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Institute for Immunology, Ludwig-Maximilians University, Munich, Germany
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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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To investigate the role of Bcl-2 in DC homeostasis, turnover, and function in vivo, we expressed the human Bcl-2 (hBcl-2) gene under control of the DC-specific murine CD11c promoter in transgenic mice. In this study, we describe the consequences of this expression and demonstrate that Bcl-2 substantially prolongs the lifespan of mature DC in vitro as well as in vivo. As a direct consequence, we find higher numbers of DC in lymphoid organs and elevated T cell and humoral responses in immunized animals. When Bcl-2 transgenic DC are used as Ag-pulsed DC vaccines, they induce CTL activation in vivo more efficiently than normal DC. Our findings indicate that the abundance and longevity of DC is directly regulated by Bcl-2 and that DC homeostasis and natural turnover regulate immune responses in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cDNA encoding for hBcl-2 was excised by restriction digestion with HindIII from pIC19-Bcl-2, a previously published vector obtained from A. Strasser (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) (16, 17). After blunt ending with Klenow fragment, the Bcl-2 cDNA was ligated into the blunt-ended EcoRI site of the previously described CD11c-pDOI-5 vector (18). The orientation of the cDNA was controlled by restriction digestion and DNA sequence analysis. The linearized transgenic construct devoid of vector sequences was injected into fertilized oozytes from (BDF1 x BDF1)F1 mice and transgenic offspring were initially identified by Southern blotting. We obtained three founders with similar copy numbers (approximately four copies) and identical transgene expression patterns. We backcrossed founder line 19, referred to in this study as CD11c-Bcl-2 mice, toward C57BL/6 for 10 generations. OT-1 mice (19) and rat insulin promoter (RIP)-OVAlow (20) mice have been described previously.
mAbs and reagents
The mAbs specific for CD4, CD8, V
5.1/5.2 TCR, V8.1/8.2
TCR, Va11 TCR, I-E, I-Ab, CD11c, CD19, hBcl-2,
isotype control MOPC.-21, and B220 were purchased from BD PharMingen
(San Diego, CA). Single cell preparation, staining, and FACS analysis
were done following standard procedures. Rabbit anti-prohibitin Ab
(21) was a gift of Dr. M. Reth (Max Planck
Institute for Immunobiology, Freiburg, Germany).
Generation of DC from bone marrow cultures and enrichment of DC
Total bone marrow was seeded in 90-mm tissue culture-treated Petri dishes at 5 x 105 cells/ml in 10 ml culture medium containing 25 ng/ml GM-CSF. Maximal yield of DC was obtained between days 7 and 9 of culture. For isolation of DC, cell suspensions of cultured DC or total splenocytes were stained with a biotinylated CD11c-specific mAb and magnetically separated with streptavidin-MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the supplier’s instructions. The purity of DC obtained by this method was controlled by flow cytometry and was usually >90%. For DC vaccination experiments, CD11c-Bcl-2 or wild-type (WT)-cultured DC were pulsed with 20 µg/ml OVA-peptide SIINFEKL (Neosystem, Strasbourg, France) for 2 h and washed extensively before immunization.
Western blot analysis
DC from GM-CSF cultures or spleens were isolated with magnetic beads as described above and lysed in 500 µl Nonidet P-40 buffer (1% Igepal CA-630 (Nonidet P-40), 137 mM NaCl, 50 mM Tris-HCl, pH 7.8, 2 mM EDTA, pH 8, 1 mM PMSF) per 107 cells for 15 min on ice. The detergent-soluble fraction was obtained by centrifugation for 10 min at 14,000 x g. Aliquots corresponding to 5 x 105 cells were suspended in 25 µl Laemmli-loading buffer, and proteins were separated by SDS-PAGE (12%) according to Laemmli (22). After transfer to nitrocellulose, proteins were visualized with primary and secondary HRP-labeled Abs and a luminescent substrate (ECL; Amersham, Piscataway, NJ).
Labeling and migration of DCs
To analyze the migratory capacities of bone marrow-derived DCs, cultured cells from CD11c-Bcl-2 or WT mice were labeled with 5-chloromethyl-fluorescein diacetate (CMFDA) according to the manufacturer’s instructions (Molecular Probes, Eugene, OR) as published previously (15). Briefly, DCs were incubated for 10 min at 37°C in CMFDA (5 µM in PBS) and then washed twice. The DCs were counted, and injected s.c. in both lower hind legs (3 x 105 per leg). The draining popliteal lymph nodes (LNs) were harvested at various time points after injection and digested with 400 U/ml collagenase (Sigma-Aldrich, St. Louis, MO); total LN cells were counted and then stained with PE-conjugated anti-CD11c mAb. After gating on live cells, migrated DC were detected as CD11c-positive and FL-1 (CMFDA) high and cells.
BrdU labeling and analysis
Mice were injected i.p. at day 0 i.p. with 1 mg BrdU dissolved in H2O and, during the pulse period, received drinking water containing 1 mg/ml BrdU. At the indicated timepoint, splenocytes were harvested and stained for extracellular surface molecules. Following fixation, staining for intranuclear BrdU incorporation was performed with a BrdU staining kit according to manufacturer’s instructions (BD Biosciences, San Diego, CA).
T cell proliferation analysis
Mice were immunized with 100 µg OVA (Sigma-Aldrich) in CFA s.c. at the tail base. Five days later, cell suspensions of draining sacral and inguinal LNs were prepared by passing LNs through a nylon mesh. LN cells (5 x 105/well) were cultured in the presence of varying concentrations of OVA in complete culture medium (IMDM, 10% FCS) for 96 h. [3H]Thymidine (1 µCi/well) was present for the last 8 h. Cells were harvested and their radioactivity content was measured with a betaplate system (1205; Wallac, Turku, Finland).
4-Hydroxy-5-iodo-3-nitrophenyl (NIP)-specific Ig responses
Mice were immunized s.c. at day 0 with 100 µg NIP-OVA (a kind gift from Dr. A. Rolink, University of Basel, Basel, Switzerland) in a 1:1 CFA emulsion and boosted s.c. at day 21 with 100 µg NIP-OVA in IFA; serum was analyzed at the timepoints indicated. ELISA plates were coated with 2.5 mg/ml NIP-BSA (kind gift of Dr. A. Rolink) in 0.02 M NaCl at 4°C for 12 h. Plates were washed extensively with PBS, and dilutions of sera (in PBS, 4% BSA, 0.2% Tween 20) were transferred to the coated plates and incubated for 2 h at room temperature. After five washes with PBS, the alkaline phosphatase-conjugated second-step reagent (goat anti-mouse IgG; Southern Biotechnology Associates, Birmingham, AL) was added and incubated for 2 h at room temperature. After washing, the alkaline phosphatase substrate p-nitrophenyl phosphate was added according to manufacturer’s instructions (N-2765; Sigma-Aldrich), and the coloration was quantified at 405 nm.
Adoptive T cell transfer and DC vaccination
Suspensions of spleen and LNs from OT-1 or LCMV-TCR transgenic
mice were prepared, and percentages of transgenic T cells were
determined by FACS analysis. A suspension of 2.5 x
106 transgenic T cells was injected i.v. into the
lateral tail vein (day 1). Recipient mice were immunized with OVA- or
gp33-pulsed DC (3 x 106 DC/mouse on day 0),
and subsequent T cell expansion was monitored by flow cytometry of
Ficoll-purified blood lymphocytes using TCR
- and -specific mAb
that recognize the transgenic TCR. For determination of diabetes
induction, 5 x 105 OT-1 cells were injected
i.v. into RIP-OVAlow-recipient mice. T cell
expansion was determined as above and the DIABUR test (Roche,
Switzerland) was performed to determine the urine glucose content of
the vaccinated animals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differential analysis of mRNA from immature and mature DC has
demonstrated that Bcl-2 expression is rapidly down-regulated upon
induction of DC maturation by an inflammatory stimulus (Ref.
5 and unpublished observations). To investigate
whether intracellular Bcl-2 protein levels reflect this decrease in
transcription, we performed intracellular stainings for murine Bcl-2
(Fig. 1
a). Spleen cell
suspensions from C57BL/6 mice were stained with mAb specific for CD11c
and MHC class II (MHC-II; I-A) as an indicator of DC maturation. Mature
DC were identified as CD11c+ MHC-II
(I-A)++ cells, whereas immature DC were defined
as CD11c+ MHC-II (I-A)+
(Fig. 1a, rectangular gates). A comparison of intracellular
staining for mouse Bcl-2 in these two populations revealed that mature
CD11c+ MHC-II (I-A)++ DC
express significantly lower Bcl-2 levels than immature
CD11c+ MHC-II (I-A)+ DC
(Fig. 1b, top and middle panels;
isotype control background excluded). Because the specific Bcl-2
stainings in DC were relatively weak, we analyzed
CD8+ T cells within the same spleen cell
suspensions as a control; CD8+ T cells have been
shown to express high levels of Bcl-2 (23), and they
demonstrate low background staining with the isotype control mAb (Fig. 1b, CD8+ T cells). Background (isotype
control) staining of DC increased with maturation (Fig. 1b);
to quantify murine Bcl-2 expression levels and accurately compare the
different DC populations, the ratio between the mean fluorescence
intensities (MFI) of murine Bcl-2 staining and isotype control staining
was calculated for each population. These "corrected" Bcl-2
expression levels (Fig. 1c) clearly demonstrate the relative
down-regulation of Bcl-2 protein in mature DC as compared with
immature DC.
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To compensate for the down-regulation of endogenous mouse Bcl-2 in
DC, we generated mice that express hBcl-2 as a transgene under the
control of the mouse CD11c promoter. This promoter region, which
regulates the expression of hBcl-2 cDNA in the context of a mini exon
derived from rabbit -globin (Ref. 24 ; Fig. 2a), has been used to drive
the DC-specific expression of various transgenes in vivo (Refs.
18 and 25). To monitor transgene
expression in DC from transgenic mice, we prepared DC from bone marrow
progenitors cultured in the presence of GM-CSF (26, 27).
After 8 days of culture, when most DC displayed a mature phenotype as
determined by high MHC-II and B7.2 expression levels (data not shown),
DC were lysed and submitted to Western blot analysis (Fig. 2b). The presence of the transgenic hBcl-2 protein (26 kDa)
was detected in DC derived from transgenic (Fig. 2b,
lane 1), but not from nontransgenic, bone marrow (Fig. 2b, lane 2). Comparable amounts of protein were
present in the two lysates as determined by loading control analysis
(data not shown). Transgenic Bcl-2 protein could also be detected in
freshly isolated splenic DC from the transgenic mice.
CD11c+ DC were enriched from total spleen cell
suspensions with magnetic beads, and both the DC-enriched fraction
(Fig. 2b, lane 3) and the DC-depleted fraction
(Fig. 2b, lane 4) were analyzed by Western blot.
Although a faint band of the expected size (Fig. 2b,
lane 3) could be detected in the DC-enriched fraction,
hBcl-2 was not detectable in the lysate from the DC-depleted cell
fraction (Fig. 2b, lane 4).
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c). In
contrast to CD11c+ cells from nontransgenic
littermates, DC from CD11c-Bcl-2 transgenic mice demonstrated
intracellular expression of hBcl-2 protein (Fig. 2c, DC).
Within the same cellular preparation, neither lymphocytes (Fig. 2b, B cells, T cells) nor macrophages (data not shown)
showed detectable expression of the transgenic hBcl-2 protein. Taken
together, the biochemical and flow cytometry analysis confirm that
CD11c-Bcl-2 transgenic mice express hBcl-2 specifically in DC. Influence of the CD11c-Bcl-2 transgene on survival of DC in vitro and in vivo
In the past, transgenic overexpression of Bcl-2 has been shown to
prolong the survival of various cell types (28, 29, 30, 31). To
investigate the effect of transgenic Bcl-2 expression in DC, we
cultured bone marrow from CD11c-bcl-2 transgenic or nontransgenic
control mice in the presence of GM-CSF. After 7–9 days of culture, the
growth factor was removed and DC survival was monitored by flow
cytometry. As shown in Fig. 3, the
transgenic DC showed significantly increased survival kinetics in the
absence of growth factor as compared with DC from nontransgenic mice,
while the latter had only 10–20% viability after 2 days of culture
without GM-CSF; DC from Bcl-2 transgenic mice were viable at elevated
levels for at least 4 days (Fig. 3a). Thus, cell death by
growth factor withdrawal was significantly inhibited by overexpression
of hBcl-2 in DC. Given the capacity of Bcl-2 to enhance DC
survival in vitro (Fig. 3a), we tested whether the longevity
of DC in vivo was also enhanced. Cultured DCs were labeled with the
fluorescent vital dye CMFDA and were then injected s.c. into recipient
mice. Draining LNs were collected 1–4 days later and analyzed by flow
cytometry for presence of CD11c+
CMFDA+ DC as shown in Fig. 3b. Most
fluorescent cells were found to express CD11c, the DC-specific marker.
As shown previously by others (32), only a small fraction
of the transferred fluorescent DC could be detected in the draining LN,
indicating that most s.c. injected DCs are either unable to find the LN
or die on route. Nevertheless, 24 h posttransfer, the percentages
of migrating DCs detected in the draining LNs of mice that had received
hBcl-2+ DCs were 
3-fold higher than the
percentages in mice injected with control DCs (Fig. 3b).
This difference was maintained at later timepoints, but decreased
dramatically in magnitude between days 1 and 4 posttransfer. The
kinetics of DC survival in this assay suggest that the survival
advantage mediated by Bcl-2 is rather short-lived when cultured DC are
adoptively transferred.
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a, right panels)
have been performed after gating on immature
(MHC-II+) and mature DC
(MHC-II++) (Fig. 4a, left
panel). As shown in Fig. 4b, mature
(MHC-II++) DC from bcl-2-transgenic mice
exhibited a reduced incorporation of BrdU as compared with DC from
nontransgenic mice. After 3 days of BrdU exposure, 44% of mature WT
DCs were BrdU positive, compared with only 26% in CD11c-Bcl-2
transgenic mice (Fig. 4b); this difference corresponds to a
40% reduced rate of BrdU incorporation by transgenic DCs. At day 6,
nearly 80% of all mature nontransgenic DC had incorporated BrdU as
compared with 60% in transgenic mice, reflecting a 25% reduction.
From these percentages, one can extrapolate to a theoretical 100% BrdU
positivity of a defined DC population, resulting in an approximate
average lifespan. For mature DC in the spleen this corresponds to 7–8
days in normal mice as compared with 10–12 days in CD11c-Bcl-2
transgenic animals. These data correspond with previously published
estimates of in vivo DC survival (3) and indicate an in
vivo survival advantage for mature (MHC-II++) DCs
in CD11c-Bcl-2 transgenic mice.
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b). After 4
days in the absence of BrdU (day 10), higher percentages of
BrdU+ DCs are detected in transgenic mice (94 vs
84% in mature DCs, 92 vs 78% in immature DCs). By day 14 (8 days
without BrdU feeding), only 17–25% of BrdU-positive DC are
detectable, with no significantly higher numbers in transgenic DC (data
not shown). In contrast to these data, differences between CD11c-Bcl-2
transgenic DC and WT DC were less pronounced in the immature population
(MHC-II+) than in the mature subset
(MHC-II++); early during the pulse period, BrdU
incorporation rates by immature DCs were 59 (nontransgenic) vs 54%
(Bcl-2 transgenic) on day 3 and 84 vs 71% on day 6 (Fig. 4). In LNs,
we could not detect any survival advantage for LN DC from Bcl-2
transgenic mice by BrdU incorporation within the same experiments (data
not shown).
The interpretation of this BrdU labeling study is complicated by the
fact that upon maturation, immature DC transit into the BrdU-positive
CD11c+ MHC-II++ DC pool.
Because BrdU can only be incorporated into cycling cells and mature DCs
are thought to be noncycling (3), we expected to first see
the BrdU take-up in the immature DC fraction (probably via their
cycling precursors) and then, after a lag, the appearance of
BrdU-positive cells in the mature DC pool. This scenario is supported
by the data shown in Fig. 4; after 3 days, the percentage of
BrdU+ cells was higher in the immature than in
the mature pool for transgene positive and WT mice. These data suggest
that presence of transgenic bcl-2 has an influence on longevity of
mature DC from spleen but does not interfere with the maturation
process itself, because BrdU metabolism was not greatly altered in the
immature DC compartment.
Influence of the CD11c-Bcl-2 transgene on lymphocyte numbers in vivo
After finding that functional Bcl-2 transgene expression in DC
affects their turnover/survival in vivo, we wanted to determine whether
this effect has an impact on overall frequencies of DCs as well as
other immune cell subsets. Therefore, we analyzed spleen, LNs, and
thymus of transgenic and nontransgenic animals and determined the
percentages and total numbers of DCs, T cells, and B cells present in
these tissues (Fig. 5). In nontransgenic
mice, DC were detected in spleen and thymus with the expected frequency
of 0.5 to 1 and 0.02%, respectively (Fig. 5a).
Approximately 2- to 3-fold higher frequencies of splenic and thymic DCs
were found in CD11c-bcl-2 transgenic mice as compared with
nontransgenic littermates (Fig. 5a). These higher
frequencies corresponded to higher total DC numbers detected in spleen
(Fig. 5a) and thymus (data not shown) and were true for all
subsets of DC, as determined by CD11c/CD11b stainings for myeloid DC
and CD11c/CD8 stainings for lymphoid DC (data not shown).
Accordingly, the ratio of myeloid to lymphoid DC was not altered in the
spleens of CD11c-Bcl-2 transgenic mice (Fig. 5a). Concurrent
analysis of the same cells demonstrated that the presence of the Bcl-2
transgene did not alter surface expression levels of MHC-II, CD86, or
CD11c (data not shown). Surprisingly, neither frequencies (Fig. 5a) nor total numbers (data not shown) of DCs were altered
in LNs of CD11c-Bcl-2 transgenic mice.
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b) as well as total numbers
(data not shown) of CD8+ and
CD4+ T cells, but not B cells, were slightly but
significantly elevated in spleens, but not in LNs. No alterations in
frequencies of monocytes or macrophages were detected (data not shown).
Because transgene expression was not detected in cell populations other
than DC (see Fig. 2), the increase of T cell numbers in CD11c-Bcl-2
mice must be an indirect effect. Higher DC numbers in the spleen could
result in enhanced T cell survival through increased exposure to MHC
molecules, as previously shown (33). Accordingly, this
effect on T cells is missing in LNs, where we do not see higher numbers
of DC. Furthermore isolated CD4+ and
CD8+ T cells from CD11c-Bcl-2 mice do not display
increased in vitro survival as compared with T cells from nontransgenic
littermates (data not shown). Taken together, these data indicate that
the expression of a bcl-2 transgene in DC leads to increased numbers of
DCs in the spleen. This has an indirect effect on
CD4+ and CD8+ T cells,
which either accumulate or survive preferentially in the presence of
more DC. Elevated humoral and cellular immune responses in CD11c-Bcl-2 mice
Because DC are important mediators of immunity, we next asked
whether mice with elevated DC numbers are able to mount stronger immune
responses as compared with normal mice. Indeed, even after taking
increased background proliferation (no OVA) into account, CD11c-Bcl-2
mice exhibited 2-fold greater proliferation than nontransgenic
controls following immunization with OVA/CFA and subsequent
restimulation of LN cells with OVA (Fig. 6a). Similarly, when the
humoral response against haptenated (NIP) OVA was monitored,
CD11c-bcl-2 transgenic mice had higher total IgG titers of NIP-specific
Abs (Fig. 6b), while IgM levels were not significantly
altered (data not shown). As shown above, the bcl-2-transgenic mice not
only harbor higher DC numbers, but also have more T cells. Therefore,
we cannot be certain of the reason for more vigorous cellular and
humoral immune responses in CD11c-Bcl-2 mice (Fig. 6); a direct effect
of higher DC numbers, which can initiate a more efficient priming,
might be augmented by the presence of more CD4+ T
cells, which specifically proliferate (Fig. 6a) or provide
more T cell help for Ab-producing B cells (Fig. 6b).
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) or naive CTL from transgenic mice
expressing a TCR specific for the LCMV glycoprotein peptide 33–41
(Ref. 34 ; data not shown). Following vaccination of
C57BL/6-mice, specific expansion of OT-1 CTL was detectable in the
peripheral blood of all recipients. However, T cell expansion in mice
that had received the Bcl-2 transgenic DC vaccine was 2- to 3-fold
greater than in mice injected with WT DC, indicating that DC expressing
transgenic Bcl-2 are more potent inducers of CTL expansion (Fig. 7).
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50% of
RIP-OVAlow mice, when they are immunized with
3 x 106 LPS-matured cultured DC i.v. (data
not shown). For the experiment shown in Fig. 7b, we used a
suboptimal number of OT-1 T cells (5 x 105
per mouse). All mice vaccinated with the Bcl-2 transgenic DC vaccine
developed diabetes within 4–8 days postvaccination and did not recover
from disease (Fig. 7b). In contrast, only one of five mice
vaccinated with normal DC developed disease and this animal
recovered completely by day 11 postvaccination. In a second
experiment (data not shown), all mice immunized with Bcl-2 DC vaccines
developed diabetes, and no animal that received a conventional DC
vaccine showed elevated blood glucose levels. We also monitored T cell
expansion in the peripheral blood of RIP-OVAlow
mice, with a similar outcome as described in Fig. 7a (data
not shown). These data indicate that DC with enhanced longevity induce
not only more efficient Ag-specific T cell expansion, but also a more
efficient cytolytic effector response as compared with
conventional DC. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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b). Henri et al.
(37) have shown that LNs harbor more DC subtypes than the
spleen. Eventually those are less sensitive to Bcl-2 as compared with
spleen DC. Another possibility would be a faster migratory turnover of
DC in LNs, which does not allow similar DC accumulations as in the
spleen, where bloodborne DC most likely terminate their life.
Altogether it remains unclear why LNs do not contain elevated DC
numbers, as the spleen does, and this is currently under investigation
in our laboratory.
The increased DC longevity has an effect on the strength of the immune
response in vivo; both humoral and cellular immune responses are
increased in mice expressing Bcl-2 in DC. The capacity of DC to induce
T cell responses is clearly affected by their longevity. Josien et al.
(15) demonstrated previously that treatment of DC with
TRANCE, which induces the up-regulation of
Bcl-xL, results in longer survival of DC vaccines
in recipients and stronger induction of T cell responses. Expression of
Bcl-2 has a similar effect on DC; as compared with conventional DC
vaccines, Bcl-2 transgenic DC induce stronger CD8 T cell responses. The
increased efficacy of CD8 T cell priming by Bcl-2 transgenic DC
vaccines was apparent from both a stronger T cell expansion in the
peripheral blood of vaccinated animals (Fig. 7a) and a more
efficient induction of cytotoxicity, as judged by induction of diabetes
in RIP-OVAlow mice (Fig. 7b). In
contrast, the effect of elevated T cell as well as humoral responses in
CD11c-Bcl-2 transgenic mice is not entirely attributable to the
increase in DC numbers; CD4 and CD8 T cell numbers are also
slightly, but significantly, increased in CD11c-bcl-2 transgenic
animals. Because T cells were not found to express the transgenic Bcl-2
and do not show increased in vitro survival (data not shown), we
speculate that the presence of higher DC numbers in lymphoid organs is
responsible for this accumulation of T cells. In previous work, we
demonstrated that DC provide sufficient signals for the survival of CD4
T cells in the periphery (33). The presence of higher DC
numbers in lymphoid organs may provide better survival conditions for T
cells. Interestingly, although B cell numbers were not affected in
CD11c-bcl-2 transgenic mice, stronger humoral immune responses were
detected. This effect is most likely the result of augmented T cell
help due to the higher CD4 T cell frequency in lymphoid organs.
In addition to intrinsic, programmed regulation of DC survival, several studies have described Ag-specific killing of DC by CTLs in vivo (32, 38), findings which were discussed as a "purposeful death" of DC (39) for the sake of immunoregulation. It has been postulated that DC resistance to perforin-mediated killing (39) may be responsible for familial hemophagocytic lymphohistiocytosis in humans (40). Similarly, insensitivity of DC to TRAIL-induced cell death, which leads to the dysregulation of T cell immune responses, is a proposed cause of human autoimmune lymphoproliferative syndrome (39, 41). The role for an active DC removal process has not yet been elucidated; the endogenously programmed short survival phase of mature DC could make additional "active" DC killing by CTL superfluous. No signs of chronic inflammation or autoimmunity could be detected in CD11c-Bcl-2 transgenic mice (data not shown). It is possible that the Bcl-2-induced increase in DC survival in these transgenic mice is not dramatic enough to induce an autoimmune status. Alternatively, the hypothesis that DC longevity plays a central role in immunoregulation may be incorrect.
DC survival is controlled by more than one mechanism. Up-regulation of
the Bcl-xL, for example, is induced by
inflammatory signals such as TNF- and LPS, which have been reported
to protect DC from apoptosis via the Fas-mediated pathway
(42), as well as anti-CD40 and TRANCE. Treatment of DC
with TRANCE has an even greater effect on survival than the induced
expression of Bcl-2; while increased survival of CD11c-bcl-2 transgenic
DC vaccines could be detected in the draining LNs until, but not later
than, 24 h postimmunization (Fig. 3b), TRANCE-treated
DC vaccines were reported to survive much longer posttransfer
(15). Stimulation of human DC by CD154, IL-12, or IL-15
has also been reported to result in increased
Bcl-xL levels and resistance of the DC to
apoptosis (43). In contrast, neither
Bcl-xL nor Bcl-2 seem to be involved in CpG-DNA
mediated DC survival, where signaling via Toll-like receptor 9 on
murine DCs leads to the activation of phosphaditylinositide-3'-OH
kinase and, most likely, the inhibition of caspase-3 activation
(44). Long-term DC cultures from TNFR1-deficient mice also
demonstrate protection from apoptosis that is independent from Bcl-2
(45). Therefore, signaling via Bcl-2 is only one mechanism
among several dedicated to DC survival. Our results show that enhanced
transgenic expression of Bcl-2 in mature DC in vivo regulates their
survival and longevity. Therefore, we conclude that Bcl-2
down-regulation is a means to restrict DC longevity in the steady
state, i.e., in the absence of inflammatory stimuli.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Brocker, Institute for Immunology, Ludwig-Maximilians University, Goethestrasse 31, 80336 Munich, Germany. E-mail address: tbrocker{at}ifi.med.uni-muenchen.de
3 Abbreviations used in this paper: DC, dendritic cell; BrdU, bromodeoxyuridin; TRANCE, TNF-related activation-induced cytokine; WT, wild type; CMFDA, 5-chloromethyl-fluorescein diacetate; LN, lymph node; NIP, 4-hydroxy-5-iodo-3-nitrophenyl; RIP, rat insulin promoter; MHC-II, MHC class II; MFI, mean fluorescence intensity; hBcl-2, human Bcl-2; LCMV, lymphocytic choriomeningitis virus.
Received for publication June 3, 2002. Accepted for publication July 12, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 |
|
 B. M. Janelsins, A. R. Mathers, O. A. Tkacheva, G. Erdos, W. J. Shufesky, A. E. Morelli, and A. T. Larregina Proinflammatory tachykinins that signal through the neurokinin 1 receptor promote survival of dendritic cells and potent cellular immunity Blood, March 26, 2009; 113(13): 3017 - 3026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Ohnmacht, A. Pullner, S. B.S. King, I. Drexler, S. Meier, T. Brocker, and D. Voehringer Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity J. Exp. Med., March 16, 2009; 206(3): 549 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Varas, C. Hernandez-Lopez, J. Valencia, S. Mattavelli, V. G. Martinez, L. Hidalgo, C. Gutierrez-Frias, A. G. Zapata, R. Sacedon, and A. Vicente Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling J. Leukoc. Biol., June 1, 2008; 83(6): 1476 - 1483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Gautier, T. Huby, F. Saint-Charles, B. Ouzilleau, M. J. Chapman, and P. Lesnik Enhanced Dendritic Cell Survival Attenuates Lipopolysaccharide-Induced Immunosuppression and Increases Resistance to Lethal Endotoxic Shock J. Immunol., May 15, 2008; 180(10): 6941 - 6946. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Illario, M. L. Giardino-Torchia, U. Sankar, T. J. Ribar, M. Galgani, L. Vitiello, A. M. Masci, F. R. Bertani, E. Ciaglia, D. Astone, et al. Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells Blood, January 15, 2008; 111(2): 723 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Obst, H.-M. van Santen, R. Melamed, A. O. Kamphorst, C. Benoist, and D. Mathis Sustained antigen presentation can promote an immunogenic T cell response, like dendritic cell activation PNAS, September 25, 2007; 104(39): 15460 - 15465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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W. L. W. Chang, N. Baumgarth, M. K. Eberhardt, C. Y. D. Lee, C. A. Baron, J. P. Gregg, and P. A. Barry Exposure of Myeloid Dendritic Cells to Exogenous or Endogenous IL-10 during Maturation Determines Their Longevity J. Immunol., June 15, 2007; 178(12): 7794 - 7804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chen, L. Huang, and J. Wang Deficiency of Bim in dendritic cells contributes to overactivation of lymphocytes and autoimmunity Blood, May 15, 2007; 109(10): 4360 - 4367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pradhan, J. Genebriera, W. L. Denning, K. Felix, C. A. Elmets, and L. Timares CD4 T Cell-Induced, Bid-Dependent Apoptosis of Cutaneous Dendritic Cells Regulates T Cell Expansion and Immune Responses J. Immunol., November 1, 2006; 177(9): 5956 - 5967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xia, S. Hao, and J. Xiang CD8+ Cytotoxic T-APC Stimulate Central Memory CD8+ T Cell Responses via Acquired Peptide-MHC Class I Complexes and CD80 Costimulation, and IL-2 Secretion. J. Immunol., September 1, 2006; 177(5): 2976 - 2984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lauterbach, A. Gruber, C. Ried, C. Cheminay, and T. Brocker Insufficient APC Capacities of Dendritic Cells in Gene Gun-Mediated DNA Vaccination. J. Immunol., April 15, 2006; 176(8): 4600 - 4607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chen, Y.-H. Wang, Y. Wang, L. Huang, H. Sandoval, Y.-J. Liu, and J. Wang Dendritic cell apoptosis in the maintenance of immune tolerance. Science, February 24, 2006; 311(5764): 1160 - 1164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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J. Yang, S. P. Huck, R. S. McHugh, I. F. Hermans, and F. Ronchese Perforin-dependent elimination of dendritic cells regulates the expansion of antigen-specific CD8+ T cells in vivo PNAS, January 3, 2006; 103(1): 147 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Celli, Z. Garcia, and P. Bousso CD4 T cells integrate signals delivered during successive DC encounters in vivo J. Exp. Med., November 7, 2005; 202(9): 1271 - 1278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lauterbach, C. Ried, A. L. Epstein, P. Marconi, and T. Brocker Reduced immune responses after vaccination with a recombinant herpes simplex virus type 1 vector in the presence of antiviral immunity J. Gen. Virol., September 1, 2005; 86(9): 2401 - 2410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Dubois, T. A. Waldmann, and J. R. Muller Survival adjustment of mature dendritic cells by IL-15 PNAS, June 14, 2005; 102(24): 8662 - 8667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Kleindienst, C. Wiethe, M. B. Lutz, and T. Brocker Simultaneous Induction of CD4 T Cell Tolerance and CD8 T Cell Immunity by Semimature Dendritic Cells J. Immunol., April 1, 2005; 174(7): 3941 - 3947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kirschnek, S. Ying, S. F. Fischer, H. Hacker, A. Villunger, H. Hochrein, and G. Hacker Phagocytosis-Induced Apoptosis in Macrophages Is Mediated by Up-Regulation and Activation of the Bcl-2 Homology Domain 3-Only Protein Bim J. Immunol., January 15, 2005; 174(2): 671 - 679. [Abstract] [Full Text] [PDF]
|
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|
|
|
|
H. Hon, E. B. Rucker III, L. Hennighausen, and J. Jacob bcl-xL Is Critical for Dendritic Cell Survival In Vivo J. Immunol., October 1, 2004; 173(7): 4425 - 4432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Quarrie and K. T. Riabowol Murine Models of Life Span Extension Sci. Aging Knowl. Environ., August 4, 2004; 2004(31): re5 - re5. [Abstract] [Full Text] [PDF]
|
|
|
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D. Homann, D. B. McGavern, and M. B. A. Oldstone Visualizing the Viral Burden: Phenotypic and Functional Alterations of T Cells and APCs during Persistent Infection J. Immunol., May 15, 2004; 172(10): 6239 - 6250. [Abstract] [Full Text] [PDF]
|
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L. Gabriele, P. Borghi, C. Rozera, P. Sestili, M. Andreotti, A. Guarini, E. Montefusco, R. Foa, and F. Belardelli IFN-{alpha} promotes the rapid differentiation of monocytes from patients with chronic myeloid leukemia into activated dendritic cells tuned to undergo full maturation after LPS treatment Blood, February 1, 2004; 103(3): 980 - 987. [Abstract] [Full Text] [PDF]
|
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 G. Bauriedel, A. Jabs, D. Skowasch, R. Hutter, J. J. Badimon, V. Fuster, U. Welsch, and B. Luderitz Dendritic cells in neointima formation after rat carotid balloon injury: coordinated expression withanti-apoptotic Bcl-2 and HSP47 in arterial repair J. Am. Coll. Cardiol., September 3, 2003; 42(5): 930 - 938. [Abstract] [Full Text] [PDF]
|
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 K. Vermaelen and R. Pauwels Accelerated Airway Dendritic Cell Maturation, Trafficking, and Elimination in a Mouse Model of Asthma Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 405 - 409. [Abstract] [Full Text] [PDF]
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E. Flano, I.-J. Kim, J. Moore, D. L. Woodland, and M. A. Blackman Differential {gamma}-Herpesvirus Distribution in Distinct Anatomical Locations and Cell Subsets During Persistent Infection in Mice J. Immunol., April 1, 2003; 170(7): 3828 - 3834. [Abstract] [Full Text] [PDF]
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0.0005, n = 4), but not in mature DC
(I-A++, middle panel; p
= 0, 4). c, To accurately compare the Bcl-2 expression
levels detected in cell types with different background staining
properties, the ratio between Bcl-2 and isotype control MFI values
(±SD) was formed for each staining. Data are representative of four
different stainings (n = 3–4).
) was placed behind a DNA
segment containing the mouse CD11c promoter (
). The rabbit
. B,
BamHI; Bs, BspHI; E,
EcoRI; H, HindIII; N,
NotI; S, SstI; X, XhoI;
restriction sites in parentheses have been destroyed by blunt-end
cloning. b, DC from 8-day bone marrow GM-CSF cultures
(lanes 1and 2) or freshly isolated
splenic DC (lanes 3 and 4) derived from
transgenic (lanes 1 and 3) or
nontransgenic mice (lanes 2 and 4) were
lysed, and the cell lysates were analyzed by Western blot as described
in Materials and Methods. A mAb specific for hBcl-2 was
used to detect presence of transgenic Bcl-2 (lanes
1–4); a mAb specific for prohibitin served as a loading
control for the bone marrow culture-derived DC (data not shown).
c, Intracellular staining for hBcl-2 in total splenocyte
suspensions from transgenic (filled histogram) or nontransgenic (open
histogram) mice. Only CD11c-positive cells meeting high forward scatter
and intermediate side scatter criteria are shown in the top
panel (DC). B cells (B220+) and T cells
(TCR
) or
nontransgenic (
, nontransgenic), and mean values are shown as a thin
horizontal line. (*, p 
