-priming. In addition, it was
consistently seen that hIg-treated animals exhibited increased
CD8+ T cell responses in the presence of
infection (
Figs. 3–5).
Cross-presentation requires bone marrow-derived DCs, which were
recently reported to be of the CD8+ or
lymphoid lineage (33). An interesting hypothesis is that
the increase in CD8+ T cell priming against OVA
in the presence of infection is due to an increase in the number of
cells or types of cells (i.e., CD8-) able to
participate in cross-priming as effective APCs. Alternatively, when
both CD40L and microbial signals are present, DCs show augmented IL-12
production in vivo, a situation that could result in increased T cell
responses (34).
In contrast, LLO- LM, which have a deletion in the hly gene, overcame CD40L blockade less efficiently in the majority of MR-1-treated animals. It is likely that the host response to LLO- LM is different from the response to other strains of LM. Infection with LLO- LM elicits lower IL-12 production compared with virulent LM, which may result in a cytokine environment that is not optimal for CD8+ T cell cross-priming in the presence of MR-1 (35, 36). Previous experiments show that IL-12 is required in at least some models of exogenous Ag presentation, although the requirement for this cytokine in cross-presentation has not been examined (37). Recent evidence also demonstrated that CD40-mediated, CD8+ T cell priming against exogenously processed heat-killed LM was dependent on endogenous IL-12 production (38).
The mechanism by which LM infection overcomes CD40L blockade of
cross-priming is still unknown. However, there is a clear requirement
for active infection, since the injection of large numbers of
heat-killed bacteria (Fig. 4B) failed to stimulate
measurable CD8+ T cell priming against OVA-loaded
splenocytes or LM Ags. This indicates that the presence of bacterial
products such as the cell wall or bacterial DNA was not sufficient to
overcome CD40L blockade at least at the levels tested. The ability of
LM infection to overcome CD40L blockade was not inhibited by treatment
with mAbs that efficiently neutralize IFN- or TNF, illustrating that
multiple signals generated during bacterial infection may stimulate the
maturation of DCs. The depletion of TNF or IFN- also does not
prevent naive CD8+ T cell priming against
secreted or nonsecreted LM Ags in the presence or the absence of MR-1
(S. E. Hamilton and J. T. Harty, unpublished observations).
Evidence has accumulated demonstrating the stimulatory properties of
members of the TNF ligand superfamily (4-1BBL, OX40L, TNF-related
apoptosis-inducing ligand, and TNF-related activation-induced
cytokine) on DCs (39). 4-1BBL can influence
CD4+ T cell and CD8+ T cell
responses, particularly under suboptimal antigenic stimulation
(40). Based on our results, it would be of interest to
examine the effect of CD40L blockade on cross-priming in
4-1BBL-/- mice in the presence or the absence
of LM infection and/or OVA-loaded splenocyte immunization. LM can also
infect DCs themselves, suggesting that this could also be route to
induce maturation during infection (41). This may be
particularly relevant in experiments in which TNF was neutralized in
mice resulting in increased levels of infection. Alternatively, another
soluble molecule, such as IL-1, could be important for the maturation
of dendritic cells during bacterial infection.
How cross-presentation results in either priming or tolerance of CD8+ T cells is still under debate. Cross-presentation is a critical mechanism both for the induction of peripheral tolerance and for immune responses to Ags without obvious access to the cytosol of APCs (reviewed in Ref. 5). Our results support a model in which tolerance is the likely result when the APC environment is lacking in inflammatory stimuli. In this situation the APC would take up and present Ag, but would lack the costimulatory molecules required to effectively stimulate naive T cells. However, under conditions of infection or inflammation, changes in the APC environment, such as cytokine secretion or pathogen products, would promote maturation of DCs for effective stimulation of naive T cells. This poses a potential problem for the host, since the uptake of peripheral self Ags would presumably continue in a T cell stimulatory environment and increase the likelihood of autoimmunity. This is consistent with models of autoimmunity in which conditions often worsen during concurrent infections (42). How tolerance to self Ags can be maintained during infection requires further investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. T. Harty, 3-512 Bowen Science Building, Department of Microbiology, University of Iowa, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu
3 Abbreviations used in this paper: CD40L, CD40 ligand; DC, dendritic cell; hIg, hamster Ig; LCMV, lymphocytic choriomeningitis virus; LLO, listeriolysin O; LM, Listeria monocytogenes; NP, nucleoprotein.
Received for publication July 6, 2001. Accepted for publication September 10, 2001.
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  M. G. H. Hernandez, L. Shen, and K. L. Rock CD40-CD40 Ligand Interaction between Dendritic Cells and CD8+ T Cells Is Needed to Stimulate Maximal T Cell Responses in the Absence of CD4+ T Cell Help J. Immunol., March 1, 2007; 178(5): 2844 - 2852. [Abstract] [Full Text] [PDF]
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M. Wuthrich, P. L. Fisette, H. I. Filutowicz, and B. S. Klein Differential Requirements of T Cell Subsets for CD40 Costimulation in Immunity to Blastomyces dermatitidis J. Immunol., May 1, 2006; 176(9): 5538 - 5547. [Abstract] [Full Text] [PDF]
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S. E. Applequist, E. Rollman, M. D. Wareing, M. Liden, B. Rozell, J. Hinkula, and H.-G. Ljunggren Activation of Innate Immunity, Inflammation, and Potentiation of DNA Vaccination through Mammalian Expression of the TLR5 Agonist Flagellin J. Immunol., September 15, 2005; 175(6): 3882 - 3891. [Abstract] [Full Text] [PDF]
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D. M. Lindell, T. A. Moore, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Generation of Antifungal Effector CD8+ T Cells in the Absence of CD4+ T Cells during Cryptococcus neoformans Infection J. Immunol., June 15, 2005; 174(12): 7920 - 7928. [Abstract] [Full Text] [PDF]
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M. J. Montfort, H. G. A. Bouwer, C. R. Wagner, and D. J. Hinrichs The Development of Functional CD8 T Cell Memory after Listeria monocytogenes Infection Is Not Dependent on CD40 J. Immunol., September 15, 2004; 173(6): 4084 - 4090. [Abstract] [Full Text] [PDF]
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 U. K. Rapp and S. H. Kaufmann DNA vaccination with gp96-peptide fusion proteins induces protection against an intracellular bacterial pathogen Int. Immunol., April 1, 2004; 16(4): 597 - 605. [Abstract] [Full Text] [PDF]
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J. C. Sun and M. J. Bevan Cutting Edge: Long-Lived CD8 Memory and Protective Immunity in the Absence of CD40 Expression on CD8 T Cells J. Immunol., March 15, 2004; 172(6): 3385 - 3389. [Abstract] [Full Text] [PDF]
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N. Ismail, L. Soong, J. W. McBride, G. Valbuena, J. P. Olano, H.-M. Feng, and D. H. Walker Overproduction of TNF-{alpha} by CD8+ Type 1 Cells and Down-Regulation of IFN-{gamma} Production by CD4+ Th1 Cells Contribute to Toxic Shock-Like Syndrome in an Animal Model of Fatal Monocytotropic Ehrlichiosis J. Immunol., February 1, 2004; 172(3): 1786 - 1800. [Abstract] [Full Text] [PDF]
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 B. O. Lee, L. Hartson, and T. D. Randall CD40-deficient, Influenza-specific CD8 Memory T Cells Develop and Function Normally in a CD40-sufficient Environment J. Exp. Med., December 1, 2003; 198(11): 1759 - 1764. [Abstract] [Full Text] [PDF]
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M. Wuthrich, H. I. Filutowicz, T. Warner, G. S. Deepe Jr., and B. S. Klein Vaccine Immunity to Pathogenic Fungi Overcomes the Requirement for CD4 Help in Exogenous Antigen Presentation to CD8+ T Cells: Implications for Vaccine Development in Immune-deficient Hosts J. Exp. Med., June 2, 2003; 197(11): 1405 - 1416. [Abstract] [Full Text] [PDF]
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B. Samten, B. Wizel, H. Shams, S. E. Weis, P. Klucar, S. Wu, R. Vankayalapati, E. K. Thomas, S. Okada, A. M. Krensky, et al. CD40 Ligand Trimer Enhances the Response of CD8+ T Cells to Mycobacterium tuberculosis J. Immunol., March 15, 2003; 170(6): 3180 - 3186. [Abstract] [Full Text] [PDF]
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D. J. Shedlock, J. K. Whitmire, J. Tan, A. S. MacDonald, R. Ahmed, and H. Shen Role of CD4 T Cell Help and Costimulation in CD8 T Cell Responses During Listeria monocytogenes Infection J. Immunol., February 15, 2003; 170(4): 2053 - 2063. [Abstract] [Full Text] [PDF]
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S. E. Hamilton and J. T. Harty Quantitation of CD8+ T Cell Expansion, Memory, and Protective Immunity After Immunization with Peptide-Coated Dendritic Cells J. Immunol., November 1, 2002; 169(9): 4936 - 4944. [Abstract] [Full Text] [PDF]
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) or CD40L Ab
MR-1 (
). On day 7 after OVA immunization intracellular staining for
IFN-
) or MR-1
(below level of detection and 
) on days -2, 0, 2, and 4. On day 0
mice were infected i.v. with 2 x 103 CFU LM-NPns (
