| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,§,§
Departments of
*
Medicine,
Pathology, and
Microbiology, University of Texas Health Science Center, San Antonio, TX 78284;
§
South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, TX 78284; and
¶
Infectious Diseases Service, Department of Medicine, Wilford Hall Medical Center, Lackland Air Force Base, TX 78236
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and increased the percentage of
activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by
retroviral gene transfer techniques to secrete high levels of
biologically active murine IL-12 augmented this immune response
further. In several different vaccination and immunotherapy protocols,
compared with sham-treated mice, animals receiving SLDA-pulsed DCs
either before or following infection had 1–3 log lower parasite
burdens, and this protection was associated with a pronounced
enhancement in the parasite-specific IFN- response. The augmentation
of this protection by IL-12-engineered DCs was striking. First, live
parasites were not detected in the liver of mice vaccinated with
IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological
response was associated with a nearly normal liver histology. In
contrast, parasites and granulomas were found in mice vaccinated with
SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the
rationale for the development of potent DC-based
immunotherapies. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We also tested the hypothesis that DCs engineered for the paracrine
delivery of gene products critical for the development of cell-mediated
immunity will amplify the protective immune responses induced by
microbial Ag-pulsed DCs. The molecular engineering of physiologically
relevant immune responses is an attractive approach to enhancing
DC-based antimicrobial immune responses, especially cell-mediated
immune responses that are critical for the control of several
intracellular infections. In this context, the cytokine IL-12 is an
attractive candidate for paracrine delivery via DCs. IL-12 is a key
link between the innate and cell-mediated Ag-specific immune responses
(8, 9, 10). In its biologically active form IL-12 is secreted
as a heterodimeric protein (p70) and the major source is DCs and other
APCs (8, 9, 10). IL-12 induces IFN- production from NK
cells and T lymphocytes, and enhances proliferation of Ag-specific T
cells. The IFN- produced by effector cells such as
CD4+ Th1 plays an important role in protection
against intracellular pathogens, whereas Th2 cells defined by their
ability to produce IL-4 but not IFN- play an important role in the
defense against extracellular organisms. This Th1/Th2 paradigm of
acquired immunity has been demonstrated in several animal models of
fungal, bacterial, parasitic, and retroviral infections emphasizing the
potential of cytokine/anticytokine therapy for recruiting
infection-specific protective Th cell responses (4, 5, 6, 11, 12). While certain aspects of this Th1/Th2 paradigm have yet to
be established in humans, there is clear evidence that IFN-
production (13), IL-12 production (14),
IFN- binding (15, 16, 17, 18), IL-12 binding (19, 20), and IFN- signaling (21) all play critical
roles in the control of intracellular infections in humans.
There are few studies that have explored the in vivo efficacy of microbial Ag-pulsed DCs in an infectious disease setting, and to our knowledge there are no studies that have evaluated the role of IL-12-transduced DCs (22, 23, 24, 25, 26, 27). This is in contrast to the cancer field where several studies in mouse models (28, 29, 30, 31, 32, 33) as well as in humans (28, 29, 30, 31, 32, 33, 34, 35, 36) have documented the efficacy of adoptive transfer of tumor Ag-pulsed DCs. To test our hypotheses, we created a retroviral-based gene transfer system to introduce IL-12 into DCs. As a model system of infection by an intracellular organism, we chose murine infection with Leishmania donovani, the causative agent for visceral leishmaniasis. This murine model mimics infection by L. donovani in humans and control of infection is known to be dependent, in part, on Th1 cell responses (37, 38). To date, effective vaccine Ags for L. donovani have not been identified. Pulsing DCs with live organisms is not an optimal choice when testing a potential vaccine or therapy. We therefore chose a mixture of unfractionated L. donovani-derived proteins (soluble L. donovani Ag (SLDA)) as a source of microbial Ags to load DCs (39). In this system, we demonstrate first that adoptive transfer of microbial Ag-pulsed DCs induces an Ag-specific Th1 response in vivo. Second, microbial Ag-pulsed DCs administered either before or following infection with L. donovani were effective in reducing the parasite burden. Third, physiologically relevant anti-infective immune responses initiated by microbial Ag-pulsed DCs can be amplified by paracrine delivery of IL-12. Finally, the anti-infective potency of microbial Ag-pulsed DCs that have been engineered to secrete high levels of IL-12 is significantly greater than that of nonengineered DCs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
L. donovani 1S strain (MHOM/SD/001S-2D) promastigotes were cultured, and SLDA was prepared as described previously (38). Stationary phase L. donovani promastigotes were inactivated by heating at 55°C for 60 min, and DCs were pulsed with heat-inactivated L. donovani at a multiplicity of infection of 10:1. Total RNA was isolated from the stationary phase L. donovani promastigotes using the RNeasy kit (Invitrogen, San Diego, CA) according to manufacturer’s instructions. For pulsing DCs with L. donovani-derived RNA, the DCs (2.5 x 106 cells/ml) were cultured in 250 µl of serum free Opti-MEM medium (Life Technologies, Gaithersburg, MD) containing 25 µg of RNA and 50 µg of N-[1-(2,3-dioleoxyloxy)propyl] N,N,N-trimethyl ammonium methylsulfate (DOTAP, Boehringer, Mannheim, Indianapolis, IN) (32). nu+/BALB/c background mice were obtained from the Veterinary Medical Unit breeding colony, Audie Murphy Veterans Affairs Hospital (San Antonio, TX). The phenotype of nu+/BALB/c mice is identical to that of BALB/c mice. Six-week-old mice were used for all studies. Mice were infected by i.v. (tail vein) injection of 0.5–1 x 107 stationary phase promastigotes.
Enrichment of DCs
Bone-marrow derived DCs were generated according to published methods (40). The femurs and tibias were flushed with 3–5 ml of PBS in 1% BSA. Particulate matter was filtered and the RBC were lysed. Bone marrow cells were differentiated into DCs by culturing in RPMI (Life Technologies) supplemented with 10% FCS, 10 µg/ml gentamicin, and recombinant murine cytokines (R & D Systems, Minneapolis, MN) GM-CSF (50 ng/ml) and IL-4 (1 ng/ml) for 7–10 days. On days 3 and 5, the nonadherent cells (granulocytes and lymphocytes) were removed and replaced with fresh medium and growth factors. On day 7, the nonadherent cells were removed and plated in either 24- or 96-well plates or TissueTek (Nunc, Naperville, IL) chamber slides for pulsing studies. In five separate experiments, bone marrow-derived DCs were stained and analyzed by FACS using DC, monocyte, and lymphocyte cell surface markers. Phenotypically bone marrow-derived DCs expressed abundant MHC class II, CD80, CD40, CD11b, NLDC145, and CD11c (data not shown). In proliferation assays, there was an increase in thymidine incorporation when SLDA-pulsed DCs were cocultured with lymphocytes derived from lymph nodes/spleens of L. donovani-infected mice, indicating that the DCs can present Ags efficiently (data not shown). Thus, the phenotypic and functional characteristics of bone marrow-derived DCs were consistent with previously published data (40).
Flow cytometry
Flow cytometry was used to define the phenotypic characteristics of DCs differentiating from bone marrow progenitors. All Abs were obtained from PharMingen (San Diego, CA) except for NLDC145 (rat anti-mouse IgG) which was from Caltag (Burlingame, CA). Consistent with other studies, DCs were identified by presence of abundant MHC class II (I-Ad haplotype), NLDC145, CD40, CD11c, CD11b, and CD86 staining, and the absence of CD3/CD2 (T cell) and B220 (B cell) staining. T lymphocyte activation in the splenocytes was confirmed by double staining for lymphocyte subsets CD4 or CD8 with CTLA-4, CD40 ligand, or CD28.
Transduction of DCs and retroviral supernatant generation
Using standard overlap PCR techniques, the two murine IL-12
subunits (cDNAs for p35 and p40 were a kind gift of Dr. U. Gubler,
Hoffmann-LaRoche, Nutley, NJ) were linked together by an amino acid
polypeptide linker and amplified as a single amplicon
(41). This fusion construct was sequenced on both strands
and then cloned into the replication-incompetent retroviral vector,
designated as MFGS. MFGS is derived from Moloney murine leukemia virus
(42, 43). The transfection/transduction protocol was as
described (42, 43). Briefly, DNA from the MFGS-murine
IL-12 (36 µg) and the SV2Neo (4 µg) constructs were transfected
into a producer packaging cell line 
cre by calcium phosphate
precipitation. The SV2Neo construct contains the neomycin-resistant
gene. Single cell colonies that were resistant to neomycin selection
were picked. Supernatants from these clones contain the retrovirus and
murine IL-12 (p70) and were used to transduce NIH-3T3 cells.
Supernatants that conferred the NIH-3T3 cells with the highest murine
IL-12 production (>20 ng/ml/24h as measured by ELISA) after one 6-h
transduction were identified as high titer clones. The sandwich ELISA
(PharMingen) used measures p(70), the bioactive form of murine IL-12.
The high titer clone was expanded, and supernatants from this clone
were frozen in aliquots of 3 ml. For transduction, the retrovirus
supernatants were thawed and DCs were transduced in fresh medium
containing 6 µg/ml polybrene (Sigma, St. Louis, MO) and growth
factors GM-CSF (50 ng/ml) and IL-4 (1 ng/ml) diluted 1:1 with viral
supernatant. DCs were transduced for 6 h on 2 or 3 consecutive
days. IL-12 levels were measured in the supernatants from DC cultures
48 h following the last transduction.
ELISA and in vitro spleen responses
Splenocytes (5 x 106/ml) were
cultured in medium alone or stimulated with 25 µg/ml of SLDA, and
supernatants were harvested at 24 and 48 h for analysis of IFN-
and IL-4 concentrations by a sandwich ELISA (PharMingen). ELISA was
used to analyze the serum Ig subtypes (PharMingen). The levels were
quantified by comparing the optical density (OD) of the sample to the
OD of known standards analyzed simultaneously in the same assay. The
cut-off for ELISA assay was 7.5–500 pg/ml for IL-4 and 15.6–500 pg/ml
for IFN-. To ensure that the OD values fall within the standard
curve, the samples for IFN- were diluted 1:10 and 1:100.
Vaccination and immunotherapy studies
DC preparations were divided into two parts, one of which was pulsed with SLDA (cocultured with 25 µg/ml of SLDA for 12 h) and the other was used as an unpulsed control. These two DC preparations were aliquoted and then injected i.v. into mice. In all studies, DC preparations (unpulsed DCs, SLDA-pulsed DCs, IL-12-transduced DCs, or IL-12-transduced, SLDA-pulsed DCs) were washed several times in PBS, and 106 DCs were resuspended in a 100 µl volume for tail vein injection at the time points specified. The Animal Committee of the University of Texas Health Science Center, San Antonio, TX, approved these studies.
Parasite burden
At various time points following infection, the parasite burden was quantified in the liver and/or spleen by limiting-dilution culture as described previously (38, 44). Briefly, liver and spleen tissue (20 mg) was homogenized between two sterile frosted end microscopic slides in 2 ml of culture medium (Graces’s insect medium enriched with 15% heat inactivated FBS). The homogenate was resuspended at 1 mg/ml and placed in 96-well flat-bottom plates in 5-fold serial dilutions and cultured for 2 wk. The wells were then scored for presence of parasites, and the reciprocal of the dilution of the final positive well was expressed as log tissue parasite burden per mg of tissue.
PCR
Genomic DNA was isolated from the spleen of each mouse, and serial 10-fold dilutions were used for PCR. The PCR primers specific to Leishmania species (forward primer, 5'-GTG GGG GAG GGG CGT TCT-3'; reverse primer, 5'-ATT TTA CAC CAA CCC CAG TT-3') amplify a 125-bp DNA fragment (45). The PCR products were analyzed on an ethidium bromide-stained, 2% agarose gel.
Histopathology
Slides of paraffin-embedded livers were stained with hematoxylin and eosin and examined for the presence, pattern, distribution, and cellular composition of inflammatory cell infiltrates within the section of liver. The slides were examined at x200 and the area within the gridlines indicating the area captured by the 35-mm Mot DX film cassette (Zeiss, New York, NY) were evaluated. Six to eight nonoverlapping microscopic fields were used to collect data. The inflammatory infiltrates (equivalent to granuloma) were characterized as either compact cluster of cells, or loosely arranged in which case the edge of the infiltrates were indistinct. The inflammatory infiltrates were scored based on the cellular mixture present and whether they contained multinucleated giant cells, lymphocytes, eosinophils, plasma cells, and intracellular parasites. The latter determination was at x400. The number of inflammatory infiltrates (granulomas) within the frame-size rectangle was counted and used in combination with the above findings to assign a grade to the lesions. Based on the number of inflammatory infiltrates observed, the sections were graded on a scale of 3+ to 0, with 0 representing fields without granulomas. The pathologist was blinded with regards to the identity of the source (vaccine groups) of the sections.
Statistical methods
All animal experiments used 4–10 animals per group and were repeated at least twice with similar results. Quantitative results are expressed as mean ± SEM. Parasite tissue burden data were compared using a Mann-Whitney U test. In vitro cytokine levels for paired SLDA-pulsed samples and unpulsed controls were compared with a paired-sample t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For paracrine delivery of IL-12, we generated a replication-incompetent retrovirus encoding the two subunits of murine IL-12 as a fusion construct. Following three transductions, the range of IL-12 detected in the supernatants of DC cultures was 6–32 ng/ml (n = 14 transductions). These levels were significantly higher than that detected in the supernatants of nontransduced, nonstimulated murine DCs (10–400 pg/ml; n = 20 transductions). The average daily rate of bioactive IL-12 produced by 106 DCs following two or three transductions was 3305 ± 399 pg and 6578 ± 437 pg, respectively. The protocol used to generate DCs from bone marrow cells results in the generation of a mixture of APCs, with the predominant subtype being DCs. Because there is no specific marker for DCs, we did not attempt to quantify the percentage of DCs that was transduced with IL-12. However, considering the high levels of IL-12 detected in the supernatants of transduced DCs, the transduction efficiency of this retroviral gene transfer system is likely to be very high.
Phenotypically, IL-12-transduced DCs and nontransduced DCs
expressed similar amounts of CD11b, CD11c, MHC I and II, the
costimulatory molecules CD54, CD80, and the DC marker NLDC145 (Fig. 1
and data not shown). However, the
expression of CD40 and CD86 was significantly higher in the
IL-12-transduced DCs (Fig. 1). Based on this expression pattern, it is
likely that the IL-12-transduced DCs contain a mixture of mature (CD86
and MHC class II positive) and immature of DCs. The percentage of
macrophages (N418 positive and NLDC145 negative) was similar in the
transduced and nontransduced DC groups. Pulsing IL-12-transduced DCs
with different forms of Leishmania-derived Ags did not alter
their ability to produce high levels of IL-12 (Fig. 2). Even after pulsing with
Leishmania-derived Ags (Fig. 2) or following in vitro
infection with live L. donovani (data not shown), the amount
of IL-12 produced by nontransduced DCs never reached the level produced
by IL-12-transduced DCs.
|
|
We first examined the function of microbial Ag-pulsed DCs after
adoptive transfer to naive mice. An Ag-specific IFN- response was
observed in splenocytes derived from mice that received SLDA-pulsed DCs
(Fig. 3, lanes 5 and
6). This response was observed as early as 2 days following
adoptive transfer of SLDA-pulsed DCs, peaked at day 14, declined by day
42, and was absent at day 180 (Fig. 3, b–d, compare
lanes 5 and 6; and data not shown). Notably, the
response at day 42 was greater than that observed at day 2 but less
than that at day 14. Unpulsed DCs or PBS did not induce an Ag-specific
IFN- response (Fig. 3, b–d, lanes 1–4). In
an additional control, spontaneous production or an Ag-induced IFN-
response was not detected in splenocytes derived from mice that
received i.v. SLDA alone but no DCs (n = 4
experiments).
|
from splenocytes derived from mice
that received unpulsed but IL-12-transduced DCs was high, and IFN-
levels did not increase following the ex vivo pulsing of the
splenocytes with SLDA (Fig. 3
, b–d, lanes 7 and
8). This spontaneous production of IFN- was evident at
both day 2 and 14 following adoptive transfer of IL-12-transduced DCs
but was absent at day 42. High spontaneous IFN- production levels
were also observed at day 2 and day 14 following adoptive transfer of
IL-12-transduced, SLDA-pulsed DCs (Fig. 3, b–c, lanes
9 and 10). However, at day 42 a different pattern
was observed. At this time point there was essentially no spontaneous
production of IFN- and a clear Ag-specific IFN- response was
evident (Fig. 3d, lanes 9 and 10).
Also, the magnitude of the Ag-specific IFN- response following
adoptive transfer of IL-12-transduced DCs was greater than that of
nontransduced DCs (Fig. 3d, compare lanes 6 and
10). Fourteen days following adoptive transfer of unpulsed or pulsed DCs, low levels of IL-4 were detected in splenocyte culture supernatants (30–100 pg/ml, no differences among groups; data not shown). IL-4 was not detected in splenocyte culture supernatants of mice sacrificed 2 and 42 days post-DC administration or in supernatants from splenocytes derived from mice that received either PBS or SLDA.
The findings presented demonstrate that microbial Ag-pulsed DCs induced
an Ag-specific IFN- response and that this response was augmented
significantly by DCs that had been transduced in vitro with IL-12 (Fig. 3). IFN- is known to induce a switch in Ig isotypes with a
preferential production of IgG2a and IgG3. Concordant with this
observation, we found that adoptive transfer of IL-12-transduced,
SLDA-pulsed DCs resulted in high serum levels of IgG2a. The Ig2a levels
were nearly 2-fold higher than those detected in mice receiving
SLDA-pulsed DCs or PBS. Adoptive transfer of IL-12-transduced and
nontransduced DCs also lead to changes in other Ig subtypes (data not
shown). However, these differences were not as striking as those
observed in IgG2a following administration of IL-12 transduced
DCs.
Compared with mice that received PBS, the activation markers CTLA-4 and
CD40 ligand were up-regulated (
200–500%) on both CD4 and CD8 T
cell splenocytes derived from mice sacrificed 14 days following
adoptive transfer of DCs. However, there were no statistically
significant differences among the various DC groups (unpulsed DCs,
SLDA-pulsed DCs, IL-12-transduced DCs, and SLDA-pulsed/IL-12-transduced
DCs; data not shown). These findings suggest that the adoptive transfer
of DCs may lead to the activation of both CD4+
and CD8+ T lymphocytes.
Microbial Ag-pulsed DCs are effective as both a vaccine and therapy
We determined next the anti-infective efficacy of SLDA-pulsed
DCs in a well-characterized murine model of visceral leishmaniasis
(38, 39). The major endpoint for protective efficacy in
our study was the level of parasite burden in the liver and/or spleen.
Compared with PBS-vaccinated mice, those vaccinated i.v. with
SLDA-pulsed DCs had an 1–3 log reduction in parasite burdens (Table I). In the first vaccination protocol
(Table I), the protective efficacy of SLDA-pulsed DCs was also observed
at 8 wk postinfection (
1 log reduction in parasite burden; data not
shown). A variable effect was observed with unpulsed DCs. In some
experiments the parasite burden following vaccination with unpulsed DCs
was similar to that of the PBS-control vaccinated group. In others, a
decline in parasite burden was observed, albeit the decrease was not
statistically different from those of the control PBS-vaccinated group
(Table I and data not shown). Two vaccinations spaced 2 wk apart with
Ag alone (SLDA) or with Ag plus an adjuvant (Cornybacterium
parvum) was also ineffective (data not shown). We also determined
whether vaccination by the s.c. route was efficacious. Mice vaccinated
by the s.c. route and then challenged with parasites i.v. had no
demonstrable protection (10 mice in vaccine and nonvaccinated
groups).
|
production following parasite
challenge was significantly higher in mice vaccinated with SLDA-pulsed
DCs than in infected, PBS-vaccinated mice (Table II). The Ag-independent production of
IL-4 following infectious challenge of PBS-vaccinated mice was
abrogated by prior vaccination with SLDA-pulsed DCs (Table II).
|
). In a different treatment protocol, starting
1 wk following infection mice were treated weekly for 3 wk. Four weeks
following the last treatment, mice treated with SLDA-pulsed DCs had 1
log lower parasite burdens in the liver and spleen (data not
shown).
Our in vivo studies in noninfected mice showed that IL-12-transduced
SLDA-pulsed DCs enhanced significantly the Ag-specific increases in
IFN-. We therefore next determined whether the protective effect of
Ag (SLDA)-pulsed DCs could be enhanced further by DCs engineered to
secrete high levels of IL-12. Parasites could not be detected in the
liver of mice that had received either one or two vaccinations with
IL-12-transduced, SLDA-pulsed DCs, whereas parasites could be detected
in mice vaccinated with SLDA-pulsed DCs or IL-12 transduced DCs (Table III and data not shown). These findings
were corroborated by a blinded histopathological analysis of the
sections from the liver of mice vaccinated with DCs (see Fig. 5 and
Table IV). Compared with the mice that
received PBS (Fig. 4A), those
vaccinated with SLDA-pulsed DCs had lower numbers of granulomas/cell
nests, giant cells, and organisms and the size of the cell nests was
also smaller (Fig. 4B). In contrast, IL-12-transduced,
SLDA-pulsed DCs had a virtually normal liver histology except for rare
granulomas identified after scanning many fields (Fig. 4C).
|
|
|
|
, lanes 5–8) or
IL-12-transduced, SLDA-pulsed DCs (Fig. 5, lanes 9–12).
However, even by this highly sensitive technique, mice vaccinated with
IL-12-transduced, SLDA-pulsed DCs had at least one log lower amounts of
L. donovani genomic DNA than mice vaccinated with
SLDA-pulsed DCs. The limitation of this PCR-based assay is that we
cannot distinguish whether the genomic DNA detected was from viable or
nonviable parasites. However, the data shown strongly suggest that the
DNA amplified is likely to be from either nonviable parasites or from a
very low level of residual infection. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In recent years, there has been a major thrust toward developing DNA vaccines for infectious diseases (48, 49). However, an alternative, and perhaps more physiologic, strategy could be one based on capitalizing on the biology of DCs. For example, because DCs rapidly home to organs of immune response, such as the liver and spleen (50, 51, 52), the protective responses induced might be generated more rapidly than those induced by DNA-based vaccines. This may be especially advantageous in the setting of an established infection that is progressing despite therapy. The scenario of relentless disease progression despite optimal therapy is seen on occasion in coccidioidomycosis and leishmaniasis. With tuberculosis, the now endemic presence of multiple drug-resistant strains makes the development of alternative therapies an urgent problem. In the context of an epidemic where the rapid induction of protective immune responses is critical, this empiric approach to a difficult vaccine development problem could be an effective initial intervention. Furthermore, in many instances the infectious agent responsible for an epidemic or an unresponsive infection is a mutant strain that has a phenotype distinct from conventional laboratory or vaccine strains. In this instance, pulsing DCs with microbial Ags derived from the mutant microbial strain may be especially advantageous because microbe-specific responses would be generated. Study of these protective responses may in turn provide insight into important immunoprotective factors that could be applied to conventional vaccines.
A major limitation of the DNA vaccine approach is that the identity of the microbial Ag(s) must be known. In the case of most intracellular microbes, their genomes are very large, and the task of identifying protective Ags is arduous. Furthermore, it is likely that a broad repertoire of microbial Ags is required to induce a potent protective immune response in vivo. Thus, the administration of DCs pulsed with unfractionated pools of microbial Ags has the advantage of inducing a protective polyclonal T cell response directed against yet-to-be identified Ags.
One of the major concepts in cytokine biology is that their activity is most potent when they are expressed in a paracrine fashion, i.e., at the site of the Ag. Cytokines differ from hormones in that they are often secreted locally and usually provide local paracrine effects rather than systemic effects. For this reason, treating patients using systemically administered cytokines may not be the most appropriate method of optimizing local Ag presentation and effector cell function at the sites where they are required. As DCs occupy such a pivotal position in the initiation of the immune response, it seems logical to target these cells for cytokine delivery. Gene transfer techniques have been used for over a decade to deliver high-level expression of cytokines and other gene products within the tumor microenvironment (53). However, the same techniques are applicable for targeting DCs to provide enhanced expression of the cytokine/chemokine of interest in the environment of Ag presentation within secondary lymphoid tissue. Conceivably, one could develop inducible-repressible promoter systems that may allow gene expression to be induced after the DC has entered secondary lymphoid tissue.
In addition to harnessing their potent Ag-presenting properties, we also targeted DCs for the paracrine delivery of IL-12 with the intent of priming the cellular microenvironment to induce a more potent response. Given the central role of IL-12 in the promotion of Th1-type cellular immunity (8, 9), IL-12 delivered directly within sites of active T-cell induction is an attractive approach in the management of serious intracellular infections. Furthermore, the efficacy of IL-12 to serve as an effective biologic adjuvant in DNA and recombinant vaccine formats reinforces the importance of this Th-1 biasing cytokine (8, 9, 54). The efficacy of IL-12 in augmenting the Ag-specific Th1 responses induced by microbial Ag-pulsed DCs in vivo is in agreement with our previous in vitro studies that showed the potential of IL-12-transduced human DCs in inducing Ag-specific Th1 responses and down-modulating a Th2 response (43).
In this study, unpulsed DCs showed some protection. However, in
contrast to vaccination or treatment with SLDA-pulsed DCs, the
reduction in parasite burden following vaccination or treatment with
unpulsed DCs was not statistically significant and the response was not
always reproducible. This effect of unpulsed DCs could be due to the
nonspecific induction of IFN- as well as the activation and
maturation of DCs in vivo. Alternatively, it is conceivable that
following adoptive transfer, unpulsed DCs process and present the Ags
presented to them in vivo, i.e., the microbial Ags of an infectious
challenge. Conceivably, the nonspecific effects of DC administration
might become less apparent if the challenge infection were given after
a longer interval than 2 wk after vaccination. Ongoing experiments are
addressing this issue as well as the durability of the protection.
Nonspecific effects of DCs have been observed in other systems also.
Shimizu et al. (55) recently showed that immunization with
DCs break the cytotoxic T lymphocyte tolerance in hepatitis B virus
transgenic mice, and was found to be more efficient than DNA
immunization in this setting.
In preliminary experiments, we found that the parasite burden in
animals vaccinated s.c. with Ag-loaded DCs was similar to that of
control mice vaccinated with PBS, and for this reason we chose to
vaccinate mice via the i.v. route. The lack of a protective response
observed in mice vaccinated with the subcutaneous route is in keeping
with the findings of Kuribayashi et al. (52). They found
that the Th1/Th2 cytokine profiles induced following subcutaneous
administration of Ag-pulsed DCs were different from those induced by
DCs administered i.v. The IL-4 levels (protein and RNA) in the spleen
cells of mice were higher in mice that received DCs s.c. Similarly, the
mRNA levels of IL-4 and IL-5 in the lymph nodes cells was higher in
mice that received DCs s.c. IFN- protein and mRNA levels were
similar following either route of DC administration. Taken together, it
appears that s.c. administration of DCs induces a dominant Th2
phenotype. In agreement with Kuribayashi et al. (52), we
show that i.v. injection of DCs induces a dominant Th1 phenotype.
Furthermore, we show that this phenotype is associated with a reduction
in parasite burden.
Despite its appeal, several limitations exist in the use of microbial Ag-pulsed and/or cytokine-transduced autologous DCs in the management of infectious diseases. First, although the techniques for generating large numbers of autologous DCs are in place, the process is laborious and expensive. This limits the ready use of DC-based anti-infective therapy in a developing nation where infections with intracellular pathogens such as Leishmania or M. tuberculosis are common. Second, such an approach may not be feasible in a large-scale setting because the DCs would have to be isolated from individual patients or the naive host. Nevertheless, we have outlined several clear-cut situations where the future use of DC-based anti-infective strategies could be highly beneficial.
In summary, DC-based anti-infective vaccines and therapies comprise a novel yet nascent and embryonic field. Despite potential limitations, our findings provide additional rationale for the future development of DC-based strategies for the prevention and treatment of serious intracellular infections. We also demonstrate that the magnitude and kinetics of the immune response to Ag-pulsed DCs can be enhanced by the paracrine delivery of IL-12 via DCs.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Seema S. Ahuja, Department of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7870. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; SLDA, soluble Leishmania donovani Ag.
Received for publication May 25, 1999. Accepted for publication July 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
: a preliminary report. N. Engl. J. Med. 330:1348. production in familial disseminated Mycobacterium avium complex infection: abnormal IL-12 regulation. J. Immunol. 157:411.[Abstract]
-receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335:1941.-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. N. Engl. J. Med. 335:1956.-receptor-1-deficient children. J. Interferon Cytokine Res. 17:583.[Medline]
receptor deficiency. Clin. Infect. Dis. 24:982.[Medline]
receptor and susceptibility to mycobacterial infection. J. Clin. Invest. 101:2364.[Medline]
: effect of interleukin-12 in experimental visceral leishmaniasis in interferon- gene-disrupted mice. J. Exp. Med. 185:1231. gene transfer. J. Immunol. 156:4345.[Abstract]
and IL-12. J. Immunol. 161:868.This article has been cited by other articles:
|
|
 |
|
  S. McCormick, M. Santosuosso, C.-L. Small, C. R. Shaler, X. Zhang, M. Jeyanathan, J. Mu, S. Takenaka, P. Ngai, J. Gauldie, et al. Mucosally Delivered Dendritic Cells Activate T Cells Independently of IL-12 and Endogenous APCs J. Immunol., August 15, 2008; 181(4): 2356 - 2367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Basu, S. Bhaumik, A. K. Haldar, K. Naskar, T. De, S. K. Dana, P. Walden, and S. Roy Hybrid Cell Vaccination Resolves Leishmania donovani Infection by Eliciting a Strong CD8+ Cytotoxic T-Lymphocyte Response with Concomitant Suppression of Interleukin-10 (IL-10) but Not IL-4 or IL-13 Infect. Immun., December 1, 2007; 75(12): 5956 - 5966. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maurer, S. L. Kostka, F. Siebenhaar, K. Moelle, M. Metz, J. Knop, and E. von Stebut Skin mast cells control T cell-dependent host defense in Leishmania major infections FASEB J, December 1, 2006; 20(14): 2460 - 2467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Woelbing, S. L. Kostka, K. Moelle, Y. Belkaid, C. Sunderkoetter, S. Verbeek, A. Waisman, A. P. Nigg, J. Knop, M. C. Udey, et al. Uptake of Leishmania major by dendritic cells is mediated by Fc{gamma} receptors and facilitates acquisition of protective immunity J. Exp. Med., January 23, 2006; 203(1): 177 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Buettner, C. Meinken, M. Bastian, R. Bhat, E. Stossel, G. Faller, G. Cianciolo, J. Ficker, M. Wagner, M. Rollinghoff, et al. Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells J. Immunol., April 1, 2005; 174(7): 4203 - 4209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kuipers, C. Heirman, D. Hijdra, F. Muskens, M. Willart, S. van Meirvenne, K. Thielemans, H. C. Hoogsteden, and B. N. Lambrecht Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization J. Leukoc. Biol., November 1, 2004; 76(5): 1028 - 1038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ghosh, C. Pal, M. Ray, S. Maitra, L. Mandal, and S. Bandyopadhyay Dendritic Cell-Based Immunotherapy Combined with Antimony-Based Chemotherapy Cures Established Murine Visceral Leishmaniasis J. Immunol., June 1, 2003; 170(11): 5625 - 5629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nakamura, T. Suda, T. Nagata, T. Aoshi, M. Uchijima, A. Yoshida, K. Chida, Y. Koide, and H. Nakamura Induction of Protective Immunity to Listeria monocytogenes with Dendritic Cells Retrovirally Transduced with a Cytotoxic T Lymphocyte Epitope Minigene Infect. Immun., April 1, 2003; 71(4): 1748 - 1754. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. A. Ansari, A. E. Mayne, J. B. Sundstrom, P. Bostik, B. Grimm, J. D. Altman, and F. Villinger Administration of Recombinant Rhesus Interleukin-12 during Acute Simian Immunodeficiency Virus (SIV) Infection Leads to Decreased Viral Loads Associated with Prolonged Survival in SIVmac251-Infected Rhesus Macaques J. Virol., February 15, 2002; 76(4): 1731 - 1743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. W. Murray Clinical and Experimental Advances in Treatment of Visceral Leishmaniasis Antimicrob. Agents Chemother., August 1, 2001; 45(8): 2185 - 2197. [Full Text] [PDF]
|
|
|
|
 |
|
 E. Handman Leishmaniasis: Current Status of Vaccine Development Clin. Microbiol. Rev., April 1, 2001; 14(2): 229 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Demangel, U. Palendira, C. G. Feng, A. W. Heath, A. G. D. Bean, and W. J. Britton Stimulation of Dendritic Cells via CD40 Enhances Immune Responses to Mycobacterium tuberculosis Infection Infect. Immun., April 1, 2001; 69(4): 2456 - 2461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Stager, D. F. Smith, and P. M. Kaye Immunization with a Recombinant Stage-Regulated Surface Protein from Leishmania donovani Induces Protection Against Visceral Leishmaniasis J. Immunol., December 15, 2000; 165(12): 7064 - 7071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Melby, G. B. Ogden, H. A. Flores, W. Zhao, C. Geldmacher, N. M. Biediger, S. K. Ahuja, J. Uranga, and M. Melendez Identification of Vaccine Candidates for Experimental Visceral Leishmaniasis by Immunization with Sequential Fractions of a cDNA Expression Library Infect. Immun., October 1, 2000; 68(10): 5595 - 5602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Quinones, S. K. Ahuja, P. C. Melby, L. Pate, R. L. Reddick, and S. S. Ahuja Preformed Membrane-Associated Stores of Interleukin (Il)-12 Are a Previously Unrecognized Source of Bioactive IL-12 That Is Mobilized within Minutes of Contact with an Intracellular Parasite J. Exp. Med., August 21, 2000; 192(4): 507 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sato, S. K. Ahuja, M. Quinones, V. Kostecki, R. L. Reddick, P. C. Melby, W. A. Kuziel, and S. S. Ahuja Cc Chemokine Receptor (Ccr)2 Is Required for Langerhans Cell Migration and Localization of T Helper Cell Type 1 (Th1)-Inducing Dendritic Cells: Absence of Ccr2 Shifts the Leishmania major-Resistant Phenotype to a Susceptible State Dominated by Th2 Cytokines, B Cell Outgrowth, and Sustained Neutrophilic Inflammation J. Exp. Med., July 17, 2000; 192(2): 205 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Sato, W. A. Kuziel, P. C. Melby, R. L. Reddick, V. Kostecki, W. Zhao, N. Maeda, S. K. Ahuja, and S. S. Ahuja Defects in the Generation of IFN-{gamma} Are Overcome to Control Infection with Leishmania donovani in CC Chemokine Receptor (CCR) 5-, Macrophage Inflammatory Protein-1{alpha}-, or CCR2-Deficient Mice J. Immunol., November 15, 1999; 163(10): 5519 - 5525. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
