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Department of Medicine and The Sam and Rose Stein Institute for Research on Aging, University of California at San Diego, La Jolla, CA 92093
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within the dermis, there are a variety of "professional" APCs such as Langerhans cells and macrophages that could be transfected by the injected plasmid DNA (6). Additionally, intradermal (i.d.)3 gene vaccination has been shown to induce expression of the gene product in cells with macrophage and dendritic cell-like morphology as well as keratinocytes and dermal fibroblasts (2). A role for directly transfected Langerhans cells was suggested by the appearance of Ag-expressing dendritic cells in draining lymph nodes after biolistic immunization (7, 8). However, professional APCs are sparse in other tissues such as muscle, although they may be recruited by the local irritation that follows injection (9, 10). Hence, it would be rare that these APCs would become directly transfected by injected DNA.
In tissues that have few resident APCs, the transfected cells may act as an antigenic reservoir. Alternative mechanisms to present exogenous Ags in the context of MHC class I have been reported (11, 12, 13, 14, 15, 16). Consequently, presentation may occur by protein transfer from transfected somatic cells to a professional APC. This potential mechanism is supported by the original cross-priming experiments in which bystander cells were shown to present MHC class I-restricted minor histocompatibility Ags in vivo (17) and in a more recent report in which a test Ag expressed by transfected myoblasts of one haplotype was able to cross-prime a CTL restricted to another MHC haplotype when transferred to F1-recipient mice (18).
Several investigators have been able to isolate dendritic cells after DNA vaccination and demonstrate their ability to present Ag in vitro to hybridomas or cell lines (8, 19, 20). After our original observation that bone marrow-derived cells present plasmid encoded Ag to the immune system (4), we initially hypothesized that these directly transfected dendritic cells were largely responsible for priming the subsequent immune responses. In this paper, we present our surprising results that although directly transfected dendritic cells are potent APCs, the magnitude of the immune response is dependent on Ag produced by transfected nonlymphoid cells.
We designed parallel avenues of investigation to determine the relative contributions of Ag produced by connective tissue or by bone marrow-derived cells after naked plasmid DNA injection. First, we used a plasmid with a monocyte-specific promoter to determine whether directly transfected APCs of monocytic cell lineage were expressing immunogenic Ag (21). Poor humoral and CTL responses were elicited after injection with this plasmid, indicating that this attempt at targeting expression to the APC did not produce the usual response.
Second, to evaluate whether transfection of migratory cells or cells distant from the site of injection was necessary for immune recognition of antigenic protein, we used an expression suppressible plasmid system to separate transport of the plasmid vs protein from the site of injection (22, 23). Mice lacking B and T cells were immunized with and without the suppressing drug. Splenocytes (APCs) from these mice were injected into recipient mice and assessed for their ability to prime a CTL response. Recipient mice that received splenocytes from mice without suppressive treatment mounted a greater CTL response, suggesting that exogenous transfer was important for the magnitude of the response. Additionally, a CTL response was not induced when the site of plasmid injection was ablated by amputation and the suppressing drug subsequently removed. The immunogenic protein was predominantly expressed locally at the site of injection and not in the draining lymph nodes or spleen.
Finally, we made chimeric mice that had either a transgenic transcriptional transactivator in their resident tissues or in their adoptively transferred bone marrow. Wild-type mice with transgenic bone marrow had weaker CTL and humoral responses than transgenic mice with wild-type bone marrow after injection of plasmid DNA that required the transactivator for high levels of expression. These data collectively indicated that nonlymphoid tissues predominantly expressed antigenic peptides encoded by plasmid DNA, which were then transferred to APCs to stimulate the bulk of the immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OVA-tcr-1 mice were a kind gift from M. Bevan (University of Washington, Seattle, WA), and Rag-1-/-, BALB/c, C57BL/6, and B6, C3-TgN(TettTALuc)1Dgs mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C3-TgN(TettTALuc)1Dgs mice express the tetracycline-controlled transactivating protein (tTA) and luciferase (24). OVA-tcr-1 mice are transgenic for a TCR that is specific for the H-2Kb-restricted epitope from OVA, SIINFEKL (25). The mice were bred and maintained under standard conditions in the University of California at San Diego Animal Facility, which is accredited by the American Association of Laboratory Care.
Plasmids
The construction of pACB and pCMVint-based vectors have been previously described (2). The vectors pUHD15-1, encoding the tTA, and pUHD10-3 were generous gifts of H. Bujard (Universität Heidelberg, Heidelberg, Germany) (22). The cDNA for hen egg OVA, a generous gift of N. Shastri (University of California, Berkeley, CA), was subcloned into pUHD10-3 (pUHD-OVA) and pACB (ACB-OVA). ACB-TPASIINFEKL was generated as described (26). A membrane-bound form of OVA fused to the transmembrane terminus of the transferrin receptor (TfROVA, a kind gift of R. Teasdale, Monash University, Melbourne, Australia) (27) was subcloned into the pCMVint vector. The pACB-Z plasmid expressed the Escherichia coli LacZ cDNA (28). The plasmid fXbaSpl-ßgal included the cis-active elements responsible for transcriptional activation of the scavenger receptor and a ß-galactosidase (ß-gal) reporter gene (kind gift of C. Glass, University of California at San Diego) (21). Plasmid DNA for injection was prepared as previously described (4).
Transfer
Adult Rag-1-deficient mice had 15 mg (21 day) slow release tetracycline pellets (Innovative Research of America, Toledo, OH) s.c. implanted 1 day before injection. These mice were injected i.d. in the proximal tail with 50 µg each of pUHD-OVA and pUHD15-1 in 50 µl of normal saline twice, 3 day apart. One week after the first injection, the animals were sacrificed, and 1.3 x 107 splenocytes were transferred i.p. into OVA-tcr-1 mice. DNA was isolated from the pooled splenocytes and the proximal and distal portions of the tails as described (20). Detection of the pUHDOVA plasmid by PCR was performed using these primers 5'-TGCTGTTGCCTGATGAAGTC-3' and 5'-TACCACCTCTCTGCCTGCTT-3'. As a control for the integrity of the DNA primers for GADPH, the following sequences were used: 5'-TCTCATGGTTCACACCCATGACGAACATG-3' and 5'-AAGAAGATGCGGCTGACTGTCGAGCCACAT-3'. PCR conditions were: 1 min at 95°C, 1 min at 50°C, and 1 min at 70°C for 30 cycles. The PCR products were analyzed by electrophoresis in 1.5% agarose gels. The detection limit of this PCR assay was 1 pg of plasmid DNA.
Ablation
Groups of adult C57BL/6 mice had 15 mg (21 day) slow release tetracycline pellets (Innovative Research of America) s.c. implanted 5 days before injection. These mice were injected i.d. in the distal tail with 50 µg each of pUHD-OVA and pUHD15-1 in 50 µl normal saline twice, 3 day apart. The wheal from injection did not extend to the proximal third of the tail. One week after the first injection, a suture was bound tightly around the tail base under metofane anesthesia and the tail was amputated near the rump with a razor blade. The tetracycline pellets were then removed. At week 2 the animals were sacrificed and their splenocytes harvested.
Bone marrow chimeras
Adult C57BL/6 and C3-TgN(TettTALuc)1Dgs were purchased from The Jackson Laboratory and bred in our colony. Mice 10–12 wk old were used as bone marrow recipients. Recipient mice were given 900 rads and then injected i.v. with 8 x 106 T-depleted bone marrow cells from young adult donors in a total volume of 100 µl of serum-free RPMI 1640 medium (BioWhittaker, Walkersville, MD) as previously described (4). After 6 wk, chimerism was verified by detecting the expression of luciferase in the peripheral blood by PCR for the luciferase gene and confirmed by assaying thymi for luciferase activity using Luciferase Assay System (Promega, Madison, WI) per the manufacturer’s recommendations.
Cell transfections
In pilot experiments, each cell type was titrated for optimal cell density for highest transfection efficiency. In 6-well plates, 2.5 x 105 COS-1 cells, 5 x 105 for RAW-264.7 cells, and 2.5 x 106/well THP-1 were seeded. Adherent cells were incubated with 5 µg of plasmid in conjunction with 15 µl of SuperFect (Qiagen, Chatsworth, CA) in 5 ml of OptiMem1 (Life Technologies, Grand Island, NY) for 8 h. The transfection solution was then removed, and the cells were washed with PBS and refed with DMEM (BioWhittaker) 10% heat-inactivated FBS, 2 mM glutamine, 50 µM 2-ME, and 1% penicillin and streptomycin. The suspension cells were transfected by continuous incubation with 5 µg of plasmid with 15 µl of SuperFect (Qiagen) in medium with 10% FCS and antibiotics. The THP-1 cells were treated with 300 nM PMA 18 h before lysing the cells as previously described (29).
Colorimetric assay for ß-gal activity
Cells were washed three times with PBS and then lysed with a 0.15% Triton X-100 and 250 mM Tris (pH 8.0) solution followed by freezing and thawing three times. The cell debris was then pelleted, and 25 µl of supernatant was added per well of a 96-well plate (Costar, Cambridge, MA). Enzyme activity was colorimetrically detected by adding 100 µl/well of a solution of 1 mg/ml chlorophenol red galactopyranoside, 60 mM sodium dibasic phosphate (pH 8.0), 10 mM KCl, and 50 µM 2-ME, and absorbance was read at 570 nm. The titration curves for each lysate were compared with a standard curve of ß-gal (Boehringer Mannheim, Indianapolis, IN) on each plate using DeltaSOFT II version 3.66 (Biometallics, Princeton, NJ).
ELISA for anti-ß-gal IgG
The enzyme-linked immunosorbent assay for Ab to ß-gal was previously described (28). The titration curves for each sera were compared with the standard sera arbitrarily set at a maximum of 108 U on each plate using DeltaSOFT II version 3.66 (Biometallics).
CTL assay
Splenocytes were harvested and restimulated in vitro with 50 U/ml rIL-2 and peptide for 5 day. Cytolytic T cell assays were performed as described using the CytoTox 96 assay kit (Promega) per the manufacturer’s instructions (4). The peptides used included the H-2Kb-restricted epitope from OVA, SIINFEKL (30); the H-2Ld-restricted epitope from ß-gal, TPHPARIGL, (31) and from influenza nucleoprotein the H-2Kd-restricted peptide TYQRTRALV; and the H-2Db-restricted peptide, ASNENMETM (32) (Molecular Research Laboratories, Durham, NC). Background controls with an irrelevant peptide for nonspecific target and effector cell lysis were included on every plate. After background subtraction lysis was calculated by: 100 x [(test release-spontaneous release)/(maximum-spontaneous release)].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether a nonsecreted Ag was able to induce a CTL
response after DNA injection, several plasmid constructs were tested.
The first construct encoded the full-length soluble form of OVA, the
second encoded OVA fused to the amino terminus of the transferrin
receptor (27), and the third plasmid encoded the
H-2Kb-restricted epitope from OVA residues
257–264 (SIINFEKL) fused to a leader sequence to facilitate transport
into the endoplasmic reticulum (33). The first two
plasmids encoded both MHC class I and class II epitopes and the third
encoded only a class I-restricted epitope. Mice that were immunized
with the membrane-bound form of OVA (TfROVA) were able to elicit a
strong CTL response, whereas the minigene-encoding plasmid elicited
only a weak response (Fig. 1
). The
presence of epitopes that could provide cognate T cell help may explain
the difference in these two plasmid systems (26, 34). Both
plasmid products should have been limited to the cell that was
expressing the plasmid, which would imply that APCs were directly
transfected. However, in a transgenic model, this membrane-bound form
of OVA has been demonstrated to induce autoreactive
CD8+ T cells by exogenous Ag transfer
(35). Hence, the immunogenic protein may have been
expressed by both APCs and other transfected cells.
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To determine whether the cells that were expressing the injected
DNA were of monocytic cell lineage, a plasmid with expression
restricted to macrophages and related cell types was used (21, 36). A plasmid with cis-active elements responsible
for transcriptional activation of the scavenger receptor and a ß-gal
reporter gene was injected i.d. in the tail base of mice
(21). This plasmid contained the scavenger receptor
proximal promoter and enhancer elements that have been demonstrated to
direct macrophage-specific expression in transgenic mice
(36). The tissue specificity of this promoter in these
transgenic mice limited expression of the reporter gene to spleen,
thymus, and testes. No expression was seen in heart, lung, liver,
kidney, muscle, or brain. In confirmatory experiments, transfected
monocyte lines expressed the ß-gal transgene in vitro (Fig. 2A). However, mice injected in
vivo generated poor anti-ß-gal IgG and CTL responses compared
with mice injected with a control plasmid with a CMV promoter, pACB-Z,
(Fig. 2, B and C). Similar results were obtained
in mice that were injected i.m. with the same plasmids (data not
shown).
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B). Similarly, the CTL
response was not improved (Fig. 2C). The weak responses
indicated that monocyte-derived cells were not expressing sufficient
antigenic protein for the humoral and cellular responses induced after
DNA injection. Directly transfected splenocytes are relatively poor at priming CTL responses
Recent studies using parent into F1 bone marrow-reconstituted mice demonstrated that the Ag was presented to the immune system in the context of the MHC on the surface of bone marrow-derived cells after plasmid injection (3, 4). It was unclear whether these bone marrow-derived cells were directly transfected or acquired soluble Ag expressed by neighboring transfected somatic cells through macropinocytosis. To separate transfection from protein expression, a tetracycline regulatable system of plasmids was used (22, 23). In this system, the tTA-activated transcription from a minimal promoter sequence from human CMV fused to the E. coli tetracycline resistance (tet) operator sequences. The tTA was unable to bind to these tet operator sequences in the presence of tetracycline, and transcription was suppressed by the drug. The transactivating protein was encoded on a separate plasmid (pUHD15-1) from the regulated transcript (pUHD-OVA). Coadministration of these plasmids allowed efficient transcription of the Ag in the absence of tetracycline.
This plasmid system was used to inject donor mice under drug
suppression to minimize the possibility of Ag expression from the
injected tissue. The splenocytes from these mice were then transferred
to recipients without the drug to allow Ag expression by directly
transfected cells that had migrated to the spleens of the donors.
Rag-1-/- mice were chosen as donors because
there would be no transfer of T or B cells, and their spleens were
relatively enriched for APCs (
17% CD11c+ and
50% MAC3+ cells by FACS). The use of TCR
transgenic mice, OVA-tcr-1, as recipients increased the sensitivity of
detection for the weaker CTL response after injection of tetracycline
suppressed splenocytes, which was not seen when wild-type recipients
were used (data not shown).
The presence of the injected plasmid was detected by PCR in the
proximal part of the tail for all of the donor mice but not the distal
tail nor in the spleen (Fig. 3A). DNA was successfully
isolated from all of the samples as shown in the GAPDH PCR
amplification (Fig. 3B). The transferred splenocytes from
Rag-1-/- mice were relatively enriched for APCs
but may not have been a sufficiently purified population to enable
amplification of the injected plasmid by PCR (20). The
inability to readily detect plasmid in these splenocytes suggested that
directly transfected APCs were rare. This paucity of transfected APCs
was consistent with reports by investigators who were able to directly
visualize such cells by microscopy (7, 8, 19, 37).
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C). These cells could
not have acquired transferred Ag in the donor mice but were able to
freely express protein from directly transfected cells in the recipient
mice. Although the splenocytes from unsuppressed donors had 1 wk longer
to express Ag, these data suggest that the predominant mechanism of
initiating a CTL response was through cross-priming rather than through
directly transfected APCs. Intact skin is required for CTL priming from i.d. gene vaccination
To evaluate the role of protein expression by the cells at the
site of DNA injection in priming an immune response, mice were injected
i.d. in the distal tail with a combination of plasmids encoding soluble
Ag and tTA with and without tetracycline suppression. After 1 wk, the
tails of some of the mice were proximally amputated and the drug
suppression was removed (Fig. 4). If CTL
were primed by cells that migrated to a draining lymph node or were
transfected at a remote site, then animals that had their tails
amputated should have been able to prime an immune response once
suppression of gene expression was removed. Control animals that were
initially on tetracycline, and had their tails left intact, were able
to mount a CTL response once the drug was discontinued. However, mice
that had their tails amputated before the removal of drug suppression
did not generate a CTL response, demonstrating that protein locally
expressed at the site of DNA inoculation was processed for presentation
by the immune system.
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Transgenic mice (TettTALuc) that expressed tTA and luciferase were
used to generate bone marrow chimeras with wild-type C57BL/6 mice
(24). These mice were lethally irradiated and
reconstituted with bone marrow from the other strain. After recovery
groups of chimeras and controls were injected i.d. in the tail with
pUHD-OVA. After injection with pUHD-OVA, the tissue that concomitantly
expressed the tTA transactivator should have been able to express high
levels of OVA. The tTA-transgenic mice made substantial anti-OVA
CTL and Ab responses regardless of their bone marrow origin (Fig. 5). Conversely, wild-type or chimeric
C57BL/6 recipients made weaker CTL and Ab responses. A strong immune
response correlated with expression of the transactivator by
nonlymphoid tissues and not to bone marrow origin. The locally
transfected cells in the tail that contained the transgenic
transactivator expressed the protein responsible for generating the
immune response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, the cellular mechanisms involved in priming a CTL response after the injection of naked plasmid DNA were further investigated. In a series of experiments, several specialized plasmid systems were used to elucidate the relative roles of resident somatic and migratory bone marrow-derived cells in priming an immune response. The tetracycline responsive promoter system has been demonstrated to tightly regulate gene expression and has enabled investigators to temporally manipulate the expression of transgenes in mice (23, 44, 45, 46). This control of expression separated the issues of protein transfer from plasmid transfer. The splenocytes from plasmid injected tetracycline treated Rag-1-/- donor mice were less immunogenic, suggesting that Ag was exogenously acquired rather than exclusively endogenously produced by APCs that migrated to the spleen.
In a follow up study, the site of injection was ablated while plasmid expression was inhibited by tetracycline. If plasmid DNA had transfected cells remote from the site of injection or had been taken up by migratory cells, then there should have been a CTL response after the site of injection was removed and drug suppression was lifted. In control mice that had their tails left intact, sufficient protein was expressed to induce an immune response after tetracycline was discontinued. The inability of mice to raise a CTL response after tail amputation and removal of drug suppression indicated that the cells in the tail skin were responsible for expressing antigenic protein to prime the CTL response.
In a previous report in which muscle and skin injection sites were rapidly ablated after DNA inoculation, the expression of the plasmid was not regulated (47). Immune responses still occurred if injected muscle tissue was excised within 1 min; however, a full response after i.d. immunization required that the injected tail skin remain in place for 72 h before excision. Our results similarly demonstrated that intact skin at the injection site was necessary for mounting an immune response to plasmid DNA encoded Ag. These data argue against CTL being primarily primed by directly transfected cells in the regional lymph nodes after i.d. needle injection of plasmid DNA. The development of an immune response after muscle ablation within 1 min after injection vs a cutaneous immunization suggested that there were likely differences in the extent of transfection outside of the target tissue with technique of DNA administration and site of injection.
Ag expressing and plasmid containing dendritic cells from the skin have been found in draining nodes and in the spleen (7, 8, 19, 37). Although few cells expressing the Ag encoded by the plasmid were visualized, a large population of untransfected dendritic cells were noted to also migrate to the draining lymph node (7, 8). These untransfected dendritic cells could have acquired Ag expressed by other cell populations in the skin and still prime immune responses. To evaluate whether the immunogen-expressing cells in the skin were exclusively of monocytic cell lineage, a plasmid was used that contained a cell type-specific promoter. The untranslated elements in this plasmid have been previously described to restrict expression primarily to macrophages in the in vitro transfection assays and in transgenic mice (21, 36). In experiments using needle-injected DNA, the cells that expressed immunologically relevant Ag in both muscle and dermally injected mice were not of monocytic cell lineage. The resident connective tissue cell population may be more susceptible to transfection and express protein that is then transferred to migratory bone marrow-derived cells.
As an alternative to endogenous protein expression by APCs, the immune system has the flexibility to acquire extracellular Ag and process it into fragments presented by both MHC class I and class II molecules (12, 13, 15, 16, 35, 48). Such cross priming has been demonstrated for ex vivo plasmid-transfected myoblasts reimplanted i.p. into F1 mice (18). Also, the skin from gene gun-treated mice stimulated a primary immune response when grafted onto naive recipients (49). There were most likely redundant mechanisms for the intercellular transport and transfer of protein. Different heat shock proteins with roles as chaperones have been shown to induce tumor-specific immunity (50, 51). As part of the reactive response to injury, these heat shock proteins may have become up-regulated after DNA injection and then performed a dual role in transferring Ag to professional APCs.
Alternatively dendritic cells have been demonstrated to engulf apoptotic cells and cross-prime their peptides (52). There may be nonlymphoid cells that expressed protein after DNA inoculation before they underwent apoptosis and were phagocytosed by immature dendritic cells. Transfected keratinocytes have been demonstrated to transfer Ag after irradiation induced cell death (19). The presence of directly transfected hematopoetic cells from DNA injection has been described as short-lived (19). However, Ag in muscle and skin has been found to continue to be expressed relatively long term. The long-lived immune responses may reflect the ability of plasmid DNA to continue to produce low levels of Ag over a prolonged period of time. The nonlymphoid cells were working in concert with professional APCs not merely as innocent bystanders but as Ag generators.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maripat Corr, Department of Medicine and The Sam and Rose Stein Institute for Research on Aging; University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663. E-mail address:
3 Abbreviations used in this paper: i.d., intradermal; ß-gal, ß-galactosidase; tTA, tetracycline-controlled transactivating protein.
Received for publication May 18, 1999. Accepted for publication August 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), nCMV-TfROVA (
), and ACB (
)
in the rear quadriceps on days 0 and 7. After 2 wk, the mice were
sacrificed and their splenocytes assayed for their ability to lyse
peptide-pulsed EL4 cells. Values plotted are average peptide-specific
lysis. The maximum lysis seen with the negative control peptide
(ASNENMETM) was 3.6%. This experiment was performed twice with four
mice per group.
), pACB-Z (
), or nothing (x). Six weeks after
immunization, their sera were tested for the presence of
anti-ß-gal Abs by ELISA (B). The IgG response of
pCMVGM-CSF- and pACB-Z-coinjected mice was statistically greater than
pACB-Z alone (p < 0.001 by Student’s
t test). At 6 wk, the animals were sacrificed and their
splenocytes were tested for their ability to lyse peptide-pulsed P815
cells after 5 day of in vitro restimulation (C). Values
plotted are the mean peptide-specific average background lysis. The
maximum lysis seen with the negative control peptide (TYQRTRALV) was
4%. These data are representative of three independent
experiments.
