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Increased Dendritic Cell Numbers Impair
Protective Immunity to Intracellular Bacteria
Despite Augmenting Antigen-Specific CD8 +
T Lymphocyte Responses
Robert C. Alaniz, Sharsti Sandall, Elaine K. Thomas and
Christopher B. Wilson
J Immunol 2004; 172:3725-3735; ;
doi: 10.4049/jimmunol.172.6.3725
http://www.jimmunol.org/content/172/6/3725
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Copyright © 2004 by The American Association of
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References
The Journal of Immunology
Increased Dendritic Cell Numbers Impair Protective Immunity
to Intracellular Bacteria Despite Augmenting Antigen-Specific
CD8ⴙ T Lymphocyte Responses1
Robert C. Alaniz,* Sharsti Sandall,† Elaine K. Thomas,‡ and Christopher B. Wilson2*
P
rotection against microbes is achieved through the coordinated actions of the innate and adaptive immune systems. Upon pathogen encounter, the innate immune system must sense a pathogen and control its early spread and
replication. If innate defense mechanisms are not successful or if
the numbers of the infecting organism are overwhelming, the adaptive arm of immunity is then required for controlling infection.
APCs bridge innate and adaptive immunity by processing and presenting Ag to T lymphocytes, thereby influencing the quality and
magnitude of the adaptive immune response and the outcome of
the infection (1).
Dendritic cells (DCs)3 form a network of motile sentinels that
play a pivotal role in initiation and modulation of the adaptive
immune response (2). Immature DCs present in peripheral tissues
have high phagocytic activity, but are unable to stimulate T cells
efficiently. As they capture microbes or microbial Ags, DCs mature in response to microbial pathogen-associated molecular patterns signaling through Toll-like receptors or in response to proin-
*Department of Immunology and †National Heart, Lung, and Blood Institute Stipends
for Training Aspiring Researchers Program, University of Washington, Seattle, WA
98195; and ‡Amgen, Seattle, WA 98101
Received for publication June 26, 2003. Accepted for publication January 6, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grant HD18184 from the National Institutes of Health
(to C.B.W.).
2
Address correspondence and reprint requests to Dr. Christopher B. Wilson, Department of Immunology, University of Washington, Box 357650, 1959 NE Pacific
Street, Seattle, WA 98195. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; Flt3-L, Flt3 ligand; LLO,
listeriolysin O; pegGM-CSF, polyethylene glycol-conjugated GM-CSF; RAG, recombination-activating gene.
Copyright © 2004 by The American Association of Immunologists, Inc.
flammatory stimuli produced by other cells responding to
pathogen-associated molecular patterns (3– 6). Mature DCs upregulate MHC and costimulatory molecules and migrate from tissues to regional lymph nodes, where they efficiently present captured Ags to naive T cells. However, DCs represent a small
population of cells in vivo (7), and thus may be a limiting factor in
the rate of development and magnitude of Ag-specific T cell responses to infection.
T cells play a critical role in cellular immunity against intracellular bacterial pathogens, such as Listeria monocytogenes and Mycobacterium tuberculosis (8). Effector CD4⫹ T cells act as Th cells
during Listeria infection by the production of Th1-type cytokines,
such as IFN-␥, which activate macrophage microbicidal activity
(9). CD8⫹ T cells, compared with CD4⫹ T cells, appear to play an
even greater role in protection against Listeria infection. Mice genetically lacking or depleted of CD8⫹ T cells show increased susceptibility to Listeria, and adoptively transferred Listeria-specific
CD8⫹ T cells provide greater protection against Listeria infection
than CD4⫹ T cells (10, 11). During primary infection, Listeriaspecific CD8⫹ T cells peak in numbers at day 7 (12) and lead to
sterilizing immunity, which is mediated by the production of
IFN-␥ (13) and by lysis of Listeria-infected cells (14, 15). Cytotoxic CD8⫹ T cell-mediated lysis of infected cells releases the
bacteria into the interstitial space in which activated macrophages
can engulf and kill them. Furthermore, protective CTL immunity
against Listeria requires priming by DCs, as demonstrated by Jung
et al. (16), in which conditional depletion of DCs in vivo completely abrogates the development of CTL during primary
infection.
Accordingly, there is great interest in manipulating DCs to augment the development of robust T cell responses to nominal as
well as tumor and microbial Ags (17–22). One approach is the
administration of Flt3 ligand (Flt3-L). When given to mice, Flt3-L
0022-1767/04/$02.00
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Dendritic cells (DCs) reside in tissues, where they function as sentinels, providing an essential link between innate and adaptive
immunity. Increasing the numbers of DCs in vivo augments T cell responses, and can cause dramatic CTL-dependent tumor
regression. To determine whether greater DC numbers promoted T cell-mediated protection in the context of host defense against
intracellular bacteria, we treated mice with Flt3 ligand (Flt3-L) to increase DCs in vivo and challenged them with Listeria
monocytogenes. Unexpectedly, after primary challenge with Listeria, the overall control of Listeria infection was impaired in
Flt3-L-treated mice, which had greater bacterial burden and mortality than controls. Similar results were obtained when DC
numbers were increased by treatment with polyethylene glycol-conjugated GM-CSF rather than Flt3-L and in mice infected with
Mycobacterium tuberculosis. Impaired protection was not due to dysfunctional T cell responses, as Flt3-L-treated mice had a
greater frequency and absolute number of Ag-specific CD8ⴙ T cells, which produced IFN-␥, exhibited cytolytic activity, and
transferred protection. The increased Listeria burden in Flt3-L-treated mice was preferentially associated with DCs, which were
unable to kill Listeria and more resistant to CTL lysis compared with macrophages in vitro. Although we cannot exclude the
possibility that other potential effects, in addition to increased numbers of DCs, are shared by Flt3-L and polyethylene glycolconjugated GM-CSF and contributed to the increase in susceptibility observed in treated mice, these results support the notion
that DC numbers must be properly controlled within physiological limits to optimize host defense to intracellular bacterial
pathogens. The Journal of Immunology, 2004, 172: 3725–3735.
3726
Materials and Methods
Mice
C57BL/6 (H-2b), recombination-activating gene (RAG)-II-deficient (RAG
null, 129/svj (H-2b)), and BALB/c (H-2d) mice used in the Flt3-L studies
were females between the ages of 2 and 6 mo, and were purchased from
The Jackson Laboratory (Bar Harbor, ME) or Taconic Farms (RAG null
mice; Germantown, NY). Mice were housed under specific pathogen-free
conditions and in accordance with the Institutional Animal Care and Use
Committee. Mice were age matched in each experiment, and BALB/c mice
were used, unless otherwise indicated.
Bacteria
Frozen (⫺80°C) 1-ml aliquots of a log-phase culture of L. monocytogenes
(ATCC 43251), which had been passaged in C57BL/6 mice to promote
virulence, were thawed and grown in trypticase soy broth for 3 h at 37°C.
Serial dilutions were then made in sterile 0.9% saline or antibiotic-free cell
culture medium to achieve the desired concentration (27).
M. tuberculosis (H37Rv strain), obtained from J. Belisle (Colorado State
University, Fort Collins, CO, through National Institutes of Health National Institute of Allergy and Infectious Diseases Contract N01 AI-75320),
was grown at 37°C to mid-log phase in Proskauer-Beckett medium. The
bacteria were passaged once in mice via aerosol to promote virulence.
After 4 wk, lung homogenates (in 0.05% Nonidet P-40 in PBS) were
spread onto 7H10 agar, and a single colony was inoculated in modified
Proskaur-Beckett medium and grown to mid-log phase at 37°C (28). Individual aliquots were frozen at ⫺80°C until use.
Experimental infections
For Listeria infections, mice were injected with bacteria (1 ⫻ 104 CFU,
unless otherwise indicated) in 100 ␮l of 0.9% saline by the i.p. route, as
previously described (27). To determine bacterial burden, livers and
spleens were aseptically removed en bloc from mice at the indicated days
and weighed. Organs were placed in sterile Nonidet P-40 (0.1% in water)
and ground in 15-ml round-bottom polypropylene tubes with a motorized
tissue homogenizer (Biospec Products, Bartlesville, OK). Rotors were
washed and flame sterilized between samples to avoid cross-contamination.
Serial 10-fold dilutions of the organ homogenates were made in 0.1%
Nonidet P-40 and spread in duplicates on trypticase-soy agar plates. Plates
were then incubated for 36 – 48 h at 37°C, and the CFU were enumerated.
The bacterial burden is expressed as the mean of the log10 CFU/g
tissue ⫾ SE.
For M. tuberculosis infections, mice were infected with M. tuberculosis
(H37Rv strain) by aerosol, which deposited ⬃100 CFU to the lungs, as
previously described (27). Lungs, livers, and spleens were removed on the
indicated days, and organs were homogenized in 0.05% Nonidet P-40 in
PBS, as above. Serial 10-fold dilutions of the homogenates were spread on
7H10 and then incubated at 37°C for 2–3 wk before CFU were enumerated.
Flt3-L and pegGM-CSF treatments
Immunex (Seattle, WA), now Amgen, provided human rFlt3-L and recombinant pegGM-CSF. Flt3-L, produced in Chinese hamster ovary cells, was
supplied as a sterile, lyophilized preparation with 40 mg of mannitol, 10
mg of sucrose, and 25 mM tromethamine (Tris) per 1.5 mg of FL. PegGMCSF was supplied in PBS. Endotoxin levels were ⬍10 pg/g protein. Sterile,
nonpyrogenic water was used to reconstitute Flt3-L and pegGM-CSF to
desired concentrations, and aliquots were frozen at ⫺80°C. Working concentrations of Flt3-L and pegGM-CSF were diluted in 10 ␮g/ml mouse
serum albumin in water and kept at 2– 8°C before injection. For in vivo DC
expansion, mice were injected i.p. with 10 ␮g of Flt3-L (9 days) or 2 ␮g
of pegGM-CSF (5 days). Control mice received mouse serum albumin
alone. All groups of mice received the same number of injections for each
experiment.
Culture medium and chemical reagents
Cell culture was performed using RPMI 1640 supplemented with 10%
heat-inactivated FCS (Life Technologies, Grand Island, NY), 2 mM Lglutamine, 50 U/ml penicillin, 50 ␮g/ml streptomycin, 40 ␮g/ml gentamicin, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 50
␮M 2-ME (RPMI-C⫹; antibiotic-free culture medium is referred to as
RPMI-C). Where indicated, RBC were lysed with RBC lysis buffer (0.15
M NH4Cl, 1.0 mM NaHCO3, 0.1 mM EDTA, pH 7.2) for 5 min at room
temperature. Cells were then washed twice in RPMI-C⫹ or RPMI-C. PBS
plus BSA (PBSA) was used for flow cytometric assays. Where indicated,
cells were exposed to the following: LPS derived from Escherichia coli
strain 0111:B4 at a final concentration of 100 ng/ml (Sigma-Aldrich, St.
Louis, MO); murine rIFN-␥ (Genzyme, MA) at a final concentration of 200
U/ml; and brefeldin A (Sigma-Aldrich) for intracellular cytokine staining
at a final concentration of 10 ␮g/ml.
Listeria peptides for the in vitro stimulation of CD8⫹ T cells were purchased from United Biochemical Research (Seattle, WA). The peptides
were for the dominant listeriolysin O91–99 (LLO91–99) (G-Y-K-D-G-N-EY-I) and the subdominant p60217–225 (K-Y-G-V-S-V-Q-D-I) H-2d MHC
class I-restricted peptide Ags.
Adoptive cell transfers
Spleens from cell donors were harvested and placed in 10 ml of RPMI-C⫹.
The spleens were ground between sterile frosted glass slides, and aspirated
through a 20-gauge needle until a single cell suspension was achieved.
RBC were lysed with filter-sterilized RBC lysis buffer for 5 min at room
temperature and then washed. Spleen cells were enriched for T cells by
depleting B cells (B220⫹) and MHC class II⫹ cells using the MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). An aliquot of this cell
preparation was stained with mAbs for CD3, CD4, CD8, NK1.1, B220, and
Mac-1 to monitor cells transferred to mice. The T cell-enriched fraction
was resuspended in HBSS (Life Technologies) to the desired concentration
in 100 ␮l and then injected in the lateral tail vein (i.v.), after which mice
were challenged with Listeria.
Flow cytometry
Analysis of lymphoid populations was accomplished by staining cell suspensions in PBSA (PBS ⫹ 0.5% BSA) with fluorochrome- or biotin-conjugated mAbs specific for surface proteins found on T and B cells, and
on macrophages, and immature and mature DCs (BD PharMingen, San
Diego, CA), as indicated. Biotin-conjugated Abs were detected with
streptavidin-fluorochrome conjugates. Stained suspensions were analyzed
on a FACScan or LSR flow cytometer and analyzed using CellQuest software (BD Biosciences, Sunnyvale, CA).
A method modified from Badovinac and Harty (29) was used to assess
CD8⫹ T cells that produce IFN-␥ in response to LLO91–99 and p60217–224
class I-restricted peptides. Briefly, spleens were dissociated and, after RBC
lysis, resuspended at 2 ⫻ 107/ml in RPMI-C, and 200 ␮l was pipetted per
well in a 96-well U-bottom tissue culture plate. For positive control wells,
PMA (0.5 ␮g/ml) and ionomycin (7.5 ␮M) were added. For peptide stimulation, 20 ␮l of synthetic peptide was added to give a 5 ␮M final concentration. Brefeldin A (10 ␮g/ml final; Sigma-Aldrich) was added to all
wells. After incubation for 6 h (37°C/5% CO2), cultures were washed twice
in cold PBSA. Splenocyte Fc receptors were blocked, and cells were
stained with PE-labeled Abs to CD8␣ at a 1/100 dilution (BD PharMingen)
or isotype control Abs. Cells were washed twice in PBSA and fixed on ice
with 2% paraformaldehyde for 10 min. Next, cells were washed twice and
allowed to permeabilize with PBS plus 0.1% saponin for 15 min on ice.
Abs to IFN-␥ conjugated to FITC (1/100 dilution) were added for 30 min
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causes the in vivo proliferation and mobilization of early hemopoietic progenitor cells in the bone marrow that result in a massive
expansion of both CD11c⫹CD8␣⫹ and CD11c⫹CD8␣⫺ DCs (23).
In tumor-bearing mice, Flt3-L treatment augments tumor-specific
T cell responses, and causes significant tumor regression (24, 25).
Similar to findings in tumor models, there is considerable experimental evidence that DCs are efficient vaccine vehicles for the
induction of protective T cell and T cell-dependent immune responses to infectious diseases (26). These results, and results in the
tumor models noted above, suggest that DC numbers may be a
limiting factor in the initiation of Ag-specific immunity, and that
treatment with agents that enhance DC numbers should augment T
cell immunity and facilitate the resolution of active infection with
intracellular pathogens. The study presented in this work addresses
this question. Contrary to this prediction, we demonstrate that increasing DC numbers through the administration of Flt3-L or polyethylene glycol-conjugated GM-CSF (pegGM-CSF) undermined
protective immunity to L. monocytogenes and M. tuberculosis despite an augmented Ag-specific T cell response. This suggests that
the immune system evolved to maintain DC numbers within limits
that are balanced to provide optimal host defense to infection with
intracellular pathogens.
DCs AND INTRACELLULAR BACTERIAL INFECTION
The Journal of Immunology
on ice. After washing twice in PBS plus 0.1% saponin, cells were resuspended in 300 ␮l of PBSA and saved at 4°C in dark until analyzed by flow
cytometry.
CTL killing assay
Results
DC numbers are increased by treatment with Flt3-L in vivo
Previously, studies using Flt3-L to expand and mobilize DCs in
vivo used either naive mice or mice challenged with tumor cells
(23, 32). To verify that Flt3-L treatment expands DC numbers in
the context of a Listeria infection, we infected mice and treated a
cohort with Flt3-L on days ⫺4 to ⫹4 of infection (standard treatment protocol). We then analyzed the splenic DC populations in
mice at various time points during infection. Listeria-infected mice
treated with Flt3-L showed increased CD11c⫹CD8␣⫹ and
CD11c⫹CD8␣⫺ DC numbers, compared with control-infected
mice, throughout treatment: DC numbers peaked on day 5 of infection, 1 day after the final Flt3-L administration (Fig. 1A). Thus,
Flt3-L increased DC numbers in Listeria-infected mice, as previously shown in uninfected mice (23).
Increasing DCs in vivo inhibits control of Listeria infection
Because Flt3-L treatment augmented the development of Ag-specific T cell-mediated protection in other models, we predicted that
Flt3-L-treated mice would exhibit enhanced bacterial clearance
during primary Listeria challenge. Surprisingly, Flt3-L-treated
BALB/c mice had increased numbers of Listeria in the liver at
days 3–7 (Fig. 1B). A lower inoculum of 103 CFU Listeria revealed similar results (data not shown). Furthermore, in one experiment in which BALB/c mice were infected with the normally
well-tolerated dose of 1 ⫻ 105 CFU Listeria, 58% (7 of 12) of
Flt3-L-treated mice, but only 8% (1 of 12) of controls died unexpectedly. Flt-L-treated C57BL/6 mice (which are more resistant to
Listeria than BALB/c mice) also had increased numbers of Listeria in the liver and spleen at days 5 and 7, but not at day 3 (Fig.
1, C and D) or day 1 (data not shown). All subsequent experiments
with Listeria were performed with the BALB/c mice.
In studies from other groups, mice were pretreated with Flt3-L
before tumor challenge or infection (33–35). The unexpected deleterious effect of Flt3-L treatment on the control of primary
Listeria infection led us to question the timing of our treatment
regimen. Therefore, we compared our standard Flt3-L regimen
(days ⫺4 to ⫹4) with a pretreatment regimen (days ⫺9 to ⫺1).
Both Flt3-L treatments inhibited the control of Listeria infection (Fig. 1E).
To determine whether the effects of Flt3-L treatment on resistance to Listeria were transient as are the effects on DC numbers,
we performed an additional experiment. In this experiment, mice
were treated with Flt3-L in the usual manner (days ⫺4 to ⫹4) or
were treated from days ⫺12 to ⫺4 and then allowed to recover for
4 days before challenge, by which time numbers of total cells and
DCs in the spleen had nearly returned to baseline (data not shown).
Mice allowed to rest after Flt3-L treatment (days ⫺12 to ⫺4)
cleared infection as well as control mice (Fig. 1F), while mice
receiving the standard Flt3-L treatment (days ⫺4 to ⫹4) had a
significant increase in Listeria CFU in both the liver and spleen.
Increasing DCs by pegGM-CSF treatment inhibits control of
Listeria infection
The deleterious effect of Flt3-L treatment on protection against
Listeria infection was surprising. To determine whether the effect
was restricted to Flt3-L treatment or was more general, we asked
whether treatment with GM-CSF, which selectively expands
CD11c⫹CD8␣⫺ DCs (23), would have effects similar to Flt3-L.
To determine this, we used GM-CSF coupled to polyethylene glycol (pegGM-CSF), which increases its t1/2. As predicted, pegGMCSF treatment increased numbers of CD11c⫹CD8␣⫺ DCs in mice
infected with Listeria (Fig. 2A). Both pegGM-CSF-treated mice
and Flt3-L-treated mice were less able to control primary Listeria
infection than control mice (Fig. 2B). Thus, high numbers of DCs
impaired host defense to Listeria infection regardless of the agent
used to increase their numbers in vivo.
Inhibition of protective immunity to M. tuberculosis by treatment
with Flt3-L and pegGM-CSF
To determine whether the harmful effect of increasing DCs in vivo
was particular to Listeria or a more general characteristic of infections with intracellular bacteria, we treated mice with Flt3-L
and pegGM-CSF and assessed their ability to control infection
with M. tuberculosis. Protective immunity to M. tuberculosis requires the host to mount a T cell-mediated immune response in
which CD4⫹ and CD8⫹ T cells, as well as IFN-␥, are important
for bacterial clearance (36). The tempo of M. tuberculosis infection and the development of T cell immunity are much slower than
for Listeria. Innate mechanisms slow bacterial replication during
the first 2 wk, after which adaptive immunity develops and acts to
control infection (36). Therefore, we assessed M. tuberculosis
CFUs at 2 and 4 wk postinfection. Increasing DCs with either
Flt3-L (given days ⫺2 to ⫹6) or pegGM-CSF (given days ⫹2 to
⫹6) inhibited M. tuberculosis clearance in all organs assessed at 2
wk (Fig. 2C). At 4 wk, the results were even more dramatic. Surviving Flt3-L-treated and pegGM-CSF-treated mice had ⬎10-fold
more CFU in all organs than control mice. Furthermore, 5 of 10
Flt3-L-treated mice and 7 of 10 pegGM-CSF-treated mice died or
had to be sacrificed by 4 wk compared with 0 of 10 controls. These
results suggest that increasing DCs in vivo provides an environment in which intracellular bacteria thrive, and that such effects are
not restricted to Listeria infection.
Increasing DC numbers in vivo augments the CD8⫹ T cell
response to Listeria
To determine the basis for the increased susceptibility of Flt3-Ltreated mice, we first asked whether the development of Listeriaspecific T cell responses was impaired by assessing the frequency
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CTL activity was determined using a standard 51Cr release assay. Splenocytes were harvested from individual Listeria-infected control or Flt3-Ltreated BALB/c (H-2d) mice at day 7 postinfection. RBCs were lysed, and
cells were resuspended at 1 ⫻ 107 per ml in RPMI-C⫹. Target cells (P815
mastocytoma cell line) were prepared by incubating with 100 ␮Ci of 51Cr
and 100 ␮M peptide for 1 h at 37°C/5% CO2. Effectors were plated in 100
␮l in 96-well U-bottom tissue culture plate to give appropriate E:T ratios.
Targets were seeded in the plate at 1 ⫻ 104 per well in triplicate. Targets
were plated in the absence of effectors for mininum (no effectors) and
maximum (0.1% Nonidet P-40) lysis controls. After 6 h at 37°C/5% CO2,
the supernatants were harvested, and the lysis was determined as cpm on
a gamma counter. CTL activity was reported as percentage of specific
lysis ⫽ (sample lysis ⫺ minimum lysis)/(maximum lysis ⫺ minimum lysis) ⫻ 100 ⫾ SE for each E:T ratio.
For evaluation of DC resistance to CTL lysis, the B9 CTL clone specific
for LLO91–99 presented on Kd was used as the effector cell (30). DCs and
macrophages pulsed with 100 ␮M LLO91–99 peptide and 51Cr were used as
targets. DCs were isolated from two sources: 1) magnetic bead-enriched
CD11c⫹ cells from Flt3-L-treated mice, or 2) bone marrow cells cultured
for 9 days in the presence of 200 ␮g/ml Flt3-L (31). Macrophages were
isolated from two sources: 1) adherent resident peritoneal exudate cells, or
2) bone marrow cells cultured for 9 days with 30% L cell conditioned
medium in RPMI-C⫹. Isolation protocols for DCs and macrophages gave
purities of ⬎80%, and each was incubated with B9 effector cells for 6 h
before supernatant harvest.
3727
3728
DCs AND INTRACELLULAR BACTERIAL INFECTION
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FIGURE 1. Treatment with Flt3-L increases DC numbers in Listeria-infected mice and impairs control of Listeria infection. A, Dot plots of spleen cells
from representative control and Flt3-L-treated mice at day 5 postinfection (1 day after the final Flt3-L treatment). Percentages of CD11c⫹CD8␣⫹ and
CD11c⫹CD8␣⫺ DCs, and total DC numbers are shown to the right of the plots. B, Listeria log CFU/g liver (mean ⫾ SEM, n ⫽ 3) of control and
Flt3-L-treated BALB/c mice after primary infection. This experiment was done twice with similar results. Listeria log CFU/g spleen (C) and liver (D)
(mean ⫾ SEM, n ⫽ 3) of control and Flt3-L-treated C57BL/6 mice after primary infection. This experiment was done twice with similar results. E, Listeria
log CFU/g liver (mean ⫾ SEM, n ⫽ 3) after primary infection of control mice and mice treated with different regimens of Flt3-L. Flt3-L pretreatment was
given on days ⫺9 to ⫺1 before Listeria challenge, and standard treatment was given on days ⫺4 to ⫹4 relative to the day of primary infection. This
experiment was performed twice with similar results. F, Flt3-L pretreat and rest group received Flt3-L on days ⫺12 to ⫺4 and was compared with control
mice and mice receiving the standard (days ⫺4 to ⫹4) Flt3-L treatment regimen. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001; ‡, p ⬍ 0.06 compared with controls, as
determined by Student’s t test.
and number of Listeria-specific CD8⫹ T cells in Flt3-L-treated and
control mice. Similar to its effects in tumor models, Flt3-L treatment enhanced T cell responses at days 5 and 7, as indicated by the
increased percentage (Fig. 3A) and total numbers (Fig. 3B) of
CD8⫹ T cells able to produce the protective cytokine IFN-␥ after
in vitro stimulation with LLO91–99 peptide (the dominant H2d
MHC class I-restricted Listeria epitope (37)). The percentage of
CD8⫹ T cells producing IFN-␥ to the subdominant p60217–225
epitope (38) was similar to controls (Fig. 3A). However, the absolute number of p60217–225-specific CD8⫹ T cells in Flt3-Ltreated mice was increased ⬃4.3-fold at day 7 (Fig. 3B) due to the
overall increased splenic cellularity in these mice.
In addition to IFN-␥ production, a crucial mechanism by which
CD8⫹ T cells protect against primary Listeria infection is the lysis
The Journal of Immunology
3729
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FIGURE 2. Treatment with Flt3-L or pegGM-CSF impairs protection against Listeria and M. tuberculosis. A, Dot plots of spleen cells from representative control, Flt3-L-treated, and pegGM-CSF-treated mice at day 5 after primary infection (1 day after final Flt3-L and pegGM-CSF treatment). Percentages of CD11c⫹CD8␣⫹ and CD11c⫹CD8␣⫺ DCs and total DC numbers are shown to the right of the plots. B, Listeria log CFU/g liver (mean ⫾ SEM,
n ⫽ 3) of control, Flt3-L-treated, and pegGM-CSF-treated mice after primary infection. This experiment was done twice with similar results. C, M.
tuberculosis log CFU/g tissue (mean ⫾ SEM, n ⫽ 10) in the lung, spleen, and liver of control, Flt3-L-treated (days ⫺2 to ⫹6), and pegGM-CSF-treated
(days ⫹2 to ⫹6) mice at 2 and 4 wk of primary aerosol infection. Five of 10 Flt3-L-treated mice and 7 of 10 pegGM-CSF mice died by 4 wk of infection.
CFU shown at 4 wk are from surviving mice only. This experiment was performed twice with similar results, except for pegGM-CSF treatment at 4 wk,
which was done once. ⴱ, p ⬍ 0.05 compared with controls, as determined by Student’s t test.
of infected cells (14, 15). To examine the cytolytic potential of T
cells from Flt3-L-treated mice, target cells were pulsed with
LLO91–99 or p60217–225 peptides in a standard 51Cr release assay.
Consistent with the increased numbers of IFN-␥-producing Agspecific CD8⫹ T cells in Flt3-L-treated mice (Fig. 3, A and B),
splenocytes from Flt3-L-treated mice consistently lysed Listeria
peptide-pulsed targets more efficiently than cells from control mice
after restimulation in vitro (Fig. 3C). In one of these two experiments, increased CTL activity was also observed directly ex vivo,
although, as expected, lytic activity was less than that observed
with cells that had been restimulated in vitro (data not shown).
We next sought to determine whether the Listeria-specific T
cells in Flt3-L-treated mice were able to mediate protection. A
classic method for determining the protective potential of T cells is
by adoptive transfer (11). We used this assay, in which T cells
from Listeria-infected mice are isolated and injected into naive
mice that are subsequently rechallenged with a high dose of Listeria. T cell-enriched splenocytes from control and Flt3-L-treated
3730
DCs AND INTRACELLULAR BACTERIAL INFECTION
mice at day 7 postinfection were pooled, divided in numbers equal
to those obtained from the spleen of a single donor mouse, and
injected into naive recipient mice that were then challenged with a
high dose (1 ⫻ 106 CFU) of Listeria (Fig. 4A). In another experiment, recipient mice in all groups received an equivalent number
of cells (3 ⫻ 107) before high dose challenge (Fig. 4B). In both
experiments, mice receiving cells from Listeria-immune, Flt3-Ltreated mice were clearly protected compared with those receiving
cells from naive mice, and protection appeared to be superior to
that achieved with Listeria-immune cells from control mice.
These data indicate that the expansion of DCs by treatment with
Flt3-L augmented the Ag-specific CD8⫹ T cell responses in mice
infected with Listeria, and that the increased susceptibility of Flt3L-treated mice was not due to impaired T cell responses.
Increasing DCs by Flt3-L treatment inhibits established and
innate immunity to Listeria
Despite the presence of increased numbers of Ag-specific CD8⫹ T
cells that produced IFN-␥, were cytolytic, and could transfer pro-
tection, Flt3-L-treated mice were unable to control primary Listeria infection as well as control mice. We reasoned that Flt3-L
treatment might be impeding an innate protective mechanism independent of the development or presence of T cell immunity.
First, we tested whether increasing DCs by Flt3-L treatment
would undermine established T cell immunity in Listeria-immune
mice. To assess this, mice were immunized with Listeria and allowed to resolve the infection at least 3– 4 wk. Immunized mice
were then treated with Flt3-L on days ⫺6 to ⫹2 and challenged
with a high dose (1 ⫻ 106 CFU) of Listeria on day 0. Flt3-L treatment
abolished the early protection at day 3 of secondary Listeria infection,
but Flt3-L-treated mice ultimately reduced the numbers of bacteria by
day 5 to values similar to controls (Fig. 5A).
Next, we tested whether increasing DCs with Flt3-L could inhibit innate immune control of Listeria. RAG-deficient mice lack
T and B lymphocytes, but have increased NK cell activity. These
mice controlled early growth of Listeria better than controls at day
3, as previously reported (39), but enhanced early control of Listeria
was abrogated in Flt3-L-treated RAG-deficient mice (Fig. 5B).
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FIGURE 3. Treatment with Flt3-L augments CD8⫹ T cell responses in Listeria-infected mice. A, At day 7 of primary infection, spleen cells from a
representative control and Flt3-L-treated mouse were stained for expression of surface CD8␣ and intracellular IFN-␥ after 6-h stimulation with the dominant
(LLO91–99) or subdominant (p60217–225) MHC class I-restricted epitopes of Listeria. B, Total numbers (mean ⫾ SEM, n ⫽ 3) of LLO91–99- and
p60217–225-specific CD8⫹ T cells in spleens of control and Flt3-L-treated mice at days 5 and 7 of Listeria infection. ⴱ, p ⬍ 0.05 determined by the Student’s
t test. This experiment was performed three times with similar results. C, Release of 51Cr from LLO91–99- and p60217–225-pulsed P815 target cells by
restimulated day 7 postinfection splenocytes from control and Flt3-L-treated mice. Shown are the mean ⫾ SEM of three individual mice assayed in
triplicate. Values of p (LLO91–99, p ⬍ 0.0001; p60217–225, p ⬍ 0.0001) were determined using the ANOVA test. This experiment was done twice with
similar results.
The Journal of Immunology
3731
Together, the results in immunized and RAG-deficient mice indicate that Flt3-L treatment undermined both established T cellmediated and innate control of Listeria infection. We next sought
to determine whether DCs provided a favorable niche for Listeria
in vivo.
DCs are a reservoir for Listeria in Flt3-L-treated mice
DCs are known to ingest Listeria (40) and other intracellular bacteria in vivo, and bacteria primarily associate with DCs in vivo
after infection with Salmonella or bacillus Calmette-Guerin (41,
42). Given that DCs are the cell type most increased in Flt3-Ltreated mice during primary and secondary Listeria challenge (data
not shown), we hypothesized that DCs were providing a reservoir
in which Listeria could replicate and evade T cell-mediated immunity. To determine whether DCs preferentially harbor Listeria,
CD11c⫹ DCs were purified from pooled spleens of control and
Flt3-L-treated mice before (day 3) and at the peak of organ CFU
(day 5) in primary. As indicated in Fig. 6A, DCs from Flt3-Ltreated mice contained more Listeria than the same number (1 ⫻
107) of DCs from control mice. To determine whether DCs, compared with other cell types, were preferentially infected with Listeria, we purified DCs, macrophages, and, for comparison, B cells
at day 5 postinfection. Both in Flt3-L-treated and control mice,
DCs contained more Listeria per cell than other cell types, and
FIGURE 5. Treatment with Flt3-L impairs established and innate protective immunity to Listeria. A, Mice were immunized with Listeria (1 ⫻
104 CFU) and allowed to recover from infection. After 3 wk, mice were
treated with vehicle (Control Secondary) or Flt3-L (Flt3 Ligand Secondary) and rechallenged with 1 ⫻ 106 CFU. Listeria log CFU/g liver (mean ⫾
SEM, n ⫽ 3) was determined at days 3 and 5 after rechallenge. Naive mice
were included for a comparison (Control Primary). ⴱ, p ⬍ 0.05 compared
with control secondary, as determined by the Student’s t test. This experiment was performed twice with similar results. B, RAG-null mice (129/sj)
treated with Flt3-L have higher spleen Listeria log CFU/g tissue (mean ⫾
SEM, n ⫽ 3) than RAG-null mice treated with vehicle at day 3 postinfection. ⴱ, p ⬍ 0.05 using the Student’s t test.
DCs from Flt3-L-treated mice contained more Listeria than DCs
from controls (Fig. 6B). Thus, DCs were preferentially infected
with and harbored viable Listeria in vivo.
DCs cannot kill Listeria in vitro and are resistant to CTL lysis
Listeria are killed in vivo inside activated macrophages, but can
grow and replicate in resting macrophages and certain other cell
types (43, 44). CD8⫹ T cells contribute to Listeria clearance, at
least in part, by lysing infected cells that are unable to kill this
organism, thereby releasing Listeria into the extracellular environment, where activated microbicidal phagocytes can engulf and efficiently kill the bacteria. We therefore hypothesized that Listeria
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FIGURE 4. T cells from Flt3-L-treated, Listeria-infected mice transfer
protection. A, Spleen equivalents (three spleens pooled and divided
equally) or B, equal numbers (1 ⫻ 107) of T cell-enriched immune or
nonimmune splenocytes from control and Flt3-L-treated groups were transferred to three naive recipient mice that were subsequently challenged with
a high dose of Listeria (1 ⫻ 106 CFU). Shown are the log CFU/g tissue
(mean ⫾ SEM, n ⫽ 3) from liver and spleen for each group at day 3
posttransfer/infection. ⴱ, p ⬍ 0.05 determined by Student’s t test compared
with control-uninfected and Flt3 ligand-uninfected groups.
3732
persist and replicate in DCs in vivo in Flt3-L-treated mice due to
decreased microbicidal action by DCs or by resistance of Listeriainfected DCs to CD8⫹ T cell lysis.
To address the first possibility, resting and activated (IFN-␥ plus
LPS) macrophages and Flt3-derived DCs were infected (multiplicity of infection ⫽ 10), and their ability to kill Listeria in vitro was
compared. Both DCs and macrophages ingested bacteria, as indicated by the numbers of Listeria present after 1 h of phagocytosis
(t ⫽ 0) (Fig. 7A). After an additional 6 h of incubation, activated
macrophages killed ⬃83% of the Listeria, while unstimulated
macrophages did not. By contrast, Listeria replicated in unstimulated DCs and, notably, DCs were unable to kill Listeria after
stimulation with IFN-␥ and LPS (Fig. 7A).
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FIGURE 6. DCs are preferentially infected in vivo. A, CD11c⫹ DCs
(1 ⫻ 107), positively selected using magnetic beads from pooled spleens of
infected Flt3-L-treated or control mice at indicated days (n ⫽ 3 per group),
were centrifuged and lysed in 0.1% Nonidet P-40 detergent. CFUs from
serial dilutions are expressed as log CFU/1 ⫻ 107 cells. This experiment
was performed twice with similar results. B, At day 5, DCs (CD11c⫹),
macrophages (Macs, CD11b⫹), and B cells (B220⫹) were serially isolated,
by positive magnetic bead selection, from pooled spleens of infected mice
treated with or without Flt3-L (n ⫽ 3 per group). Cells were pelleted and
lysed in 0.1% Nonidet P-40 detergent. Results are expressed as log CFU/
107 cells. This experiment was performed twice with similar results. ⴱ, p ⬍
0.001 by Student’s t test.
DCs AND INTRACELLULAR BACTERIAL INFECTION
FIGURE 7. DCs do not kill Listeria and are resistant to CTL lysis. A,
Macrophages (M␾) and DCs were cultured overnight in the presence or
absence of IFN-␥ (200 U/ml) and LPS (100 ng/ml) and then infected with
Listeria (multiplicity of infection ⫽ 10). t ⫽ 0 represents CFUs present
after extracellular bacteria were removed by incubation for 1 h with gentamicin (5 ␮g/ml). t ⫽ 6 represents CFUs 6 h after gentamicin wash. Cells
were lysed in 0.1% Nonidet P-40 detergent at the indicated times. Results
are expressed as log CFU ⫾ SEM of triplicate cultures. This experiment
was performed two times with similar results. ⴱ, p ⬍ 0.001 by Student’s t
test. Resident peritoneal macrophages and CD11c⫹ DCs positively selected from splenocytes of Flt3-L-treated mice (B), total splenocytes from
control and Flt3-L-treated mice (C), or bone marrow-derived macrophages
and DCs derived from culturing bone marrow in vitro with Flt3-L (D) were
pulsed in vitro with LLO91–99 peptide, then used as target cells for B9 effector
CTL in standard 51Cr release assays. Values of p determined using the
ANOVA test. These experiments were performed twice with similar results.
To determine whether DCs were more resistant to CTL lysis
compared with macrophages, the B9 CD8⫹ CTL line, specific for
the dominant LLO91–99 peptide (37), was used as the effector cell
The Journal of Immunology
against peptide-pulsed DC targets labeled with 51Cr. When purified splenic CD11c⫹ DCs from Flt3-L-treated mice were used as
targets compared with resident peritoneal macrophages, DCs were
more resistant to CTL lysis than macrophages (Fig. 7B). Similarly,
splenocytes from Flt3-L-treated mice were more resistant to lysis
than were splenocytes from control mice (Fig. 7C), and DCs prepared by culturing bone marrow with Flt3-L were more resistant
than bone marrow-derived macrophages (Fig. 7D).
Discussion
Increased numbers of DCs enhance T cell responses, yet can
undermine T cell immunity to intracellular bacteria
The finding that Flt3-L treatment enhanced the CD8⫹ T cell response to Listeria infection is consistent with other reports using
DCs to augment T cell immunity. DCs, pulsed in vitro with nominal, microbial, or tumor Ags, are potent inducers of Ag-specific
CD4⫹ and CD8⫹ T cell responses in vivo (21, 45, 46). Furthermore, DCs infected in vitro with M. tuberculosis and used to immunize mice can induce a high level of protection to challenge
with virulent M. tuberculosis (47). Increasing DCs in vivo with
Flt3-L treatment enhanced CD4⫹ and CD8⫹ T cell responses to
normally nonimmunogenic tumor Ags. In fact, Flt3-L treatment of
tumor-bearing mice caused complete tumor regression that was
both CD4⫹ and CD8⫹ T cell dependent (24, 25, 32, 35).
The unexpected observation in our study is that despite augmented T cell responses, the presence of DCs in numbers greater
than normal undermined protective immunity to Listeria. The deleterious outcome may have to do with key differences between
intracellular bacterial infection and tumor challenge, as well as
differences between immunizing with Ag-pulsed DCs and increasing DCs in vivo. Tumor immunity, similar to immunity for intracellular pathogens, is cell mediated (32). However, tumor cells do
not occupy an intracellular compartment, and therefore can be di-
rectly recognized and lysed by tumor-specific CD8⫹ T cells induced by DCs. Intracellular Listeria have a reduced exposure to
the immune system, but are released after infected cells are lysed
by CD8⫹ T cells (14). Under selective pressure of an immune
response, and in the presence of DCs that do not kill bacteria and
that resist CD8⫹ T cell lysis, Listeria may preferentially replicate
in this more favorable cellular niche (48).
Other differences between our studies and those that used DCs
as an immunizing vehicle are the numbers of DCs and the period
during which DC numbers are increased relative to the time of
microbial challenge. In our studies, DC numbers increased during
an ongoing infection were available as reservoirs for Listeria. In
the previous studies, DC numbers were either relatively low, or
DC numbers would have returned to baseline before infectious
challenge (21, 47). However, three groups reported that mice
treated with Flt3-L before primary infection (days ⫺9 to ⫺1) had
better outcomes when challenged with Listeria, Leishmania, or
HSV (33, 34, 49). For these studies, a high number of DCs would
have been present during the first few days of infection. Still, when
we examined Listeria clearance and Listeria-specific CD8⫹ T cell
responses with this same regimen and our standard Flt3-L regimen
in parallel, both treatment regimens increased bacterial burden at
days 5 and 7 postinfection, despite increasing the numbers of Listeria-specific CD8⫹ T cells (data not shown). By contrast, resistance to Listeria in mice treated from days ⫺12 to ⫺4 did not
differ from controls, indicating that the deleterious effect of Flt3-L
treatment resolved in parallel with the decline in numbers of DCs
after treatment was stopped.
In the study by Gregory et al. (33), similar numbers of Listeria
were found 6 h after primary infection in Flt3-L-treated and control C57BL/6 mice, but later time points were not assessed. We
found that Flt3-L-treated C57BL/6 mice had similar or slightly
decreased numbers of Listeria early (days 1 and 3; data not
shown), but much higher Listeria numbers at later time points than
control C57BL/6 mice (Fig. 1C). Thus, the deleterious effects of
Flt3-L treatment are not apparent early in C57BL/6 mice (unlike
BALB/c mice) and would not have been apparent in the study by
Gregory et al. Kremer et al. (34) reported that control of cutaneous
Leishmania major infection was enhanced in Flt3-L-treated
BALB/c mice. Protective immunity against Leishmania infection
is dependent on NO and Th1 T cells, which activate macrophages.
Unlike Listeria, Leishmania remains within the phagosome after
infection, and no direct role for CD8⫹ T cells has been described
(50). DCs in our system did produce NO levels as high as macrophages under similar stimuli (data not shown), but did not kill
Listeria. However, the role of NO in killing of Listeria remains
unclear (43). Therefore, DCs may be able to control Leishmania
using NO, and lysis of infected DCs by CD8⫹ T cells may not be
required as with Listeria. Similarly, the beneficial effects of Flt3-L
treatment in mice infected with HSV (49) most likely reflect the
fact that it infects and replicates in epithelial and neural cells rather
than in macrophages and DCs. Consistent with this, Vollstedt et al.
(51) found that treatment of neonatal C57BL/6 mice with Flt3-L
enhanced innate and adaptive immunity to HSV and enhanced innate resistance of neonates to Listeria during the first 5 days of
infection, but provided no long-term protection to Listeria, because the mice all subsequently died.
DCs: immune sentinels without armament
Our finding that Listeria preferentially resides in DCs in vivo is not
unexpected because immature DCs reside precisely where they can
encounter pathogens from the external environment. During oral
Salmonella infection, DCs acquire the bacteria from M cells, or
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As predicted, we found that Flt3-L treatment dramatically increased DC numbers and augmented the Ag-specific T cell response during primary Listeria infection. Listeria-specific CD8⫹ T
cells were present in greater numbers in Flt3-L-treated mice compared with controls, and produced IFN-␥, were cytolytic, and
could transfer protection. Despite the enhanced CD8⫹ T cell responses, Flt3-L-treated mice did not control bacterial replication as
well as control mice during primary or secondary infection with
Listeria. The increased Listeria replication in Flt3-L-treated RAG
null mice suggested that Flt3-L treatment impaired innate immune
mechanisms. Consistent with this, Listeria were preferentially associated with DCs in vivo, and DCs were unable to kill Listeria
and were relatively resistant to lysis by Listeria-specific CTL in
vitro. This suggests that the higher numbers of DCs in Flt3-Ltreated mice created a favorable environment for Listeria in vivo
that could not be overcome by an augmented Listeria-specific
CD8⫹ T cell response. We cannot exclude the possibility that
treatment with Flt3-L impaired protection in part through other
mechanisms. However, treatment with pegGM-CSF also impaired
protection from Listeria. The common feature of Flt3-L and
pegGM-CSF treatment is that they increase DC numbers, supporting the notion that this was the principal mechanism by which they
increased susceptibility to Listeria. Similar to the findings with
Listeria, both Flt3-L and pegGM-CSF inhibited control of M. tuberculosis infection. Together, these data show that the impaired
immunity is not particular to one therapeutic cytokine, treatment
regimen, or pathogen, but is a more general phenomenon, in which
intracellular bacteria can evade an otherwise protective Ag-specific immune response when DCs are present in exaggerated numbers in vivo.
3733
3734
may directly sample the gut lumen for microbes (52, 53). Salmonella preferentially infect and activate DCs, but DCs are unable to
kill Salmonella, which persist in vivo for several days (54). Similarly, Mycobacterium bovis bacillus Calmette-Guerin resides in
splenic DCs for 2 wk following infection, suggesting that DCs do
not kill this bacterium in vivo (41), and Bodnar et al. (55) demonstrated that murine bone marrow-derived DCs do not kill, but
are bacteriostatic for M. tuberculosis in vitro, whereas bone marrow macrophages kill M. tuberculosis efficiently. Human DCs are
also not microbicidal for M. tuberculosis (56). Thus, the inability
of DCs to kill intracellular bacteria in vitro is not likely to reflect
infection-induced impairment of DC function, but rather to be a
normal feature of this cell type. Listeria-infected DCs did function
as potent APC, because CD8⫹ T cell responses were enhanced in
Flt3-L-treated mice. In fact, DCs are induced to mature and express high levels of MHC class I and II molecules, costimulatory
molecules, and protective cytokines in response to Listeria, M.
tuberculosis, or Salmonella spp. (57–59).
Why are DCs less efficient killers of intracellular bacteria and how
might this be beneficial to the host under normal circumstances?
Several dominant intracellular bacterial Ags are only made when
the bacteria are inside host cells, e.g., LLO from Listeria and
␣-crystallin from M. tuberculosis (60, 61). The low microbicidal
ability of DCs may be an intrinsic mechanism to allow the production, expression, and presentation of the full panel of pathogenic intracellular Ags, which would enable DCs to better access
and present these Ags to T cells.
When DCs are present in normal numbers, the system of reduced bacterial killing benefits the host. CD8⫹ T cells need only a
short encounter with Ag and APC to divide, and to differentiate
into effector cells that are lytic and produce IFN-␥ (62, 63). Thus,
a few infected DCs are capable of activating hundreds of T cells
and would not provide a meaningful niche for an intracellular bacterium to occupy. Wong and Pamer (64) have recently demonstrated that CD8⫹ T cells are primed during the first 72 h of Listeria infection and regulate their expansion by lysing the priming
DC in vivo. When DC numbers are artificially increased, their
greater presence may recruit and serially stimulate more naive
CD8⫹ T cells to give a larger magnitude response (65), as we
observed in Flt3-L-treated mice. Bousso and Robey (66) have addressed this by tracking the dynamics of CD8⫹ T cell priming by
DCs in intact lymphoid tissue. They have demonstrated that CD8⫹
T cells and DCs have lengthy and stable interactions that, due to
limiting DC numbers in vivo, may sterically inhibit further engagement of Ag-specific CD8⫹ T cells. Expanding DC numbers in
vivo may overcome this limitation. However, treatment-induced
increases in DC numbers may create an “Achilles’ heel” for the
host by providing a reservoir in which intracellular bacteria survive in vivo. The relative resistance of DCs to CTL lysis, also
observed by others (67, 68), may contribute to Listeria persistence
by preventing the release of intracellular bacteria from infected
cells, which normally enables more microbicidal phagocytes to
acquire and kill the bacteria (14, 48).
The data presented in this work suggest that the numbers of DCs
in vivo are delicately balanced for good reason. When DC numbers
are increased through Flt3-L or pegGM-CSF in naive mice, strong
homeostatic pressure exists in vivo such that DC numbers rapidly
return to normal over a period of 2– 4 days when the cytokine
regimen is discontinued (69) (our unpublished observations). Increased numbers of DCs do enhance the development of Ag-specific T cell responses. Nonetheless, because DCs are poorly mi-
crobicidal and relatively resistant to CTL lysis, properties that may
be important for their primary role as APC, maintaining DC numbers within tightly controlled limits may be essential for overall
host defense to intracellular bacterial pathogens.
Acknowledgments
We thank Immunex (Amgen) for Flt3-L and pegGM-CSF; Heidi Harowicz
for animal husbandry; Sherilyn Smith for assistance with studies of M.
tuberculosis; and David Fitzpatrick, Charlie Maliszewski, Tobias Kollmann, Sing Sing Way, and members of the Wilson laboratory for advice
and suggestions.
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