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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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of Ag-specific CD8+ T cells results in
proliferation and differentiation into effector cells which are able to
secrete inflammatory cytokines (IFN-
and TNF-
) and cytotoxic
compounds like perforin and granzymes (7, 8). These
effector cells will contract in a programmed fashion (9),
resulting in stable memory levels related to the peak of initial
expansion (7, 9). Although a pathogen may contain many
epitopes that stimulate CD8+ T cell precursors,
populations of CD8+ T cells that differ in Ag
specificity expand and contract with tightly regulated coordinate
kinetics regardless of the magnitude of response (7, 9, 10, 11, 12). Also upon activation, CD8+ T cells
appear to acquire as yet undefined characteristics enabling them to
migrate to and enter nonlymphoid tissues (13). These
characteristics may have important ramifications for the ability of
memory CD8+ T cells in different locations to
perform effector functions and protect the host from subsequent
infections.
Our goal was to assess in quantitative fashion the ability of peptide-coated DCs to stimulate CD8+ T cell memory and protective antibacterial responses. Thus, we determined the kinetics of primary CD8+ T cell responses to DCs pulsed with several MHC class I-restricted peptides derived from Listeria monocytogenes (LM). Next, we examined the relationship between DC immunization and protective immunity against LM.
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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) mice were purchased from the National Cancer Institute (Frederick, MD). Virulent LM strains used in this study were recombinant strain HSL236 (listeriolysin O (LLO)+, nuclear protein (NP)+) expressing a secreted fusion protein containing the lymphocytic choriomeningitis virus NP118–126 epitope and HSL235 (LLO+, NP-) which expresses the fusion protein without NP118–126 (14). Growth and maintenance of all LM strains were as described (15). Mice were infected i.v. with the number of bacteria indicated in figure legends.
Generation of peptide-coated BMDCs
CD11c+ BMDCs were generated as previously
described with modifications (16). Briefly, RBC-depleted
BALB/c (H-2d) bone marrow was subjected to
complement depletion after incubation with mAbs 3.168 (CD8-specific),
34-5-3 (I-Ad specific), RL172 (CD4-specific),
RA3-3A1/6.1 (B220/CD45R-specific), and RB6-8C5 (neutrophil-specific).
All mAbs were purified from culture supernatant by protein G (Amersham
Pharmacia, Piscataway, NJ). Remaining cells were plated at 1 x
106/ml in RP10 (17) supplemented
with 1000 U/ml rGM-CSF (BD PharMingen, San Diego, CA) and 25 U/ml rIL-4
(PeproTech, Rocky Hill, NJ) and incubated for 5–7 days with 75% media
replacement every other day. LPS (500 ng/ml; Sigma-Aldrich, St. Louis,
MO) was then added for 1–2 days to induce maturation, and 1 µM
peptide (except as indicated in Fig. 1
D) was added to
cultures 3 h before harvest. Cells were then washed extensively
before injection. The resulting cell populations consisted of 40–80%
CD11c+ cells. These cells were also
H-2Ld+, B7.1+,
B7.2+, CD8-,
I-Ad+, and CD11b+. Based on
the percentage of CD11c+ cells (determined before
injection), 2–2.5 x 105 mature BMDCs were
injected i.v. per mouse unless otherwise indicated in figure
legends.
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Detection of Ag-specific CD8+ T cells
The number of CD8+ T cells specific for
NP118–126 or murine CMV (MCMV)168–176 in the context of
H-2Ld and LLO91–99 or p60 217–225 in the
context of H-2Kd was determined by intracellular
cytokine staining (ICS) for IFN-. 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 (eBioscience, San Diego, CA), fixed, permeabilized,
and stained with PE-conjugated anti-IFN- mAb according to
manufacturer’s instructions (eBioscience). Data was acquired on a
FACScan flow cytometer (BD Biosciences, San Jose, CA) using
Summit software. One-hundred thousand events were collected for
analysis with FlowJo software (TreeStar, San Carlos, CA). The
gate for IFN-+ cells was selected on an
unstimulated sample for each mouse. 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 percent of CD8+ T cells in each
sample, and the total number of splenocytes per animal.
51Cr-release assays
LLO91–99 or p60 217–225 specific CD8+ T cell-mediated cytotoxicity was determined in a 6-h 51Cr-release assay using BMDC (H-2d MHC) targets coated with or without 1 µM peptide as described for cell injection. 51Cr-labeled (PerkinElmer, Boston, MA) target cells (104) were plated with titrations of in vitro restimulated, peptide-specific T cell lines (18) to generate a series of E:T ratios. Total release was determined by incubating targets in 0.5% Triton X-100 (Fisher Scientific, Pittsburgh, PA). Spontaneous release was determined by incubating targets in medium alone. Percent-specific release was determined as (experimental release - spontaneous release)/(total release - spontaneous release) x 100.
Isolation of lymphocytes from the lung and liver
Before tissue removal, mice were perfused in the left ventricle with PBS containing 70 U/ml heparin (Sigma-Aldrich). Tissues were cut into small pieces, and were then shaken for 90 min at 37°C in collagenase (150 U/ml) (Life Technologies, Rockville, MD). The resulting mixture was made into a single cell suspension, filtered, and washed. Liver cell suspensions were underlayed with a 35% Percoll (Sigma-Aldrich) solution and lung cell suspensions were underlayed with both a 40% Percoll solution and then a 75% Percoll solution. Samples were centrifuged at 2500 rpm for 20 min at room temperature. The single interface of liver cells and the lower interface of lung cells were collected and treated with ammonium chloride to removed RBCs. Cells were then washed, counted, and stimulated for ICS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mature, peptide-coated DCs can stimulate primary
CD8+ T cell responses in vivo (3, 5, 19), however, it is not known whether the
CD8+ T cell response to peptide-coated DC is
similar in duration or protective capacity to that obtained after
infection. We first examined the CD8+ T cell
response after i.v. injection of 2 x 104 to
2 x 106 LPS-matured BMDCs coated with 1
µM MHC class I peptides LLO91–99 (H-2Kd
restricted) or NP118–126 (H-2Ld restricted). For
comparison, other mice were infected i.v. with rLM strains HSL235
(LLO+, NP-) or HSL236
(LLO+, NP+). Because others
have shown that injection of BMDC i.v. results in homing to T cell
areas of the spleen (19), we measured the Ag-specific
CD8+ T cell response in the spleen 7 days after
immunization by ICS for IFN-. The Ag-specific
CD8+ T cell response of representative mice,
either infected with LM strain HSL236 or immunized with 2 x
105 peptide-coated BMDCs, is shown in Fig. 1
A. As shown previously
(20), few CD8+ T cells from immune
mice make IFN- in the absence of peptide stimulation. In contrast,
0.6–3.3% of CD8+ T cells from LM- or
peptide-coated BMDC-immunized mice produced IFN- in response to
LLO91–99 or NP118–126 stimulation. This response is Ag-specific as
seen by the lack of IFN- production following stimulation with
peptides not present during the in vivo immunization. The total number
of Ag-specific cells ranged from 3 x 104 to
6 x 105 per spleen at 7 days after
immunization with peptide-coated BMDC. Interestingly, the magnitude of
response to LLO91–99 after immunization with peptide-coated BMDCs was
comparable to that seen after LM infection (6 x
105 compared with 7–8 x
105 LLO91–99 CD8+ T
cells/spleen). However, the response to NP118–126 was consistently
lower than the response to LLO91–99 (ranging from 2- to 10-fold in
different experiments) (Fig. 1C). This is in contrast to LM
infection, where LLO91–99- and NP118–126-specific
CD8+ T cell responses are similar in magnitude
(Fig. 1B). The hierarchy of response after BMDC immunization
was not a result of insufficient quantities of peptide, as coating of
BMDC with 1 µM of either LLO91–99 or NP118–126 was found to be
saturating for stimulating CD8+ T cell responses
in vivo (Fig. 1D). The magnitude of the
CD8+ T cell responses to BMDCs was also not
enhanced by the addition of an MHC class II epitope (OVA 323–339)
(data not shown).
The Ag-specific CD8+ T cell response after
delivery of 2 x 104 to 2 x
106 peptide-coated BMDCs was dose-dependent (Fig. 1C). Because the spleen is normally composed of 1–2 x
106 DCs, it is likely that injection of 2 x
106 peptide-coated BMDCs is saturating given the
available space in the lymphoid compartment. Based on this rationale,
mice in the following experiments were immunized with a nonsaturating
dosage of 2–2.5 x 105 peptide-coated BMDCs
(Fig. 1C).
BMDCs can stimulate CD8+ T cells of multiple Ag specificities provided that peptides are presented by different MHC class I molecules
Due to efficient Ag capture ability, and the high number of MHC
class I molecules on the cell surface, DCs most likely present a number
of different antigenic determinants simultaneously in vivo. We asked if
the presentation of multiple epitopes by DCs impacted the magnitude of
the CD8+ T cell response. BMDCs were coated with
LLO91–99, p60 217–225 (both H-2Kd restricted),
or NP118–126 (H-2Ld restricted) alone or in
combination, and the CD8+ T cell response in the
spleen was determined 7 days after immunization of BALB/c mice. We
found that multiple peptides could be presented effectively by the same
BMDC populations in the case of LLO91–99 and NP118–126 (Fig. 2A) or p60 217–225 and
NP118–126 (Fig. 2C). Importantly, the magnitude of the
CD8+ T cell response against BMDC coated with
single or multiple peptides was similar. Because the number of injected
BMDCs was not saturating (Fig. 1C), this data suggests that
the same DC is capable of stimulating CD8+ T cell
responses of more than one Ag specificity.
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B). This result was not due to
exclusion of the p60 217–225 epitope from binding to
H-2Kd on the BMDC surface, because both p60
217–225- and LLO91–99-specific CD8+ T cell
lines caused significant 51Cr release from BMDC
targets (Fig. 3, A and
B). Although there was a reduction in p60 217–225-specific
51Cr release when BMDC targets were also coated
with LLO91–99, this data still suggests that both p60 217–225 and
LLO91–99 epitopes were presented by peptide-coated BMDCs. However,
because the Ag density required for activation of naive
CD8+ T cells may be much greater than for
previously activated T cell lines (21), the requisite
number of p60 217–225:Kd complexes may not have
been available to stimulate naive CD8+ T cells in
vivo. The in vitro data also suggests that if peptide titrations were
performed, conditions might be found in which both p60 217–225 and
LLO91–99 specific CD8+ T cells may be stimulated
in vivo. However, dilution of LLO91–99 could also decrease the
CD8+ T cell response to this epitope (Fig. 1D).
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C). However, responses to both epitopes were reduced in
these mice which may be due to a limitation in the space available in
the spleen for this number of peptide-coated BMDCs (twice that injected
in previous experiments). This notion is supported by data
demonstrating a decrease in the number of LLO91–99 and NP118–126
specific CD8+ T cells if mice received a second
injection of "cold" BMDC which were not coated with peptide (Fig. 2C). Although this experimental design prevents peptide
transfer between BMDC loaded with either p60 217–225 or LLO91–99
during in vitro peptide loading and injection, it is a possibility that
some degree of peptide transfer occurs in vivo after BMDC migrate to
the spleen. Together, the data in Figs. 2C and 3B
suggest that competition for the H-2Kd molecule
during in vitro loading as well as competition for access to
peptide-loaded DC in vivo may influence the eventual
CD8+ T cell response. Although studies with
TCR-transgenic systems suggest that in vivo
CD8+ T cells may compete for access to the APC or
for peptide:MHC complexes on the cell surface (4, 22, 23),
our results demonstrate that BMDCs coated with multiple peptides can
effectively stimulate CD8+ T cells provided that
the peptides are presented by different MHC class I molecules. Differential kinetics of CD8+ T cells responding to BMDCs coated with peptides presented by H-2Kd and H-2Ld
During bacterial or viral infection in wild-type mice,
CD8+ T cells with different Ag specificities and
MHC restriction display coordinate expansion and contraction leading to
stable memory (7, 10, 11, 12). We examined the pattern of
CD8+ T cell expansion and contraction after
immunization with BMDCs coated with LLO91–99
(H-2Kd restricted) or NP118–126
(H-2Ld restricted) (Fig. 4, A and B). Five
days after injection, the NP118–126 specific response had peaked at
3.4 x 105 CD8+ T
cells per spleen. Although the number of LLO91–99-specific
CD8+ T cells was similar on day 5 (3.0 x
105 CD8+ T cells), this
response did not reach peak levels until day 7. In contrast, the number
of NP118–126-specific CD8+ T cells dropped
between 3- and 5-fold by day 7. These kinetics were observed in both
mice injected with single (Fig. 4) and double (Fig. 5) peptide-coated BMDCs. Between days 12
and 14, both populations of CD8+ T cells had
declined to memory levels that could be detected for at least 100 days
following immunization (Fig. 4 and data not shown).
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, C and
D). We found that although the magnitude of Ag-specific
CD8+ T cell responses depended on the epitope
examined, similar kinetic patterns were measured in response to
NP118–126, p60 217–225, and MCMV168–176, but not LLO91–99. Although
this experiment examines only four peptides, it demonstrates that
noncoordinate kinetics after peptide-coated BMDC immunization are not
restricted to peptides presented by H-2Ld.
Another possible mechanism for the noncoordinate responses observed in
Fig. 4 may be that the NP118–126:Ld complex is
less stable than the LLO91–99:Kd complex.
H-2Ld has a shorter surface half-life compared
with other H-2d MHC class I molecules due to an
unusually weak association with
2-microglobulin and peptide (24).
We compared the surface half-life of peptide:MHC class I molecule
complexes by coating BMDCs with peptide, washing away unbound peptide,
and combining these cells with Ag-specific T cell lines over the next
three days (Fig. 4E). No additional peptide was added to
BMDCs after day 0, and on each day BMDCs were washed to remove any free
peptide before addition to Ag-specific CD8+ T
cell lines. This experiment is designed to functionally measure the
number of peptide:MHC class I molecule complexes on the BMDC surface by
measuring CD8+ T cell activation. Surprisingly,
the ability of peptide-coated BMDCs to stimulate NP118–126
and LLO91–99 specific CD8+ T cell responses
decreased similarly over time, while a stronger decline in T cell
stimulation was seen in BMDC coated with p60 217–225. However, we do
not know from this experiment if the initial number of
LLO91–99:Kd and
NP118–126:Ld complexes formed was different,
which may impact the rate of decline in the CD8+
T cell response. Because this experiment also uses in vitro
restimulated CD8+ T cell lines, it is also
possible that the Ag level for activation of naive
CD8+ T cells in vivo differs from that measured
with previously activated T cell lines. However, this experiment
suggests that the stability of the NP118–126:Ld
complex is not responsible for the differential kinetics measured in
vivo between NP118–126- and LLO91–99-specific
CD8+ T cell responses.
LM infection does not alter CD8+ T cell kinetics in response to BMDCs presenting multiple MHC class I peptides
CD8+ T cell expansion kinetics can be
affected by the inflammatory response to infection (25).
This may be due to bacterial products and/or proinflammatory cytokines
that promote the maturation of DCs to a highly stimulatory state for
naive T cells or to the production of growth-promoting cytokines that
drive CD8+ T cell proliferation upon activation
(20, 25). We asked whether LM infection would alter the
magnitude or kinetics of the CD8+ T cell response
to peptide-coated mature BMDCs. Mice were either immunized with mature
BMDCs coated with LLO91–99 and NP118–126, infected with LM strain
HSL235 (LLO+, NP-), or
were coinjected with LM and peptide-coated mature BMDCs. Because strain
HSL235 does not express the NP118–126 epitope, an enhanced
CD8+ T cell response to BMDC presenting this
epitope in infected mice would reflect a role for inflammation. In
contrast to this notion, the NP118–126-specific response was similar
in magnitude and kinetics in mice that received BMDCs alone or in the
presence of LM infection (Fig. 5A, filled symbols). This was
also true if animals received LM infection 1 day before peptide-coated
BMDC immunization, which would allow more time for a vigorous
inflammatory response to be generated (data not shown). These
experiments suggest both that LPS-matured BMDCs are optimal for
stimulating naive CD8+ T cells, and that the
primary purpose of infection-induced inflammation in vivo is to
establish the maturation of APCs (20).
In contrast, the LLO91–99-specific CD8+ T cell
response peaked on day 7 at 8.3 x 105
cells for both BMDC- and LM-immunized animals. This response was
enhanced (2.3 x 106 LLO91–99 specific
cells) in animals which were immunized with peptide-coated BMDCs and
LM. This increased response is presumably due to increased levels of Ag
in these mice, although the measured response was more than additive.
When the same experiment was performed using LM strain HSL236
(LLO+, NP+), both
LLO91–99- and NP118–126-specific CD8+ T cell
responses were enhanced in animals which received peptide-coated BMDC
and LM (Fig. 5, B and C). These experiments
demonstrate that infection-induced inflammation is not sufficient to
cause the coordinate regulation of CD8+ T cell
kinetics.
Ag-specific CD8+ T cells are present in the lung and liver after DC or LM immunization
One explanation for the inability of inflammation to alter
Ag-specific CD8+ T cell numbers in the spleen is
that infection with LM may change the migratory properties of
CD8+ T cells, resulting in their accumulation in
nonlymphoid organs. Previous work has shown that populations of
Ag-specific CD8+ T cells are present in many
nonlymphoid tissues after infection with bacteria or viruses, and that
these cells may contain unique properties compared with Ag-specific T
cells isolated from the spleen or lymph nodes (13, 26). To assess the magnitude of Ag-specific
CD8+ T cell populations in nonlymphoid
organs, mice were injected with BMDCs coated with LLO91–99 and
NP118–126 or were infected with LM strain HSL235. Seven days after
immunization, Ag-specific CD8+ T cells were
quantitated by intracellular staining for IFN- in the spleen, liver,
and lung (Fig. 6A). We found
that immunization with peptide-coated BMDC or infection with LM caused
trafficking of Ag-specific CD8+ T cells to
tissues. The magnitude of LLO91–99- and NP118–126-specific
CD8+ T cells was similar between organs, although
the frequency of CD8+ T cells isolated from each
organ was significantly different as previously reported
(13) (data not shown). As a control, immature BMDC defined
as being CD11c+ but B7.2 low or negative (Fig. 6B), were also injected into mice after coating with
LLO91–99 and NP118–126. Five days after immunization, no
Ag-specific response could be detected in any of the organs examined
(Fig. 6C). If immature BMDCs were plated for 24 h in
LPS, peptide-coated, and then injected into mice,
CD8+ T cell responses were detected in the
spleen, lung, and liver (data not shown) demonstrating that these cells
are capable of stimulating a response when matured in vitro. This
experiment shows that immature BMDC do not stimulate
CD8+ T cell responses in vivo, and that detection
of Ag-specific CD8+ T cells in the spleen
correlates with detection in nonlymphoid organs. Together, these
experiments demonstrate that infection is not required for Ag-specific
CD8+ T cells to migrate into nonlymphoid organs,
suggesting that peptide-coated BMDC immunization is likely to generate
both central and effector memory CD8+ T cell
populations (13, 27).
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We next asked whether the CD8+ memory T
cells generated by immunization with peptide-coated BMDC were capable
of protecting mice from high dose challenge with LM. Twenty-eight days
after injection with either mature BMDCs coated with LLO91–99 and
NP118–126 or LM, mice contained 1.5 x
105 LLO91–99 (LM- and BMDC-immunized) and
3.0 x 104 NP118–126- (BMDC-immunized
only) specific CD8+ T cells in the spleen (Fig. 4). On day 30, mice were infected with 10 LD50
(for a naive mouse) of HSL235 (LLO+,
NP-) or HSL236 (LLO+,
NP+). These strains of LM are isogenic,
demonstrate equivalent virulence (14), and equivalent
sensitivity to CD8+ T cell protective immunity in
vivo (8). Bacterial numbers in the liver and spleen 3 days
after infection showed that BMDC-immunized animals challenged with
HSL235 (LLO+, NP-) had
1000- and 25-fold fewer organisms in the liver and spleen,
respectively, compared with naive animals (Fig. 7, A and B). Mice
challenged with HSL236 (LLO+,
NP+) had a 600-fold or more decrease in
bacterial numbers compared with naive mice in both the spleen and
liver. LM-immune animals had numbers of bacteria similar to
BMDC-immunized mice challenged with HSL236 (LLO+,
NP+) (Fig. 7, A and B).
These results demonstrate that stimulation of NP118–126-specific
memory CD8+ T cells in addition to
LLO91–99-specific memory T cells (after HSL236 challenge only)
resulted in enhanced immunity. This was particularly true in the spleen
where a 50-fold decrease in bacteria load was measured between
BMDC-immunized animals challenged with HSL236
(LLO+, NP+) compared with
those challenged with HSL235 (LLO+,
NP-) (Fig. 7B). Enhanced immunity in
this situation is somewhat surprising, considering that the same low
number of NP118–126-specific CD8+ T cells alone
were unable to consistently protect mice from HSL236 infection (data
not shown). However, a large expansion of LLO91–99- (all animals) and
NP118–126- (HSL236 challenge of BMDC-immunized mice only) specific
CD8+ T cells was observed at 5 days after
infection (Fig. 7C). Interestingly, both populations of
specific CD8+ T cells expanded to 10 x
106 cells per spleen despite the difference in
the type of primary immunization (BMDC vs LM) and the number of memory
T cells before infection (Fig. 7C). These experiments
demonstrate strong protection of mice from a high dose of LM infection
by prior immunization with peptide-coated BMDCs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because DCs efficiently acquire Ag and have numerous MHC class I
molecules on their surface, it is likely they have the ability to
present multiple peptide determinants simultaneously in vivo. We have
shown that multiple peptides can be presented by peptide-coated BMDCs
provided these peptides are not presented by the same MHC class I
molecule (Fig. 2, A and B). Support for this
conclusion has recently been published by Probst et al.
(28), in which no competition was seen between
CD8+ T cells of differing Ag specificities after
adoptive transfer of DCs presenting multiple Ags. Data examining
CD8+ T cell priming in H-2b
mice has also indicated that there is no significant difference in the
primary host response to single- vs double-peptide-coated DC
immunization (4). Because our experiments were conducted
with a dose of BMDCs that is not saturating, the results
suggest that DCs can engage in stimulation of naive T cells of multiple
Ag specificities, either simultaneously or in sequence.
In contrast to this data, we saw strong inhibition of p60
217–225-specific responses when BMDCs were simultaneously coated with
LLO91–99 (both presented by H-2Kd). Both p60
217–225 and LLO91–99 have been reported to bind with high affinity to
the H-2Kd molecule and to stabilize the
peptide:Kd complex similarly (29, 30). However, our data suggest that there is competition for the
Kd molecule in vitro between these two peptides.
In addition to this competition, we also measured competition in vivo
when double the normal number of CD11c+ cells
were injected (half of which were not coated with peptides) (Fig. 2C). This experiment suggests competition for access to
peptide-coated BMDCs because we did not detect any inhibition of the
CD8+ T cell response when multiple epitopes were
loaded onto the entire population of BMDCs. However,
additional experiments would need to be done to assess the contribution
of this mechanism compared with the previously demonstrated competition
for peptide:MHC complexes on the cell surface (4, 22, 23).
After infection with pathogens, there is coordinate expansion and
contraction of Ag-specific CD8+ T cell
populations regardless of the amount of epitope generated during
infection (7, 9, 10, 12). Intriguingly, we observed
noncoordinate responses between LLO91–99- and NP118–126-specific
CD8+ T cells after peptide-coated BMDC
immunization (Fig. 4, A and B). Although the
number of naive CD8+ T cell precursors specific
for LLO91–99 and NP118–126 could be different, it is unlikely that
there are significantly fewer NP118–126 precursors because the number
of LLO91–99- and NP118–126-specific CD8+ T
cells after HSL236 (LLO+,
NP+) infection is comparable and follows similar
kinetics (Fig. 1B and data not shown). Secondly, the largest
CD8+ T cell response measured is to the
NP118–126 epitope after LCMV infection (7). Thus, it is
unlikely that the precursor frequency for this epitope is abnormally
low. The instability of H-2Ld compared with other
MHC class I molecules may limit the duration of
CD8+ T cell stimulation in vivo
(31). However, examination of other peptides bound by
H-2Ld and H-2Kd showed that
noncoordinate kinetics were not restricted to
H-2Ld-presented peptides (Fig. 4). Additionally,
when MHC:peptide stability was examined in vitro there was no
measurable difference in the rate at which BMDC coated with LLO91–99
or NP118–126 lost the ability to stimulate CD8+
T cell lines (Fig. 4E). Although these experiments suggest
that there is no gross difference in the stability of
LLO91–99:Kd and
NP118–126:Ld, we do not know if a difference in
the on rate of these peptides causes a difference in the number of
peptide:MHC class I complexes formed. Similarly, we do not know
precisely how the sensitivity to epitope levels of naive
CD8+ T cells in vivo differs from our T cell
lines. The noncoordinate response observed between LLO91–99- and
NP118–126-specific CD8+ T cells was unchanged by
either LM or LCMV infection unless additional Ag was present in the
infecting pathogen (Fig. 5A and data not shown). Therefore,
the presence of a robust innate inflammatory environment does not alter
the Ag-specific CD8+ T cell kinetic after
peptide-coated BMDC immunization. An additional difference between
immunization with peptide-coated BMDC and infection may relate to the
duration of Ag presentation. Peptide-coated BMDC immunization results
in uniformly high level presentation which will last as long as the
BMDC survive and maintain surface peptide:MHC expression. In contrast,
DC-mediated presentation after infection may occur over a longer
interval because it may take a week or longer to clear the infection.
Increasing the duration of Ag presentation by DC could alter both the
number of precursors recruited and time to achieve a peak response.
However, despite these differences in CD8+ T cell
kinetics, both infection and peptide-coated BMDC immunization result in
stable memory populations.
It has been clearly demonstrated that numerous Ag-specific
CD8+ T cells are present in nonlymphoid organs
after infection (13). Recently, it was shown that specific
Ag is itself not required in peripheral organs for recruitment of
previously activated CD8+ T cells after influenza
virus infection (26). We show that Ag-specific
CD8+ T cell responses can be observed in the lung
and liver after peptide-coated mature BMDC immunization. In contrast,
no responses were observed in any organs after vaccination with
immature peptide-coated BMDC (Fig. 6). This demonstrates that infection
is not a requirement for CD8+ T cell trafficking
to peripheral organs and suggests that migration to all nonlymphoid
tissues may be programmed upon initial activation. Additional
experiments will be needed to determine whether organ-specific
differences exist in the CD8+ T cell response to
peptide-coated BMDCs, what characteristics allow
CD8+ T cell entry into these organs, and what
impact individual populations have on protective immunity.
Finally, because CD8+ T cells play a critical
role in protection against many viral and intracellular bacterial
pathogens, we examined the protective capacity of memory
CD8+ T cells generated by immunization with
peptide-coated BMDCs. Memory cells specific for a single
CD8+ T cell epitope protected mice from high dose
challenge with virulent LM, demonstrating that
CD4+ T cell responses are not required for robust
protective immunity in this system. Diversification of the memory
compartment did result in enhanced immunity despite small increases in
the absolute number of Ag-specific CD8+ memory T
cells (Fig. 7). These results indicate that in the design of
vaccination strategies using Ag-loaded DC, strong consideration should
be given to the unique characteristics of the specific peptide:MHC
complex(es) of interest and to creating a diverse memory T cell
compartment in the host.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, University of Iowa, 51 Newton Road, 3-512 Bowen Science Building, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu
3 Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; LM, Listeria monocytogenes; LLO, listeriolysin O; NP, nuclear protein; MCMV, murine CMV; ICS, intracellular cytokine staining.
Received for publication April 1, 2002. Accepted for publication August 27, 2002.
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2 microglobulin. Proc. Natl. Acad. Sci. USA 94:6880.This article has been cited by other articles:
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