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*
Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, Instituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy;
Section of Microbiology, Department of Molecular Biology, University of Siena, Siena, Italy; and
Department of Biotechnology and Bioscience, University of Milan-Bicocca, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release. Compared with DCs treated with soluble Ag, DCs fed
with recombinant bacteria required 102- to
103-fold less Ag and were at least 102 times
more effective on a per-cell basis for activating specific T cells.
S. gordonii was internalized in DCs by conventional
phagocytosis, and cytochalasin D inhibited presentation of
bacteria-associated Ag, but not of soluble Ag, suggesting that
phagocytosis was required for proper delivery of recombinant Ag.
Bacteria were also very potent inducers of DC maturation, although they
enhanced the capacity of DCs to activate specific CD4+ T
cells at concentrations that did not stimulate DC maturation. In
particular, S. gordonii dose-dependently up-regulated
expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40,
CD83) and reduced both phagocytic and endocytic activities.
Furthermore, bacteria promoted in a dose-dependent manner DC release of
cytokines (IL-6, TNF-
, IL-1ß, IL-12, TGF-ß, and IL-10) and of
the chemokines IL-8, RANTES, IFN--inducible protein-10, and monokine
induced by IFN-. Thus, recombinant Gram-positive bacteria appear a
powerful tool for vaccine design due to their extremely high capacity
to deliver Ags into DCs, as well as induce DC maturation and secretion
of T cell chemoattractans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given their central role in the immune system, DCs can represent an important target for vaccine development, and the possibility of generating large numbers of autologous DCs from precursors has made feasible and very attractive the use of DCs as natural adjuvants for inducing or boosting anti-tumor immune responses (2, 3, 4, 5). A major problem in using DCs in immunotherapy is to optimize Ag administration so that MHC molecules are properly loaded. In addition, it is highly desirable to stimulate DC maturation to maximize their Ag-presenting capacity and render them resistant to the inhibitory effects of products (e.g., IL-10) released by tumor cells (6, 7). Bacteria or their components, including cell wall constituents and unmethylated CpG-containing DNA, are among the strongest inducers of DC maturation (8, 9, 10, 11, 12). Because bacteria can directly stimulate DC maturation and can be easily modified, they appear very good candidates as delivery systems for vaccine Ags. Bacteria in fact can be engineered to express the gene product at the cell surface, in the cytosol or as secreted protein (13, 14, 15), and can also serve as carriers for introducing Ag-encoding DNA into APCs (16, 17, 18). These different approaches have been employed for inducing protective immune responses against viral and tumor Ags in mouse models (15, 19).
Recombinant strains of Streptococcus gordonii have been successfully used as vectors for parenteral and mucosal delivery of vaccine Ags and have been shown to induce both local and systemic Ab responses in mice (14, 20, 21, 22). More recently, the efficacy of such vaccination approach has been confirmed in primates against viral Ags (23). However, the cell populations and mechanisms involved in these bacteria-based immunostimulating systems have been only marginally investigated. We have previously shown that mouse DCs very efficiently present a MHC class I-restricted heterologous Ag expressed on the surface of recombinant S. gordonii to T lymphocytes and that S. gordonii induces neo-biosynthesis and membrane stabilization of MHC class I and class II molecules (10). In the present study, human DCs generated from peripheral blood monocytes were investigated for their capacity to present a MHC class II-restricted model Ag, the C-fragment of tetanus toxin (TTFC), expressed on the surface of recombinant S. gordonii to specific CD4+ T cells. Results demonstrate that DCs pulsed with recombinant bacteria stimulate specific CD4+ T cell response with high efficiency and that S. gordonii is a potent stimulus for human DC maturation and induces DC release of chemokines active on T cells. These findings can be of value for the design of new DC-based vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recombinant strain of S. gordonii expressing TTFC (named GP1253) was constructed using the host-vector system GP1221-pSMB55, as already described in detail (24). Surface expression of TTFC on GP1253 was achieved using the M6 protein as fusion partner (14, 25). Recombinant S. gordonii GP1253 and control strain GP1221 were grown at 37°C in tryptic soy broth without dextrose (Difco, Detroit, MI) and harvested by centrifugation at the end of the exponential phase of growth. Bacterial cells were then washed and resuspended in fresh medium containing 10% glycerol at 1:500 of the original culture volume. Aliquots were stored frozen at -80°C until use. Surface expression of TTFC was determined by flow cytometry analysis on whole cells and Western blotting of cell fractions (not shown). The concentration of TTFC expressed on bacteria was determined by Dot blot analysis of whole cells using a standard curve of purified TTFC. Two-fold dilutions of recombinant S. gordonii GP1253, control strain GP1221 (from 4 x 109 to 6.2 x 107 CFU), and 200–3.12 ng soluble TTFC (Boerhinger Mannheim, Mannheim, Germany) in a volume of 100 µl were transfered onto the nitrocellulose membrane using a HYBRI.DOT instrument (Life Technologies, Gaithersburg, MD). The membrane was than reacted with a TTFC-specific rabbit serum (Calbiochem, San Diego, CA; 1:1,000 dilution) followed by anti-rabbit serum conjugated to alkaline phosphatase (Sigma, St. Louis, MO; 1:5,000 dilution). The concentration of TTFC expressed on the bacterial surface was calculated by densitometric analysis using the Fluor-s Multimager (Bio-Rad, Hercules, CA)
Bead preparations
TTFC was bound to 1-µm latex beads (Polysciences, Warrington, PA) by passive absorption, according to the manufacturer’s instructions. The amount of TTFC coupled to beads was measured by difference in optical densities at 595 nm of the protein solution before and after the linkage using Bradford assay (Bio-Rad). Bead preparations were washed with culture medium before use.
DCs
DCs were prepared from peripheral blood monocytes of healthy individuals, as described by Sallusto and Lanzavecchia (26). Briefly, PBMC isolated by standard density gradient centrifugation were further separated on multistep Percoll gradients (Pharmacia, Uppsala, Sweden). Cells from the light density fraction (42.5–50%; >90% CD14+) were recovered and cultured at 1 x 106 cells/ml in RPMI 1640 (Life Technologies) complemented with 10% FBS (HyClone, Logan, UT), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies), and 0.05 mM 2-ME (Merck, Darmstadt, Germany) (complete medium) at 37°C with 5% CO2 in the presence of 200 ng/ml recombinant human GM-CSF (Mielogen; Schering-Plough, Milan, Italy) and 200 U/ml recombinant human IL-4 (Genzyme, Cambridge, MA). Medium was changed after 3 days, and at day 6 of culture, cells were recovered and depleted of CD2+ and CD19+ cells by means of immunomagnetic beads coated with specific mAbs (Dynal, Oslo, Norway). This procedure resulted in >97% pure CD1a+ and CD14- DC preparation.
Ag presentation assay
CD4+ T cell clones specific for TTFC (CP7,
ALS4) were prepared by limiting dilution of TTFC-specific
CD4+ T cell lines generated from PBMC of two
healthy individuals. These T cell clones were
CD4+, CD8-, TCR
ß+, TCR 
-, and
CD28+ and secreted high IFN-, but no IL-4,
upon activation with anti-CD3 and anti-CD28 mAbs or in
Ag-specific stimulation assay. TTFC-specific CD4+
T cell clones were strictly MHC class II-dependent, as determined by
inhibition studies with anti-HLA-DR or anti-MHC class I mAb
(not shown). Autologous DCs were pulsed at 37°C with S.
gordonii GP1253, control bacteria GP1221, TTFC-conjugated beads,
or soluble TTFC in complete medium at the indicated concentrations.
After 18 h, APCs were washed, examined for cell viability by the
trypan blue exclusion test, and then cocultured with T cells (2–3
x 104 cells/well) in triplicate wells.
Cocultures were pulsed with 1 µCi/well
[3H]thymidine at day 2 or 3. In some
experiments, DCs were pretreated for 30 min at 37°C with 10 µg/ml
cytochalasin D (CCD; Sigma), and then washed twice with PBS before Ag
pulsing. Radioactivity was measured in a beta counter (Topcount,
Packard Instruments, Groningen, The Netherlands). Results are given as
mean cpm ± SD of triplicate cultures. Where indicated,
supernatants from cocultures were analyzed for IFN- contents by
ELISA (R&D Systems, Minneapolis, MN).
Transmission electron microscopy
DCs were incubated with live S. gordonii GP1253 (cells-to-bacteria ratio, 1:50) in complete medium at 37°C with 5% CO2. After 2–18 h, cells were pelleted, washed, fixed in 1% glutaraldehyde in a Tyrode buffer, postfixed in 1% osmium tetroxide, dehydrated in graded alcohols, and finally embedded in Durcopan ACM (Fluka, Buchs, Switzerland). Thin sections were stained with uranyl acetate and lead citrate and then examined under a Philips CM100 electron microscope.
Flow cytometry analysis
After 18 h incubation with S.
gordonii, lipoteichoic acid (LTA; Sigma), or medium alone, cells
were washed and then incubated in PBS added of 2% FBS and 0.01%
NaN3 with the following mAbs: FITC-conjugated
anti-HLA-DR (L243, IgG2a) and FITC-conjugated anti-CD14
(M
P9, IgG2b) from Becton Dickinson (San Jose, CA); FITC-conjugated
anti-CD1a (HI149, IgG1) and FITC-conjugated anti-CD86 (FUN-1,
IgG1) from PharMingen (San Diego, CA); FITC-conjugated anti-CD40
(BB20, IgG1) from Ylem (Avezzano, Italy); FITC-conjugated anti-CD54
(84H10, IgG1), FITC-conjugated anti-CD80 (MAB104, IgG1), and
anti-CD83 (HB15, IgG2b) from Immunotech (Marseille, France);
anti-CSF-1R (CD115) (204A5-4, rat IgG1) from Calbiochem, and
anti-MHC class I (W6/32, IgG1) from Dako (Glostrup, Denmark).
FITC-conjugated anti-mouse Ig F(ab')2,
FITC-conjugated anti-rat IgG, and control rat IgG came from
Southern Biotechnology Associates (Birmingham, AL). In control samples,
the mAb was substituted with matched isotype control mouse or rat Ig
(Becton Dickinson). Cells were analyzed in a FACScan equipped with Cell
Quest software (Becton Dickinson, Mountain View, CA).
Phagocytosis and endocytosis assays
DCs incubated with S. gordonii or 10 µg/ml LTA for 18 h were washed, resuspended in complete medium, and finally pulsed with 1 mg/ml Texas red-conjugated BSA or FITC-labeled 2-µm latex beads (Molecular Probes, Eugene, OR). DCs were then incubated at 37°C or 4°C, and, at selected time points, uptake was stopped by adding cold PBS containing 2% FBS and 0.01% NaN3. Cells were then washed four times and analyzed in a FACScan. Surface binding values obtained by incubating cells at 4°C were subtracted from values measured at 37°C.
Cytokine and chemokine secretion
DCs treated as described above were cultured in 6-well plates
(1 x 106 cells/well) for 18–24 h at
37°C, and then supernatants were collected and stored at -80°C.
IL-1ß, TNF-, IL-6, IL-12 (p70), TGF-ß, IL-10, IL-8, and RANTES
were measured by commercially available ELISA kits (R&D Systems),
according to the manufacturer’s instructions. IP-10 (IFN--inducible
protein of 10 kDa) and MIG (monokine induced by IFN-) were detected
with sandwich ELISAs performed with Abs pairs from R&D (anti-IP-10:
mouse mAb 33036.211 and goat polyclonal Ab) and PharMingen
(anti-MIG: mouse mAbs B8-11 and B8-6) following the manufacturers’
protocol. Adsorbance (450–540 nm) was read in an ELISA reader model
3550 UV (Bio-Rad).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. gordonii, a Gram-positive coccus and a normal
commensal present in the human oral cavity, has been used in previous
studies as vector for mucosal delivery of heterologous vaccine Ags
(20, 21, 22). The strain "Challis" allows easy genetic
manipulation because it is naturally competent for genetic
transformation and is capable of colonizing the mouse oral and vaginal
mucosa (14). In the present study, we used a recombinant
strain of S. gordonii (GP1253) expressing TTFC on the cell
surface as fusion with the M6 protein (25). The S.
gordonii parental strain GP1221 served as control. Western blot
analysis of bacterial cell fractions showed the presence of a 84-kDa
protein band reactive with the TTFC-specific Ab only in the envelope
fraction (data not shown). The amount of TTFC expressed on bacteria was
determined by Dot blot analysis and resulted in 50 ng/5 x
108 CFU as calculated by densitometric analysis
with a standard curve of purified TTFC (Fig. 1
). Thus, incubation of DCs
(106 cells/ml) with GP1253 at a bacteria-to-DC
ratio of 50:1 corresponded to 5 ng/ml of TTFC.
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shows that specific
CD4+ T cells were much more readily activated
when the same amount of Ag was administered to DCs as a surface
bacterial protein than as soluble Ag, with the T cell response
determined as cell proliferation (Fig. 2A) and IFN-
release (Fig. 2B). When DCs were pulsed with recombinant
bacteria in respect to soluble TTFC,
102-103 times less Ag was
required to obtain a comparable T cell response, and incubation of DCs
with as few as one bacterium per DC (corresponding to 0.1 ng/ml TTFC)
was sufficient for inducing a significant T cell proliferation. On a
per-cell basis, DCs fed with recombinant bacteria were at least
102 times more effective than DCs pulsed with an
equal dose of soluble Ag (Fig. 2C). Control strain GP1221
did not induce a significant T cell response in any case.
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Next, we determined whether and how DCs internalize S.
gordonii. Immature DCs were incubated with live bacteria at
a bacteria-to-DC ratio of 50:1, and then analyzed by transmission
electron microscopy. As soon as 2 h after incubation at 37°C
(the shortest time tested), bacteria were found in a small proportion
(<20%) of DCs within membrane-bound phagosomal organelles (not
shown). After 18 h incubation, bacteria were visualized in most
(>90%) DCs and appeared at various stages of degradation always
included in vacuoles and phagosomes and not free in the cytosol (Fig. 3). The number of bacteria present in
each DC section varied, with the majority of DCs having phagocytosed
5–20 bacteria. In phagosomes, cell membranes were tightly or loosely
opposed to bacteria, and in many instances a single organelle contained
multiple bacteria. S. gordonii GP1253 and GP221 were
internalized similarly, and no bacteria were found when DCs where
incubated at 4°C. Finally, no bacteria enclosed by cell membrane
protrusions or pseudopod coils were observed, suggesting that S.
gordonii was taken up exclusively by conventional phagocytosis, as
previously observed with mouse DCs (10).
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A), but did not affect
presentation of soluble TTFC (Fig. 4B). Thus, presentation
of bacteria-associated Ag by DCs required phagocytosis and was not the
result of membrane processing.
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To dissect the mechanisms by which TTFC on S. gordonii
is presented with higher efficiency by DCs, T cell responses to DCs fed
with S. gordonii GP1253 and to DCs pulsed with equal doses
of soluble TTFC or TTFC-conjugated to latex particles and concomitantly
treated with control strain GP1221 were compared. Fig. 5A shows that pulsing DCs with
5 ng/ml soluble TTFC in the presence of strain GP1221 or LTA did not
change the magnitude of the T cell response. In contrast, incubation of
DCs with GP1221 or LTA increased their capacity to present a higher
dose (100 ng/ml) of soluble TTFC (Fig. 5B). Strain GP1253
could not be included in this last experiment because incubation with
1000 bacteria/DC drastically reduced cell viability. In contrast, DCs
treated with 5 ng/ml of TTFC bound to 1-µm particles showed a better
T cell stimulating activity compared with DCs pulsed with an equal dose
of soluble Ag, and incubation of DCs with TTFC-conjugated beads
together with control bacteria further augmented the T cell
proliferation, although not to the levels measured with strain GP1253
(Fig. 5C). The results indicated that particulate TTFC was
more efficiently presented than soluble TTFC, and that S.
gordonii could effectively exert an adjuvant activity when
suboptimal doses of soluble Ag or minute amounts of Ag in particulate
form were provided. Therefore, in the following experiments, we tested
whether S. gordonii was capable of stimulating DC maturation
in terms of cells-surface marker expression, phagocytic and endocytic
functions, and cytokine release.
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Cells treated with S. gordonii (either GP1253 or
GP1221) for 18 h showed a dramatic increase in the expression of
molecules involved in Ag presentation such as MHC class I and class II
molecules, CD80 and CD86 costimulatory molecules, and CD54. In
addition, CD40 and CD83 were both up-regulated (Figs. 6 and 7).
In contrast, expression of CD1a was slightly but consistently
diminished in DCs treated with S. gordonii, whereas
expression of the M-CSF receptor (CD115) was abolished (not shown).
These effects of S. gordonii on DCs were strictly
dose-dependent (Fig. 7) and could be detected also using heat-killed
bacteria or LTA, but not following phagocytosis of 2-µm latex beads
(not shown). Interestingly enough, doses of bacteria (bacteria-to-DC
ratio of 1:1) that allowed an efficient presentation of the recombinant
Ag did not induce significant changes in the surface phenotype of
DCs.
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A) and 2-µm FITC-conjugated
latex beads (Fig. 8B). To control for potential nonspecific
association of BSA or beads with the exterior of DCs, experiments were
performed in parallel at 4°C.
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To analyze whether the observed phenotypic maturation was
associated with cytokine production, supernatants from 24-h DC cultures
performed in the presence of different doses of bacteria were tested by
ELISA. Both strains of S. gordonii induced a dose-dependent
release of large amounts of TNF- and IL-6 and less IL-1ß and
TGF-ß. As previously observed in mouse DCs (10), and in
human DCs exposed to other stimuli that promote DC maturation
(29, 30), S. gordonii also caused the release
of IL-10, an important DC regulatory factor that maintains DC in an
immature state and inhibits their Ag-presenting activity. In addition,
S. gordonii promoted substantial production of IL-12
heterodimer, with levels up to 4 ng/ml at the maximum dose of bacteria
(Fig. 9). Finally, monocyte-derived DCs
were found to release constitutively significant amounts of IL-8 and
low levels of IP-10. Upon incubation with S. gordonii, DCs
increased secretion of IL-8 and IP-10 as well as started to
dose-dependently release RANTES and MIG (Fig. 10). Similar amounts of cytokines or
chemokines were detected in supernatants from DC cultures stimulated
with LTA (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release. Compared with soluble TTFC, a
markedly reduced amount of Ag or much less DCs were necessary to obtain
similar T cell responses. Two principal explanations can account for
these findings: first, recombinant bacteria can allow a more efficient
Ag delivery into the class II pathway of Ag processing, and, second,
bacteria can stimulate DC maturation and thus potentiate their
Ag-presenting function. Results indicate that both mechanisms cooperate
in rendering heterologous Ag on recombinant bacteria very effectively
presented by DCs to CD4+ T cells. S. gordonii was internalized by DCs via conventional phagocytosis and delivered in membrane-bound phagosomes, and phagocytosis was required for effective Ag presentation to specific T cells. Although DCs have been considered poorly phagocytic compared with macrophages, the capacity of mouse or human DCs and epidermal Langerhans cells to phagocytose bacteria has been documented for a variety of microorganisms including Corynebacterium parvum, Staphylococcus aureus, Borrelia burgdorferi, Chlamydia species, Mycobacterium tubercolosis, and Leishmania and has been confirmed both in vitro and in vivo (3, 31, 32, 33). The administration of exogenous class I-restricted Ags in a form that requires phagocytosis is essential for an efficient presentation to T cells (10, 34, 35). Our results show that phagocytosis of particulate Ag may represent a very effective means to supply Ags to the class II processing pathway, and this can partly explain why TTFC expressed on the bacterial surface was presented better than soluble TTFC. In keeping with this hypothesis is the observation that DCs presented bacteria-associated TTFC with a greater efficiency when low numbers of recombinant bacteria not sufficient for inducing DC maturation were used. In addition, Ag adsorbed to latex particles was presented better than soluble Ag. The fact that adsorptive phagocytosis is very effective for the generation of MHC class II-peptide complexes from Ag-carrying bacteria has been previously reported for mouse DCs (35) and recently confirmed in DCs phagocytosing apoptotic or necrotic allogeneic B cell blasts (36).
In addition, bacteria effectively induced DC maturation and enhanced
the APC function of DCs. In fact, treatment of DCs with control
bacteria markedly increased T cell proliferation to either 100 ng/ml of
soluble Ag or 5 ng/ml of Ag bound to latex particles. However,
cotreatment of DCs with particulate Ag and control bacteria partially
reconstituted (30–60%) the efficiency of recombinant bacteria, most
likely because DCs possess specific receptors for recognizing bacteria
and their delivery in the Ag-processing pathway may be favored
(35). DCs are critical for the induction of primary immune
responses against pathogens, but to perform this function they need to
undergo a series of changes, collectively known as "maturation,"
which enables DCs to migrate to lymphoid organs and to acquire high T
cell-stimulating capacity (1, 2). In this study, we show
that S. gordonii is very effective in inducing DC
maturation, although this bacterium is a normal commensal of the oral
cavity and considered nonpathogenic. In particular, S.
gordonii dose-dependently induced increased membrane expression of
MHC, CD80, CD86, ICAM-1, CD40, and CD83 and, in parallel, considerably
increased the capacity of DCs to activate allogeneic T cells in the
primary MLR assay (data not shown). In addition, DCs exposed to
S. gordonii released higher amounts of proinflammatory
cytokines such as IL-6, IL-1ß, and TNF-, which can mediate DC
maturation in an autocrine fashion. However, incubation of DCs with
bacteria in the presence of neutralizing anti-TNF- Ab had only
marginal effects on DC membrane molecule expression (data not shown),
indicating that autocrine release of TNF- is not relevant to DC
maturation in this system, as previously reported in mouse DCs
(10) or in human DCs incubated with bacillus
Calmette-Guérin (37). S. gordonii was
effective in inducing the release of IL-12, a cytokine pivotal for the
induction of Th1 cell differentiation, as observed for human
blood-derived DCs stimulated with M. tubercolosis or
B. burgdorferi (8, 31). Stimulation with
bacteria also induced secretion of IL-10, which may serve as a
counter-regulatory signal for blocking DC activation and thus
exaggerated T cell responses. All these effects could be reproduced by
using LTA, suggesting that this cell wall component play a crucial role
in Gram-positive bacteria-induced DC maturation. Following incubation
with bacteria or LTA, DCs decreased their capacity to endocytose a
soluble protein and phagocytose latex beads, confirming that mature DCs
down-regulate mechanisms of Ag capture and switch from an "Ag
capturing" to an "Ag presenting" mode (2). To
maximize their Ag-presenting potential, mature DCs transiently increase
the biosynthesis of MHC class II molecules, and, most strikingly, MHC
molecules are massively exported to the cell membrane where their
half-life is prolonged because the rate of endocytosis is lowered
(10, 38, 39). The accumulation of high numbers MHC class
II molecules on the cell membrane together with increased expression of
costimulatory molecules allow a highly efficient Ag presentation to T
lymphocytes.
An interesting finding of this study was that DCs stimulated with bacteria released chemokines very active in attracting T cells. Indeed, recent results in the mouse system demonstrated that DCs produce an array of chemokines in response to bacterial stimulation, and that the pattern of chemokines expressed changes during the maturation stages of DCs (40). Here, we show that blood-born human DCs constitutively secrete IL-8 and IP-10 and, upon maturation, high levels of IL-8, IP-10, RANTES, and MIG. RANTES can bind to different chemokine receptors and is active on a variety of leukocytes, including memory lymphocytes, NK cells, monocytes, and immature DCs (40, 41). IP-10 and MIG are selective and efficient chemotactic stimuli for memory and activated T lymphocytes, and their main receptor, CXCR3, is preferentially expressed by Th1 cells (42, 43). Other than functioning as chemoattractants, these chemokines can directly mediate an Ag-independent activation signal on T cells and potentiate Th1 as well as CTL responses (44, 45). Thus, DC release of these chemokines, together with the secretion of IL-12, can be very important during the development of an immune response for recruiting and favor the functions of Th1 cells.
In conclusion, recombinant bacteria appear very good candidates for the design of new DC-based vaccine strategies. In fact, compared with soluble Ag or Ag conjugated to inert particles, recombinant bacteria combine a high efficiency of delivering Ag into DCs with the capacity to induce a robust DC maturation. The use of mature DCs in vaccination procedures can offer several advantages: in vivo experiment using mouse bone marrow-derived DCs have clearly shown that the ability of DCs to induce protective antitumor immune responses correlates with their degree of maturation (46). Additionaly, mature DCs may be resistant to the suppressive effects of IL-10 or other factors released by tumor cells (7, 47, 48) and may have a higher propensity to home to lymphoid organs (49). Finally, mature DCs have an impaired capability to acquire and process exogenous Ags and thus may carry a reduced potential of inducing undesirable autoimmune responses (5, 50).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint request to Dr. Silvia Corinti, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, Instituto di Ricovero e Cura a Carattere Scientifico, Via dei Monti di Creta 104, 00167 Rome, Italy. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; CCD, cytochalasin D; LTA, lipotheicoic acid; TTFC, C-fragment of tetanus toxin; IP-10, IFN--inducible protein; MIG, monokine induced by IFN-.
Received for publication January 26, 1999. Accepted for publication June 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 179:1109. over IL-4 responses. FASEB J. 12:705.This article has been cited by other articles:
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M. Nagl, L. Kacani, B. Mullauer, E.-M. Lemberger, H. Stoiber, G. M. Sprinzl, H. Schennach, and M. P. Dierich Phagocytosis and Killing of Bacteria by Professional Phagocytes and Dendritic Cells Clin. Vaccine Immunol., November 1, 2002; 9(6): 1165 - 1168. [Abstract] [Full Text] [PDF]
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 G. Gorgun, K. B. Miller, and F. M. Foss Immunologic mechanisms of extracorporeal photochemotherapy in chronic graft-versus-host disease Blood, July 18, 2002; 100(3): 941 - 947. [Abstract] [Full Text] [PDF]
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A. Unkmeir, U. Kammerer, A. Stade, C. Hubner, S. Haller, A. Kolb-Maurer, M. Frosch, and G. Dietrich Lipooligosaccharide and Polysaccharide Capsule: Virulence Factors of Neisseria meningitidis That Determine Meningococcal Interaction with Human Dendritic Cells Infect. Immun., May 1, 2002; 70(5): 2454 - 2462. [Abstract] [Full Text] [PDF]
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 S. Corinti, L. Chiarantini, S. Dominici, M. E. Laguardia, M. Magnani, and G. Girolomoni Erythrocytes deliver Tat to interferon-{gamma}-treated human dendritic cells for efficient initiation of specific type 1 immune responses in vitro J. Leukoc. Biol., April 1, 2002; 71(4): 652 - 658. [Abstract] [Full Text] [PDF]
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H. C. Heystek, C. Moulon, A. M. Woltman, P. Garonne, and C. van Kooten Human Immature Dendritic Cells Efficiently Bind and Take up Secretory IgA Without the Induction of Maturation J. Immunol., January 1, 2002; 168(1): 102 - 107. [Abstract] [Full Text] [PDF]
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C. W. Cutler, R. Jotwani, and B. Pulendran Dendritic Cells: Immune Saviors or Achilles' Heel? Infect. Immun., August 1, 2001; 69(8): 4703 - 4708. [Full Text] [PDF]
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A. Kolb-Maurer, S. Pilgrim, E. Kampgen, A. D. McLellan, E.-B. Brocker, W. Goebel, and I. Gentschev Antibodies against Listerial Protein 60 Act as an Opsonin for Phagocytosis of Listeria monocytogenes by Human Dendritic Cells Infect. Immun., May 1, 2001; 69(5): 3100 - 3109. [Abstract] [Full Text] [PDF]
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S. Corinti, C. Albanesi, A. la Sala, S. Pastore, and G. Girolomoni Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions J. Immunol., April 1, 2001; 166(7): 4312 - 4318. [Abstract] [Full Text] [PDF]
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C. J. Hertz, S. M. Kiertscher, P. J. Godowski, D. A. Bouis, M. V. Norgard, M. D. Roth, and R. L. Modlin Microbial Lipopeptides Stimulate Dendritic Cell Maturation Via Toll-Like Receptor 2 J. Immunol., February 15, 2001; 166(4): 2444 - 2450. [Abstract] [Full Text] [PDF]
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M. Svensson, C. Johansson, and M. J. Wick Salmonella enterica Serovar Typhimurium-Induced Maturation of Bone Marrow-Derived Dendritic Cells Infect. Immun., November 1, 2000; 68(11): 6311 - 6320. [Abstract] [Full Text] [PDF]
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A. la Sala, S. Corinti, M. Federici, H. U. Saragovi, and G. Girolomoni Ligand activation of nerve growth factor receptor TrkA protects monocytes from apoptosis J. Leukoc. Biol., July 1, 2000; 68(1): 104 - 110. [Abstract] [Full Text]
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A. Kolb-Maurer, I. Gentschev, H.-W. Fries, F. Fiedler, E.-B. Brocker, E. Kampgen, and W. Goebel Listeria monocytogenes-Infected Human Dendritic Cells: Uptake and Host Cell Response Infect. Immun., June 1, 2000; 68(6): 3680 - 3688. [Abstract] [Full Text] [PDF]
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S. Corinti, D. Medaglini, C. Prezzi, A. Cavani, G. Pozzi, and G. Girolomoni Human Dendritic Cells Are Superior to B Cells at Presenting a Major Histocompatibility Complex Class II-Restricted Heterologous Antigen Expressed on Recombinant Streptococcus gordonii Infect. Immun., April 1, 2000; 68(4): 1879 - 1883. [Abstract] [Full Text] [PDF]
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) and then used to
stimulate a TTFC-specific Th1 clone (CP7; 30,000 cells/well). At each
Ag dose, the amount of soluble TTFC and bacteria-associated TTFC were
equivalent. DCs incubated with wild-type S. gordonii
GP1221 (
) served as control. Cocultures were carryed out for 3 days,
and the T cell response measured as [3H]thymidine uptake
and IFN-
) or CCD
(10 µg/ml) pretreated (
) for
18 h at 37°C. Thereafter, DCs were collected and then incubated
with 1 mg/ml Texas red-BSA at 37°C. At various time points, cells
were washed with cold PBS, and the BSA accumulation was measured by
flow cytometry. Dashed line with open squares represents the
fluorescence of untreated DCs pulsed with Texas red-BSA at 4°C.
Fluorescence of cells incubated at 4°C was subtracted from the
fluorescence of cells incubated at 37°C. B, DCs
treated as described above were incubated with 2-µm FITC-conjugated
latex beads for 24 h at 37°C or 4°C and then were analyzed by
flow cytometry.
