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*
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, and Departments of
Medicine,
Cell Biology and Physiology,
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Molecular Genetics and Biochemistry, and
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Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1), whereas mature DC switch
expression to CCR7 and respond exclusively to MIP-3ß and 6Ckine.
Mature DC transduced to express a marker gene localized to lymph nodes
after intradermal injection, constituting 1.5% of lymph node DC. In
contrast, cutaneous DC transfected in situ via gene gun were detected
in the draining lymph node at a 20-fold lower frequency. Unexpectedly,
the state of maturation at the time of injection had no influence on
the proportion of DC that localized to draining lymph nodes, as labeled
immature and mature DC were detected in equal numbers. Immature DC that
trafficked to lymph nodes underwent a significant up-regulation of CD86
expression indicative of spontaneous maturation. Moreover, immature DC
exited completely from the dermis within 36 h of injection,
whereas mature DC persisted in large numbers associated with a marked
inflammatory infiltrate. We conclude that in vitro maturation is not a
requirement for effective migration of DC in vivo and suggest that
administration of Ag-loaded immature DC that undergo natural maturation
following injection may be preferred for DC-based
immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immature DC express CC chemokine receptor 1 (CCR1), CCR2, and CCR5 that bind inflammatory chemokines, which is believed to promote the accumulation of Ag-acquiring immature DC at sites of tissue injury. In contrast, mature DC switch receptor expression to CCR7 and migrate in response to constitutive chemokines that are expressed in lymphatics and lymphoid tissues. This interaction induces migration of Ag-stimulated and maturing DC to lymph nodes (2, 3, 4, 5, 6, 7). Based on this pattern of receptor expression it is thought that DC-based vaccination protocols should focus on using ex vivo-matured DC, as without maturation DC may not traffic to lymph nodes and, hence, may not contribute to induction of T cell responses. However, in vivo studies by us and others in mice and chimpanzees suggest that the state of DC maturation before administration does not accurately predict the capacity of DC to migrate to lymph nodes (8, 9, 10, 11, 12). One explanation for this discrepancy is that immature DC undergo maturation upon injection in skin, resulting in responsiveness to constitutive chemokines that induce homing to lymph nodes.
To test this hypothesis in a preclinical setting, we have studied the role of maturation on DC trafficking in the rhesus macaque model. We first characterized the phenotype and function of immature and CD40L matured monocyte-derived DC and correlated this with chemokine receptor expression and chemotactic responses in vitro. We then used rare event analysis by flow cytometry to quantify DC trafficking to lymph nodes following intradermal (i.d.) injection, using both membrane dye and gene transduction to mark cells. To provide relevance to other methods of DC-based vaccination, we compared the migration of gene-transduced DC with that of cutaneous DC transfected in situ with gene gun.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal adult rhesus macaques (Macaca mulatta) were used in this study. Animals were housed and all in vivo experiments were performed at the Infectious Disease Primate Research Facility, University of Pittsburgh (Pittsburgh, PA), using protocols approved by the institutional animal care and use committee.
Media and reagents
The medium used to culture DC was RPMI 1640 medium supplemented
with 10% FCS (BioWhittaker, Walkersville, MD),
L-glutamine, sodium pyruvate, nonessential amino acids,
penicillin-streptomycin, 10 mM HEPES buffer (Life Technologies, Grand
Island, NY), and 2-ME (Sigma, St. Louis, MO). Recombinant human GM-CSF
and IL-4 were provided by Schering-Plough (Kenilworth, NJ), and
trimeric human CD40L was provided by Immunex (Seattle, WA). Recombinant
human MIP-1, 6Ckine (BioSource, Camarillo, CA), and MIP-3ß (R&D
Systems, Minneapolis, MN) were used in chemotaxis assays. DQ Green BSA
was a gift from Mingjie Zhou (Molecular Probes, Eugene, OR). The
lipophilic carbocyanine dye
1,1'-dioctadecyl-3,3,3',3'-tetramethylindodi-carbocyanine perchlorate
(DiD; excitation/emission spectra = 644 nm/663 nm) used in
labeling studies was purchased from Molecular Probes.
DC culture
PBMC were isolated from heparinized blood by centrifugation through sodium diatrizoate and Ficoll (Sigma), and CD14+ cells were positively selected using Ab-coated microbeads and magnetic separation (Miltenyi Biotec, Auburn, CA). The purity of CD14+ cells using this technique was consistently >90%. Cells were cultured at a density of 3 x 105 cells/ml in six-well plates. GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) were added at the initiation of culture, and media and cytokines were replenished at regular intervals for 7 days, depending on the experiments. CD40L (3 µg/ml) was added on day 5 of culture for 24–48 h to mature cells, and cells were harvested following incubation with 10 mM EDTA.
Flow cytometric analysis
DC were stained for flow cytometric analysis as previously
described (13). Cross-reactive mAb specific for human
HLA-A, -B, and -C (clone B9.12.1); CD83 (HB15A; both from
Coulter-Immunotech, Miami, FL); HLA-DR (L243), CD14 (m
P9), CD80
(L307.4; all from Becton Dickinson, San Jose, CA); CD86 (FUN-1;
PharMingen, San Diego, CA); CD40 (EA-5; Ancell, Bayport, MN); and CCR5
(FAB182; gift from Frank Mortari, R&D Systems) were used as conjugates
with FITC or PE. In some experiments biotinylated CD86 was used with
streptavidin-Cy5 (Amersham, Arlington Heights, IL). The CCR7 mAb was a
gift from Lijun Wu (LeukoSite, Cambridge, MA) and was used in
association with a goat anti-mouse secondary Ab (BioSource).
Analyses related to the characterization of immature and mature DC were
performed on a FACSCalibur flow cytometer (Becton Dickinson) using
CellQuest software. For analyses of DC and lymph node suspensions in
trafficking studies, 1.5 x 106 events were
acquired in triplicate on a FACS Vantage flow cytometer (Becton
Dickinson), using a second argon laser emitting at 647 nm to excite DiD
fluorescence. Data were acquired and analyzed using LYSIS II and
WinList software, respectively. Fluorescence and side scatter
measurements for in vivo data are shown in four-decade log scales.
Assessment of Ag uptake
For analysis of macropinocytosis by DC we used DQ Green BSA, a self-quenched dye conjugate of BSA. Degradation of protein in this reagent results in dequenching and elaboration of green fluorescence (14). DC were incubated with 10 µg/ml DQ Green BSA at 4 or 37°C for 30 min with and without prior incubation with 10 µg/ml cytochalasin D (Sigma) followed by labeling with mAb to CD86. Fluorescence in this assay is indicative of uptake and proteolytic cleavage of BSA.
Analysis of cytokine production
Supernatants were collected from cultures on days 4 and 7, with
and without addition of CD40L (3 µg/ml) on day 5, and stored at
-70°C until assayed. IL-12 (p40 and 70 forms) and TNF- were
detected using ELISA kits specific for rhesus monkey (BioSource)
according to the manufacturer’s instructions.
Chemotaxis assays
Cell migration was measured using a 96-well chemotaxis chamber with a 5-µm pore polycarbonate membrane (Neuro Probe, Gaithersburg, MD). Thirty microliters of chemokine in RPMI, 0.5% BSA, and 10 mM HEPES medium was added to appropriate wells, which were then covered by the membrane. Twenty-five thousand cells at a density of 1 x 106 cells/ml in the same medium were placed on the membrane. Plates were incubated for 90 min at 37°C in a humidified incubator, and cells remaining on the topside of the membrane were then removed by gentle washing with PBS and wiping. Migrated cells that had attached to the underside of the membrane were fixed in 2.5% glutaraldehyde and stained with 0.1% toluidine blue (Fisher Scientific, Pittsburgh, PA). To quantify migration, cells in four nonoverlapping areas were counted under x400 magnification and summed. Results are expressed as the mean ± SEM for duplicate wells.
Transduction of monkey DC using recombinant adenovirus encoding a marker gene
DC were transduced with a replication-defective recombinant adenoviral vector expressing enhanced green fluorescence protein (EGFP) under the control of the CMV immediate-early promoter/enhancer (15), which was provided by Andrea Gambotto (University of Pittsburgh). Briefly, virus was added at a multiplicity of infection of 100 directly to wells containing day 4 cells and incubated for 1 h at room temperature. Medium was then replenished, and cells were matured with addition of CD40L for 24 h.
DC trafficking in vivo
Immature day 4 or mature CD40L-treated day 7 DC were labeled with DiD as previously described (12) or were transduced with recombinant adenovirus and resuspended in 400 µl of PBS for injection. Between 2.7 x 106 and 5.2 x 106 DC were injected i.d. into anesthetized animals from which cells were derived in a region lateral to the proximal inguinal lymph node chain. In some experiments EGFP gene was administered via gene gun to skin before injection of labeled DC. Animals were anesthetized 36 h later, and inguinal and axillary lymph nodes and skin were biopsied. Lymph node tissues were disrupted using digestion with collagenase D as previously described (16). Unseparated lymph node cell suspensions were labeled with mAb to CD83 and CD86 before flow cytometric analysis. Skin biopsies were treated with 4% paraformaldehyde and 30% sucrose infusion before freezing in isopentane. Sections were cut and stained with hematoxylin and eosin or were examined directly for fluorescent cells as previously described (12).
DNA-based immunization
Genetic immunizations were performed by biolistic delivery as previously described (17) using the pEGFP-C2 plasmid (Clontech, Palo Alto, CA), which contains the EGFP gene under the control of the CMV promoter. Animals received a total of four shots given to two overlapping regions of shaved skin in the inguinal region. In all cases the gene gun was applied before and at least 0.5 in. away from injection of cells. In experiments in which the gene gun was applied, injected DC were labeled with DiD so as to distinguish in situ transfected cells (green) from injected DC (red).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the monkey DC system we performed extensive
phenotypic and functional studies in vitro. DC cultured from
CD14+ peripheral blood monocytes of rhesus
macaques had characteristic dendritic morphology (data not shown),
similar to that of human CD14-derived DC (18) and DC
generated by us and others from PBMC of the chimpanzee and macaque
(13, 19). DC cultured for 4 days in GM-CSF and IL-4
retained expression of CD14, but were negative for the mature DC marker
CD83 (Fig. 1
). The day 4 DC expressed
moderate levels of costimulatory molecules CD80, CD86, and CD40 and
high levels of MHC class I and class II (Fig. 1). In contrast, monkey
DC treated by ligation of CD40, which is known to induce rapid
maturation of DC in the human system (20, 21), uniformly
expressed CD83 and high levels of costimulatory molecules CD80, CD86,
and CD40 (Fig. 1). Expression of MHC class I and class II was
unaltered, whereas expression of CD14 was down-regulated. Cells
cultured for 7 days without CD40 ligation had an intermediate phenotype
(Fig. 1).
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). This required actin
polymerization and, hence, was an active process, as treatment with
cytochalasin D substantially inhibited uptake (22). In
contrast, DQ Green BSA fluorescence was almost completely absent at
37°C when CD40L-matured day 7 cells were used (Fig. 2). Cells
cultured for 7 days in the absence of CD40L had an intermediate
function. The loss of ability to internalize and cleave protein upon
maturation was correlated with increased expression of CD86, as can be
seen by the small proportion of day 4 DC that did not process DQ Green
BSA and expressed high levels of CD86 (Fig. 2). Hence, in this system
CD86 up-regulation is a reliable indicator of functional maturation. In
cytokine release assays, day 4 and day 7 DC secreted negligible levels
of IL-12 and TNF-, whereas CD40L-treated day 7 DC markedly
up-regulated secretion of these cytokines (Table I).
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Immature and mature monkey DC have distinct patterns of chemokine receptor expression and chemotactic responses
We next determined the chemokine receptor expression and
chemotactic responsiveness of monkey DC at different stages of
maturation. Chemokine receptor expression of human DC is highly
polarized, with immature DC expressing receptors for inflammatory
chemokines and switching expression to receptors for constitutive
chemokines upon maturation (2, 3, 4, 5, 23). Consistent with the
human DC literature, immature monkey DC expressed low levels of CCR5,
but not CCR7. Conversely, CD40L-matured DC were negative for expression
of CCR5, but expressed CCR7 at high levels (Fig. 3). When monkey DC were tested in
chemotaxis assays, day 4 immature DC migrated in response to CCR5
ligand MIP-1, but not at all to the CCR7 ligands 6Ckine and
MIP-3ß. Upon maturation, chemotactic responses were completely
switched to MIP-3ß and 6Ckine (Fig. 3). The weaker response of
immature DC to chemokine correlated with the low level expression of
CCR5 on these cells. These data indicate that in vitro chemotactic
responses of monkey monocyte-derived DC are tightly regulated as a
function of maturation.
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To test the capacity of in vitro-derived DC to traffic to lymph
nodes, we first transduced DC ex vivo with a gene expressing the
cytoplasmic Ag EGFP using recombinant adenovirus and then matured the
cells with CD40L. Transduced cells were injected i.d. into the inguinal
region, and the draining lymph node was excised 36 h later.
Approximately 99% of DC expressed EGFP before injection, and a
proportion of these cells could subsequently be identified in the
draining lymph node (Fig. 4). No EGFP
fluorescence was detected in control axillary lymph nodes (data not
shown). When lymph node cells were stained for expression of CD83 and
CD86, a discrete population of double-labeled cells constituting 
1%
of the total lymph node was identified, representing interdigitating DC
(24, 25). The EGFP+ DC that
localized in the lymph node were also
CD83+CD86+, as expected
from our in vitro analyses of mature DC, and represented 1.5% of total
lymph node DC (Fig. 4). A proportion of CD83-
cells was also identified within the region describing
EGFP+ cells in the lymph node. This may reflect
endogenous cells that have a level of autofluorescence overlapping that
of EGFP+ cells. To place this result in the
context of other DC-based vaccine protocols, in additional experiments
we administered EGFP gene via gene gun to the inguinal skin of monkeys
and excised lymph nodes 36 h later. Skin DC transfected in situ
with EGFP gene were not detected in the draining lymph nodes of two
animals using flow cytometric analysis. This is not surprising, as in
previous murine studies fluorescence microscopy has been required to
identify the small number of transfected cells in lymph nodes
(17, 26). When draining lymph nodes from four other
animals were sectioned in their entirety and examined, occasional
EGFP+ cells were identified (data not shown).
Whereas few EGFP+ cells could be detected in
draining lymph nodes, cells containing gold particles and expressing
EGFP could be identified in the superficial dermis (Fig. 5). An acute inflammatory response was
present in the epidermis as a result of gene gun bombardment (Fig. 5).
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The above experiments indicated that mature Ag-stimulated monkey
DC could efficiently traffic to lymph nodes after i.d. injection, as
was predicted from our in vitro chemotaxis assays. We next wanted to
directly compare the migration of immature and mature DC in vivo. We
elected to switch labeling techniques from the adenovirus system to
eliminate any potential for inadvertent maturation of immature DC
before injection. We therefore used an inert lipophilic membrane dye to
label cells as in our previous studies (12). In separate
experiments, DiD-labeled immature and mature DC were injected i.d. into
the inguinal region of donor monkeys, and the draining inguinal lymph
nodes were excised 36 h later and analyzed by flow cytometry.
Labeled DC were stained for expression of CD83 and CD86 immediately
before injection to confirm the maturation state of cells. Immature
cultured DC were
CD83-CD86+, as expected by
our previous analyses (Fig. 6). In
contrast, mature cultured DC were CD83+
CD86bright, expressing significantly higher
levels of CD86 than immature DC (Fig. 6). Following injection,
DiD+ cells were identified in the draining lymph
node of animals receiving both immature and mature DC (Fig. 6). No
DiD+ cells could be identified in control
axillary lymph nodes (data not shown). The DiD+
cells constituted 1% of total lymph node DC regardless of the in
vitro maturation state (Fig. 6). The proportion of injected cells that
were identified in the lymph node at 36 h ranged from 0.07 to
0.12% over five experiments.
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From our in vitro data we know that immature DC do not migrate in
response to constitutive lymphoid chemokines MIP3ß and 6Ckine,
yet the in vivo trafficking results clearly indicate migration of these
cells to lymph nodes in significant numbers. One explanation for this
difference is that upon injection the immature DC undergo spontaneous
maturation. To evaluate this possibility we compared expression of CD86
on immature DC before injection with these cells following trafficking
to lymph nodes. CD86 up-regulation is a valid marker of maturation, as
we have shown in experiments in vitro (Fig. 2
). The mean fluorescence
intensity of CD86 on immature DC that localized to lymph nodes was
3.6-fold higher than that of immature DC before injection, suggestive
of in vivo maturation (Fig. 7).
Surprisingly, CD86 expression on mature DC that had localized in lymph
nodes was, on the average, 3.9 times lower than that of the same cells
before injection. As a result, expression of CD86 on DC that localized
to lymph nodes was essentially the same regardless of the maturation
state at the time of injection (Fig. 7).
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To determine the relative efficiency of migration of immature and
mature DC from the dermal injection site, we examined skin biopsied
36 h after injection of cells. Injection of immature DC resulted
in a minor localized acute inflammatory response. No fluorescently
labeled cells could be identified at this time, suggesting complete
migration of immature DC from the injection site (Fig. 8). In marked contrast, a severe acute
inflammatory infiltrate was present at the site of injection in two of
three animals that received injections of mature DC. A large number of
fluorescently labeled mature DC were detected in the dermis at 36
h in these animals (Fig. 8). The accumulation of DC was not dependent
on labeling with DiD, as mature DC that expressed EGFP by adenoviral
transduction were also identified in large numbers in a separate
experiment (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. These are recognized properties of
immature and mature DC as defined in the human system (1).
Monkey DC had the expected chemokine receptor profiles, switching from
expression of CCR5 on immature cells to CCR7 following maturation with
CD40L (2, 3, 4, 23). Accordingly, immature DC migrated
exclusively in response to the inflammatory chemokine MIP-1, whereas
mature DC migrated to constitutive chemokines 6Ckine and MIP-3ß that
are important in attracting DC to T cell areas of lymph nodes
(5, 6, 7). The clear in vitro similarities between
monocyte-derived DC in the human and monkey suggest that conclusions
drawn from in vivo experiments using this model can safely be
extrapolated to the human system.
For the in vivo trafficking experiments, we focused on flow cytometric
methods to accurately determine the number and phenotype of DC that
migrated to lymph nodes following labeling and injection. Unexpectedly,
we found that immature and CD40L matured DC trafficked to lymph nodes
with similar efficiency despite the polarized chemokine receptor
expression and in vitro chemotactic responses of these cells. In
several experiments using two different labeling techniques, the
proportion of lymph node DC that were derived from the injected
population was 1% at 36 h postinjection regardless of the
state of maturation at the time of injection. Given this relatively
large number, it is likely that Ag-loaded DC will readily contact and
stimulate Ag-specific T cells that migrate through the paracortex,
indicating that DC-based vaccination should be effective at stimulating
immune responses in large animals and humans. Using histological
techniques in a similar chimpanzee model, we have previously
demonstrated that injected DC localize to the lymph node paracortex,
with a peak migration occurring from 24–48 h post injection
(12). Injected DC intimately associate with T cells in
this region and maintain high levels of HLA-DR, CD40, and CD86
expression similar to interdigitating DC (12). In
preliminary studies in monkeys we noted that immature and mature DC
both localized to T cell-rich areas of lymph nodes when administered
together in equal numbers, using different fluorochromes to track cells
(data not shown). From these analyses we can conclude that labeled DC
detected in tissues by flow cytometry have actively trafficked to lymph
nodes and taken up residence as interdigitating DC.
Our findings are consistent with data from the mouse using purified
splenic DC of the myeloid lineage (CD8-),
which are analogous to human monocyte-derived DC. These cells traffic
to lymph nodes following s.c. injection in the absence of any in vitro
maturation stimulus, representing about 1% of total lymph node DC
(27). A recent report in the murine system indicates that
immature bone marrow-derived DC migrate poorly to lymph nodes following
s.c. injection, relative to migration of GM-CSF transfected immature DC
(28). Interestingly, GM-CSF transfection did not induce
maturation of DC in this system, based on phenotypic and functional
analysis (28). When the migration of immature and
CD40L matured DC was compared in the same system, injected DC
that trafficked to lymph nodes constituted from 1–2% of lymph node DC
48 h after s.c. injection regardless of maturation state
(11).
Two potential explanations exist for the trafficking of immature DC to lymph nodes. The first is that immature DC respond to as yet unknown chemokines that do not use the CCR7 receptor. This is unlikely, as 6Ckine, which binds CCR7, appears to be required for DC homing to lymph nodes. Mice lacking expression of 6Ckine have a paucity of DC in lymph nodes (7), and administration of Ab to 6Ckine blocks the migration of mature DC to lymph nodes when injected s.c. into mice (5). A more likely explanation is that immature DC undergo spontaneous maturation upon injection into skin. Consistent with this hypothesis was the finding that labeled DC that trafficked to lymph nodes had a statistically significant increase in CD86 expression compared with cultured immature DC, which is suggestive of functional DC maturation on the basis of our in vitro experiments and other reports in mice (29). A mild inflammatory response was present in the skin at the site of injection at 36 h, and hence injected DC would be exposed to proinflammatory cytokines that induce maturation (30, 31). Interestingly, while CD86 expression was up-regulated on immature DC following localization to lymph nodes, a decrease in expression of this costimulatory molecule was noted on mature DC that migrated to lymph nodes, although this change was not statistically significant. Moreover, the relative changes in CD86 expression of injected immature and mature DC resulted in an almost identical expression of CD86 by these cells in lymph nodes even though the data were collected from different animals in different experiments. Removal of DC from in vitro exposure to GM-CSF and IL-4 is not likely to be responsible for the decrease in CD86 expression by mature DC, given the up-regulation of CD86 by immature cells subjected to the same conditions. However, it is possible that withdrawing cells from high dose exogenous CD40L resulted in modulation of CD86 expression. The data raise the possibility that homeostatic mechanisms exist in vivo that regulate the degree of activation of exogenously supplied DC upon localization to the lymph node.
A second unexpected finding from the in vivo studies was that immature DC migrated completely from skin following i.d. injection in monkeys, whereas mature DC tended to remain in the dermis. Our previous studies in the chimpanzee also demonstrated that in vitro-derived DC, labeled with DiD but administered without Ag or prior maturation, migrated away from an s.c. injection site within 48 h of injection (12). Fossum (32) reported that purified lymph DC injected into footpads of mice mostly were retained at the site of injection. Lymph DC represent a maturing DC population (33), and hence these findings may be consistent with our results. More recently, Ag-stimulated blood-derived human DC used therapeutically in human cancer patients were only partially cleared from an i.d. injection site and apparently not at all following s.c. injection (34). Labeur et al. (11) also found that the majority of mouse DC remained at the site of s.c. injection regardless of the in vitro maturation state before administration. The differences between our report and others may be due in part to the types of labels used and the different species studied. However, our findings raise the question of whether there is a fundamental difference between the capacity of immature and mature DC to be cleared from a skin injection site. Mature DC produce large quantities of proinflammatory cytokines and probably induced the severe inflammatory response observed in the dermis. Therefore, while a mild inflammatory response may induce maturation of immature DC and promote migration, it is possible that a more severe inflammatory response impairs migration of mature DC out of skin.
Despite the finding that 1% of lymph node DC were derived from the
injected population, migration was relatively inefficient, as only
0.1% of injected cells could be accounted for in the draining lymph
node at 36 h postinjection. Similar quantitative studies in the
murine system also indicate that only 0.3% of injected DC can
subsequently be detected in the draining lymph node (27).
The reasons for this apparent inefficiency are not known. A large
number of mature DC in our study could be accounted for in the dermis;
however, the fate of immature DC that migrated completely from the skin
but were not detected in the draining lymph node is uncertain. We are
able to rule out the possibility that DC localized in substantial
numbers to other nodes in the inguinal chain, as secondary nodes were
collected and found to contain few, if any, labeled cells (data not
shown). It is possible that injected cells trafficked via blood to
distant lymphoid sites. Labeled cells were not detected in venous blood
samples at 30 min and 36 h postinjection in several experiments
(data not shown), although massive dilution by circulating leukocytes
would make such cells very difficult to detect.
Our studies with membrane-labeled DC are limited by the fact that the dye does not represent a model Ag, and hence the trafficking results do not relate directly to DC-based immunotherapy. However, we showed, using a recombinant adenovirus, that DC transduced in vitro with EGFP traffic to lymph nodes relatively efficiently and retain high level expression for at least 36 h. These data clearly support the application of adenoviral vectors as a means of loading DC with Ag for vaccination protocols (35, 36, 37). By comparison, DC transfected in situ with EGFP using a gene gun were not detectable in draining lymph nodes by flow cytometry, although single EGFP-transfected cells could be detected in skin and lymph node by fluorescence microscopy. This would suggest a frequency of EGFP+ DC that migrated to lymph nodes following gene gun administration to be <0.05% of total lymph node DC, at least 20-fold lower than that of ex vivo transduced DC. Although the number of directly transfected DC that localize in lymph nodes is relatively small, as has been shown by others in the murine system (26), we and others have shown that this method clearly represents a potent means of inducing Ag-specific immune responses (17, 26). Our data indicate that it is possible to generate substantially larger numbers of gene-transfected DC in draining lymph nodes using ex vivo manipulation compared with gene gun administration. Whether this difference translates into a stronger immune response is yet to be determined.
Our results suggest that DC-based vaccine protocols can be substantially more flexible with respect to in vitro maturation than was previously thought. It may be preferable to administer immature DC immediately following Ag pulsing, especially when Ag is supplied as exogenous peptide that may have a limited half-life. Factors in the dermis will induce maturation of injected cells, leading to responsiveness to constitutive chemokines and migration into lymphatics and lymph nodes. In contrast, our finding that mature DC remain at the site of injection in such large numbers suggests that a majority of in vitro matured DC may not contribute effectively to the immune response. It will be important to determine in this model the relative immunogenicities of vaccines using immature and mature DC as well as cutaneous immunization via gene gun to establish the optimal DC-based vaccine system for human use.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Simon M. Barratt-Boyes, Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cells; CCR, CC chemokine receptor; CD40L, CD40 ligand; MIP, macrophage inflammatory protein; i.d., intradermal; DiD, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodi-carbocyanine perchlorate; EGFP, enhanced green fluorescence protein.
Received for publication October 14, 1999. Accepted for publication December 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node. J. Exp. Med. 189:593. induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans’ cell migration. Immunology 75:257.[Medline]
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 M. von Bergwelt-Baildon, A. Shimabukuro-Vornhagen, A. Popov, N. Klein-Gonzalez, F. Fiore, S. Debey, A. Draube, B. Maecker, I. Menezes, L. M. Nadler, et al. CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants Blood, April 1, 2006; 107(7): 2786 - 2789. [Abstract] [Full Text] [PDF]
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 G. P. Linette, D. Zhang, F. S. Hodi, E. P. Jonasch, S. Longerich, C. P. Stowell, I. J. Webb, H. Daley, R. J. Soiffer, A. M. Cheung, et al. Immunization Using Autologous Dendritic Cells Pulsed with the Melanoma-Associated Antigen gp100-Derived G280-9V Peptide Elicits CD8+ Immunity Clin. Cancer Res., November 1, 2005; 11(21): 7692 - 7699. [Abstract] [Full Text] [PDF]
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B. Morandi, R. Costa, M. Falco, S. Parolini, A. De Maria, G. Ratto, M. C. Mingari, G. Melioli, A. Moretta, and G. Ferlazzo Distinctive Lack of CD48 Expression in Subsets of Human Dendritic Cells Tunes NK Cell Activation J. Immunol., September 15, 2005; 175(6): 3690 - 3697. [Abstract] [Full Text] [PDF]
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A. Machlenkin, O. Goldberger, B. Tirosh, A. Paz, I. Volovitz, E. Bar-Haim, S.-H. Lee, E. Vadai, E. Tzehoval, and L. Eisenbach Combined Dendritic Cell Cryotherapy of Tumor Induces Systemic Antimetastatic Immunity Clin. Cancer Res., July 1, 2005; 11(13): 4955 - 4961. [Abstract] [Full Text] [PDF]
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N. Teleshova, J. Kenney, J. Jones, J. Marshall, G. Van Nest, J. Dufour, R. Bohm, J. D. Lifson, A. Gettie, and M. Pope CpG-C Immunostimulatory Oligodeoxyribonucleotide Activation of Plasmacytoid Dendritic Cells in Rhesus Macaques to Augment the Activation of IFN-{gamma}-Secreting Simian Immunodeficiency Virus-Specific T Cells J. Immunol., August 1, 2004; 173(3): 1647 - 1657. [Abstract] [Full Text] [PDF]
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 N. Teleshova, J. Jones, J. Kenney, J. Purcell, R. Bohm, A. Gettie, and M. Pope Short-term Flt3L treatment effectively mobilizes functional macaque dendritic cells J. Leukoc. Biol., June 1, 2004; 75(6): 1102 - 1110. [Abstract] [Full Text] [PDF]
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K.-J. Liu, C.-C. Wang, L.-T. Chen, A.-L. Cheng, D.-T. Lin, Y.-C. Wu, W.-L. Yu, Y.-M. Hung, H.-Y. Yang, S.-H. Juang, et al. Generation of Carcinoembryonic Antigen (CEA)-Specific T-Cell Responses in HLA-A*0201 and HLA-A*2402 Late-Stage Colorectal Cancer Patients after Vaccination with Dendritic Cells Loaded with CEA Peptides Clin. Cancer Res., April 15, 2004; 10(8): 2645 - 2651. [Abstract] [Full Text] [PDF]
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B. L. Colvin, A. E. Morelli, A. J. Logar, A. H. Lau, and A. W. Thomson Comparative evaluation of CC chemokine-induced migration of murine CD8{alpha}+ and CD8{alpha}- dendritic cells and their in vivo trafficking J. Leukoc. Biol., February 1, 2004; 75(2): 275 - 285. [Abstract] [Full Text] [PDF]
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K. Brown, W. Gao, S. Alber, A. Trichel, M. Murphey-Corb, S. C. Watkins, A. Gambotto, and S. M. Barratt-Boyes Adenovirus-Transduced Dendritic Cells Injected into Skin or Lymph Node Prime Potent Simian Immunodeficiency Virus-Specific T Cell Immunity in Monkeys J. Immunol., December 15, 2003; 171(12): 6875 - 6882. [Abstract] [Full Text] [PDF]
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S. Watanabe, H. Kagamu, H. Yoshizawa, N. Fujita, H. Tanaka, J. Tanaka, and F. Gejyo The Duration of Signaling through CD40 Directs Biological Ability of Dendritic Cells to Induce Antitumor Immunity J. Immunol., December 1, 2003; 171(11): 5828 - 5836. [Abstract] [Full Text] [PDF]
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N. Teleshova, I. Frank, and M. Pope Immunodeficiency virus exploitation of dendritic cells in the early steps of infection J. Leukoc. Biol., November 1, 2003; 74(5): 683 - 690. [Abstract] [Full Text] [PDF]
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 J Plumb, M A Armstrong, M Duddy, M Mirakhur, and S McQuaid CD83-positive dendritic cells are present in occasional perivascular cuffs in multiple sclerosis lesions Multiple Sclerosis, April 1, 2003; 9(2): 142 - 147. [Abstract] [PDF]
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L. H. Butterfield, A. Ribas, V. B. Dissette, S. N. Amarnani, H. T. Vu, D. Oseguera, H.-J. Wang, R. M. Elashoff, W. H. McBride, B. Mukherji, et al. Determinant Spreading Associated with Clinical Response in Dendritic Cell-based Immunotherapy for Malignant Melanoma Clin. Cancer Res., March 1, 2003; 9(3): 998 - 1008. [Abstract] [Full Text] [PDF]
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L. A. Harshyne, M. I. Zimmer, S. C. Watkins, and S. M. Barratt-Boyes A Role for Class A Scavenger Receptor in Dendritic Cell Nibbling from Live Cells J. Immunol., March 1, 2003; 170(5): 2302 - 2309. [Abstract] [Full Text] [PDF]
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L. Tailleux, O. Neyrolles, S. Honore-Bouakline, E. Perret, F. Sanchez, J.-P. Abastado, P. H. Lagrange, J. C. Gluckman, M. Rosenzwajg, and J.-L. Herrmann Constrained Intracellular Survival of Mycobacterium tuberculosis in Human Dendritic Cells J. Immunol., February 15, 2003; 170(4): 1939 - 1948. [Abstract] [Full Text] [PDF]
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 I. J. M. de Vries, D. J. E. B. Krooshoop, N. M. Scharenborg, W. J. Lesterhuis, J. H. S. Diepstra, G. N. P. van Muijen, S. P. Strijk, T. J. Ruers, O. C. Boerman, W. J. G. Oyen, et al. Effective Migration of Antigen-pulsed Dendritic Cells to Lymph Nodes in Melanoma Patients Is Determined by Their Maturation State Cancer Res., January 1, 2003; 63(1): 12 - 17. [Abstract] [Full Text] [PDF]
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R. Ignatius, K. Tenner-Racz, D. Messmer, A. Gettie, J. Blanchard, A. Luckay, C. Russo, S. Smith, P. A. Marx, R. M. Steinman, et al. Increased Macrophage Infection upon Subcutaneous Inoculation of Rhesus Macaques with Simian Immunodeficiency Virus-Loaded Dendritic Cells or T Cells but Not with Cell-Free Virus J. Virol., September 3, 2002; 76(19): 9787 - 9797. [Abstract] [Full Text] [PDF]
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M. I. Zimmer, A. T. Larregina, C. M. Castillo, S. Capuano III, L. D. Falo Jr, M. Murphey-Corb, T. A. Reinhart, and S. M. Barratt-Boyes Disrupted homeostasis of Langerhans cells and interdigitating dendritic cells in monkeys with AIDS Blood, April 15, 2002; 99(8): 2859 - 2868. [Abstract] [Full Text] [PDF]
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C. D. Richters, I. Mayen, C. E. G. Havenith, R. H. J. Beelen, and E. W. A. Kamperdijk Rat monocyte-derived dendritic cells function and migrate in the same way as isolated tissue dendritic cells J. Leukoc. Biol., April 1, 2002; 71(4): 582 - 587. [Abstract] [Full Text] [PDF]
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A. E. Chang, B. G. Redman, J. R. Whitfield, B. J. Nickoloff, T. M. Braun, P. P. Lee, J. D. Geiger, and J. J. Mule A Phase I Trial of Tumor Lysate-pulsed Dendritic Cells in the Treatment of Advanced Cancer Clin. Cancer Res., April 1, 2002; 8(4): 1021 - 1032. [Abstract] [Full Text] [PDF]
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I. Frank, M. Piatak Jr., H. Stoessel, N. Romani, D. Bonnyay, J.D. Lifson, and M. Pope Infectious and Whole Inactivated Simian Immunodeficiency Viruses Interact Similarly with Primate Dendritic Cells (DCs): Differential Intracellular Fate of Virions in Mature and Immature DCs J. Virol., February 22, 2002; 76(6): 2936 - 2951. [Abstract] [Full Text] [PDF]
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 H. Jebbari, A. J. Stagg, R. N. Davidson, and S. C. Knight Leishmania major Promastigotes Inhibit Dendritic Cell Motility In Vitro Infect. Immun., February 1, 2002; 70(2): 1023 - 1026. [Abstract] [Full Text] [PDF]
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J. D. Geiger, R. J. Hutchinson, L. F. Hohenkirk, E. A. McKenna, G. A. Yanik, J. E. Levine, A. E. Chang, T. M. Braun, and J. J. Mule Vaccination Of Pediatric Solid Tumor Patients with Tumor Lysate-pulsed Dendritic Cells Can Expand Specific T Cells and Mediate Tumor Regression Cancer Res., December 1, 2001; 61(23): 8513 - 8519. [Abstract] [Full Text] [PDF]
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D. O'Hagan, M. Singh, M. Ugozzoli, C. Wild, S. Barnett, M. Chen, M. Schaefer, B. Doe, G. R. Otten, and J. B. Ulmer Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines J. Virol., October 1, 2001; 75(19): 9037 - 9043. [Abstract] [Full Text] [PDF]
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T. Takayama, A. E. Morelli, N. Onai, M. Hirao, K. Matsushima, H. Tahara, and A. W. Thomson Mammalian and Viral IL-10 Enhance C-C Chemokine Receptor 5 but Down-Regulate C-C Chemokine Receptor 7 Expression by Myeloid Dendritic Cells: Impact on Chemotactic Responses and In Vivo Homing Ability J. Immunol., June 15, 2001; 166(12): 7136 - 7143. [Abstract] [Full Text] [PDF]
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S. Weijzen, S. C. Meredith, M. P. Velders, A. G. Elmishad, H. Schreiber, and W. M. Kast Pharmacokinetic Differences Between a T Cell-Tolerizing and a T Cell-Activating Peptide J. Immunol., June 15, 2001; 166(12): 7151 - 7157. [Abstract] [Full Text] [PDF]
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L. A. Harshyne, S. C. Watkins, A. Gambotto, and S. M. Barratt-Boyes Dendritic Cells Acquire Antigens from Live Cells for Cross-Presentation to CTL J. Immunol., March 15, 2001; 166(6): 3717 - 3723. [Abstract] [Full Text] [PDF]
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P. Hjelmström Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines J. Leukoc. Biol., March 1, 2001; 69(3): 331 - 339. [Abstract] [Full Text]
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S. Dunzendorfer, A. Kaser, C. Meierhofer, H. Tilg, and C. J. Wiedermann Cutting Edge: Peripheral Neuropeptides Attract Immature and Arrest Mature Blood-Derived Dendritic Cells J. Immunol., February 15, 2001; 166(4): 2167 - 2172. [Abstract] [Full Text] [PDF]
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J. J. Campbell, K. E. Murphy, E. J. Kunkel, C. E. Brightling, D. Soler, Z. Shen, J. Boisvert, H. B. Greenberg, M. A. Vierra, S. B. Goodman, et al. CCR7 Expression and Memory T Cell Diversity in Humans J. Immunol., January 15, 2001; 166(2): 877 - 884. [Abstract] [Full Text] [PDF]
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