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* Center for Biomedical Inventions, Department of Internal Medicine, and Departments of
Dermatology and
Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Introduction |
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To address the mechanism of LC activation and Ag loading by GI, we characterized the impact of gene-gun treatment on LC function. First, LC migratory capacity from skin and LC activation state were examined. Second, we examined transgene expression in migratory LC and their potential to initiate Ag-specific responses in recipient animals. Third, the relative contribution of direct vs indirect Ag loading was investigated through the use of an inducible vector system that permits exogenous control of transgene expression.
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Materials and Methods |
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Specific pathogen-free, age-matched (4–8 wk), female A/J mice were obtained from Harlan Laboratories (Indianapolis, IN) and maintained under specific pathogen-free conditions at University of Texas Southwestern Medical Center (Dallas, TX). Both the XS106 LC line and NF46 fibroblasts were established from the epidermis of newborn A/J mice and maintained in vitro, as described previously (12, 13, 14).
Gene gun immunization
Standard GI vectors used in this study have been described previously (12). The standard vectors pCMVi-Luc and pCMVi-AAT encode the reporter gene luciferase+ (Luc) and the model secreted Ag human 
-1-antitrypsin (AAT), respectively (1, 12). The pTracer-GFP reporter vector, used for visualizing transfected LC, used the SV40 promoter to drive green fluorescent protein (GFP) expression (Clontech Laboratories, Palo Alto, CA). Plasmid DNA, purified with Qiagen Endo-free columns (Valencia, CA), was precipitated onto gold particles (2 ± 1 µm diameter; Metz Metallurgical, South Plainfield, NJ) at 4 µg/mg. A biolistic gene gun (Rumsey-Loomis, Ithaca, NY) was used to deliver a total of 1 µg of DNA at the pressure of 1200 pounds per square inch into the skin of mouse ears and into cell lines (15, 16). In some experiments, the Helios gene gun (Bio-Rad, Hercules, CA) was compared with the Rumsey-Loomis model. Helios bullet preparation and delivery at 900 pounds per square inch was done following the manufacturer’s instructions. XS106 cells were transfected by gene gun as previously described (12). The level of gene expression achieved by gene gun delivery of pCMVi-Luc DNA was determined by assaying for luciferase (Luc) activity 24 h after bombardment, as described previously (12).
Phenotypic analysis
Migratory cells (5 x 105 cells per sample) were washed and incubated in 10 µl with a mixture of Fc-Block (anti-CD16/CD32, clone 2.4G2; BD PharMingen, San Diego, CA) and 10 µg of rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were stained with a mixture of FITC-conjugated anti-Ia (clone 2G9 is specific for I-Ad and I-Ed and cross-reacts with multiple haplotypes including H-2a of the A/J strain; BD PharMingen) and PE-conjugated anti-CD86 (clone GL1; BD PharMingen) or PE- and FITC-rat IgG2a isotype control per manufacturer’s instructions. In some experiments, cells were additionally stained with combinations of FITC-anti CD3 (clone 17A2), PE or allophycocyanin-anti-CD11c (clone HL3), FITC-anti-CD11b (clone M1/70), FITC or PE-anti-CD40 (clone 3/23) which were all purchased from BD PharMingen. PE-anti-Dec205 (clone NLDC-145) was purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). FITC-anti-
TCR (clone GL3) was a generous gift from Dr. H. Xu (Department of Dermatology, University of Alabama, Birmingham, AL). Viable cells were by gated by forward angle and side scatter characteristics and analyzed using a FACScan flow cytometer and CellQuest Software (BD Immunocytometry Systems, San Jose, CA.).
Migratory LC from gene gun-transfected skin
The dorsal and ventral sides of mouse ears were each subjected to two gene gun shots, delivering a total of 1 µg of DNA per side and harvested 1 h after bombardment. Harvested ear specimens were pulled apart with forceps and split into dorsal and ventral halves placed on droplets of sterile PBS dermal side down. Using the rounded end of curved forceps, collagen was gently scraped from the bottom of the ventral half to enhance the passage of migrating LC through the epidermal and dermal layers into surrounding medium. Prepared ear halves were floated dermal side down and cultured on complete RPMI 1640 medium (containing 10% FBS) for 2 days in 24-well plates (17). The cells that migrated from the skin specimens into the culture medium were harvested, passed through a nylon screen to rid floating debris and examined for cell counts and viability by trypan blue exclusion. Migratory cells routinely contained 46 ± 9% Ia-positive cells as determined by flow cytometry. In some experiments, further enrichment or depletion of Ia-positive cells was performed using the MACS purification system (Miltenyi Biotec, Sunnyvale, CA) per manufacturer’s directions. The percent of Ia-positive cells in each fraction was determined by flow cytometry. LC-enriched populations were 75% Ia-positive while LC-depleted populations were only 6% Ia-positive.
Immunocytochemistry
Surface densities of epidermal LC were examined by whole mount staining with anti-Ia mAb. Ear specimens were split and cartilage was removed from the dermal side of the dorsal half. Ear epidermis was lifted from the dermis after floating the specimens dermal side down in 0.5 M ammonium thiocyanate (Sigma-Aldrich, St. Louis, MO) PBS for 15 min. Epidermal sheets were fixed with cold acetone and washed in PBS. Blocking Ab solution was added and incubated 1 h at 4°C. After three washes with PBS, resident LC were labeled with FITC-anti Ia Ab (clone 2G9; BD PharMingen) diluted 1/200 at 4°C overnight. The next day, excess Ab was washed from sheets by three 30-min incubations with cold PBS. Images at x200 magnification were captured (IP LabSpectrum, Spectral Engine Software; 4pi, Durham, NC) and the number of positive cells were counted by a third experimenter blinded to sample identity.
RT-PCR
Migratory LC were harvested and the RNA was extracted using TriPure isolation reagent (Roche Molecular Biochemicals, Indianapolis, IN). Single reverse-strand synthesis was performed on 5 µg of mRNA using M-MLV-RT (Promega, Madison, WI). Input RNA (50 ng) from this reverse transcriptase (RT)-treated sample was used for PCR amplification. The PCR conditions were optimally performed for AAT- or 
-actin-specific primer pairs (5 or 2.5 µM, respectively). Primer pairs used for detecting mRNA expression levels of the housekeeping -actin gene or the human AAT gene were as follows: AAT-specific primers, forward (5'-GCACTGGGCAG/GTGTCCA-3') and reverse (5'-GGCTTCTGAGTGGTACAA-3') gave rise to a 590-bp product; -actin-specific primers, forward (5'-ACCTGACAGACTACCTCATGAA-3') and reverse (5'-ACTTGCGGTGCACGATGGAGG-3') gave rise to a 570-bp product. The forward primer for AAT bridges a processed spliced sequence that is generated after splicing out 133 bp of our vector’s intron region 5' of the AAT gene and is indicated by the slash in the sequence. Thus, the resultant 590 PCR product can only be amplified from spliced mRNA. Using this AAT sequence-specific primer set we were unable to amplify PCR products from plasmid DNA (data not shown). PCR conditions were: denaturation at 93°C for 1 min followed by 35 amplification cycles, each comprising denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C in a PerkinElmer 9600 PCR machine (Wellesley, MA).
Adoptive transfer experiments
Ear specimens inoculated with pCMVi-AAT were cultured for 2 days to obtain migratory LC. As an adoptive transfer control, XS106 cells were transfected in vitro with pCMVi-AAT by gene gun as described previously (12) and cultured in parallel for 2 days. Naive syngeneic A/J mice were injected s.c. in the ear with a single dose of 2 x 105 LC or transfected XS106 cells and the humoral and cellular responses generated were examined by ELISA and cytotoxicity assays. As a positive control, we inoculated mice by standard GI. To serve as a parallel negative control group, some of the GI-treated mice were subjected to ear removal 1 h after gene gun treatment (van Gogh (VG) experiments).
ELISA was performed on sera obtained at 3 wk, as described previously (12). Serial dilutions of human AAT-specific mouse mAb (Calbiochem, La Jolla, CA) provided a standard curve for determining the reported microgram per milliliter values.
To test for the generation of AAT-specific CTL, mice were boosted at 3 wk with pCMVi-AAT by standard GI and their spleens were harvested after 8 days. Spleen cells from individual mice were cultured for 5 days in the presence of 30 µg/ml heat-denatured AAT protein and 5% rat Con A-stimulated spleen cell supernatant as a source of IL-2 (T-Stim; Collaborative Biomedical Products, Bedford, MA). Viable cells were harvested from these cultures by centrifugation over Ficoll-Paque (r = 1.083 gm/ml; Pharmacia, Uppsala, Sweden) and examined for their cytotoxic activity. For target cells, we used NS46 fibroblasts, derived from A/J mice, pulsed with or without heat-denatured AAT protein at 1 mg/ml (18) and labeled 1 h in 51Cr solution. Washed target cells, at 5 x 103 per well, were incubated for 4 h at 37°C with effector cells in triplicate. Maximum and spontaneous releases were determined from wells containing 2% Triton X-100 or medium alone, respectively. Specific lysis was calculated as (experimental 51Cr-release - spontaneous 51Cr-release)/(maximum 51Cr-release - spontaneous 51Cr-release) x 100%.
Inducible genetic vector system
A mifepristone (RU486)-inducible plasmid system (GeneSwich Systems) was a generous gift from J. L. Nordstrom (Valentis, The Woodlands, TX). The parent two plasmid system originally described by Wang et. al. (19), was modified by Valentis and ourselves for better control of gene expression in transferred LC. The effector plasmid, GS1158 (referred to in this report as pCMV-Gal4), expresses a chimeric RU486-regulated transactivator through a CMV promoter. The regulated expression plasmids, p4U-AAT or p4U-Luc, express AAT or Luc, respectively, in the presence of RU486. The chimeric transcription activator is a three-domain fusion protein consisting of an N-terminal DNA binding domain of yeast Gal4, a RU486 ligand-binding domain from the human progesterone receptor, and a transcription activation domain from human Rel (p65) NF-
B (20). This chimeric molecule binds to the yeast-specific Gal4 upstream activation sequence elements on the regulated plasmid and in the presence of RU486 activates AAT or Luc gene expression. Gene gun inoculation of 2.0 µg of DNA, containing both plasmids pCMV-Gal4 and p4U-AAT or p4U-Luc at a ratio of 1:10, was administered to dorsal and ventral sides of the ear. Migratory LC were harvested as described previously. For maximal gene induction in vivo, RU486 was given i.p in 100 µl of sesame oil (dilution vehicle) at 0.5 mg/kg body weight. Mock-treated animals received vehicle alone. For in vivo induction, recipient mice received three doses of RU486 per week over 3 wk, with the first dose given 1 h before LC transfer.
Statistical analysis
A one-tailed Student’s t test was applied and the p values are indicated in the text and figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether gene gun treatment would enhance the migration of LC, we studied the effect of biolistic gene gun bombardment on LC migration. Gene gun-treated or -untreated ear skin samples were cultured over 2 days, and the numbers of LC that remained in the epidermis were counted after immunofluorescence staining with anti-Ia mAb. We observed a dramatic reduction of LC density as a result of gene gun treatment (Fig. 1A). The few remaining LC were larger in size than those in untreated epidermal sheets and stained with higher intensity with anti-Ia Abs, indicating an activated phenotype. In the absence of bombardment we observed 30% reduction in LC surface density during culture, which is in agreement with reports by others (17) (Fig. 1B). Surprisingly, LC densities dropped by 86–92% in skin treated by gene gun, either our standard gene gun (Rumsey-Loomis) or the Helios, as compared with the levels found in fresh skin. This reduction in LC density from gene gun-treated ears was also seen when animals were sacrificed after 24 h and then examined (data not shown), indicating that the in vitro culture closely mimics in vivo conditions. These results indicate that the introduction of gold into skin tissue plays an important role in eliciting the local LC migration effect. These data are consistent with two possible interpretations, either LC at the GI site migrated out of the epidermis or they died due to gene gun-mediated events. To distinguish between these two possibilities, we enumerated the number of Ia-positive cells that migrated out of skin into the culture medium. We observed that concomitant with gene gun-mediated LC disappearance from the epidermis, 3-fold higher numbers of Ia-positive migratory cells were recovered from the medium as compared with untreated skin cultures (Fig. 2A).
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TCR, but was negative for B220 and Ia molecules (data not shown). The remainder of cells were large and coexpressed CD11c, CD11b, Dec205, CD40, and Ia molecules. In addition 14–27% of total cells were large CD11b-positive but Ia-negative cells, suggesting that they represent a subset of inflammatory granulocytes. The maturation state of migratory LC was examined by measuring the expression levels of DC-specific activation markers CD86 and CD40. Interestingly, while migratory LC from control skin cultures exhibited uniformly high levels of Ia and CD86 on their surface, a large proportion of the migratory LC from gene gun-treated skin expressed low levels of CD86 (Fig. 2B) but higher levels of DC-specific markers and activation marker CD40 (Table I). Collectively these data indicate that LC disappearance from the epidermis is not due to significant levels of cell death, but is due to their activation and enhanced migration out from the epidermis. Further, the identification of a subset of Ia-positive cells that are low in CD86 suggest that the ability to migrate from the epidermis can precede a fully activated phenotype, or they may represent a distinct subset of cells that are also activated by gene gun treatment derived from the dermis such as dermal DC or macrophages.
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To determine whether any migratory cells were transduced by gene gun, ear skin specimens inoculated with a GFP reporter plasmid were cultured over 2 days and migratory cells were examined for GFP expression. A faint green fluorescent signal was detected in cells that had migrated from skin inoculated with the GFP reporter plasmid but not in cells from control skin inoculated with the Luc-encoding plasmid (Fig. 3A). The GFP-positive cells were large and demonstrated dendritic or veiled LC morphology. Further, the GFP-positive cells contained a gold bead in the cytoplasm consistent with the hypothesis that these cells were directly transfected.
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Adoptive transfer of migratory LC leads to AAT-specific humoral responses
The gene gun (Rumsey-Loomis model) covers an area of 
1 cm2 of skin. Because the average density of LC in ear epidermis of A/J mice is 103 cells/mm2 (see Fig. 1B), the theoretical number of LC affected by each gene gun treatment would be 105. Based on this theoretical calculation we chose an inoculation dose of 2 x 105 cells (equivalent to the number of LC affected by two gene gun treatments) to compare the outcome of adoptive transfer to GI. Plasmid DNA that encoded either the model Ag human AAT or negative control Luc was inoculated into the skin by gene gun. Transfected LC were obtained from gene gun-treated ear skin (LC-AAT) after a 2-day culture. Serving as a positive control, XS106 cells were transfected in parallel by gene gun and also cultured over 2 days (XS-AAT). A single dose (2 x 105) of migratory cells or XS106 cells was transferred s.c. into the ears of naive mice (i.e., the same site as that used for gene gun treatment). Significant levels of AAT-specific Abs were detected in mice receiving adoptive transfer of either LC or XS106 cells (transfected with AAT encoding plasmid) as compared with the negative control mice inoculated by gene gun with irrelevant Luc encoding plasmid (p < 0.025) (Fig. 4A). These data indicated that migratory LC alone were sufficient to initiate humoral immune responses. However, the Ab levels were >10-fold lower than those observed in positive control mice receiving the AAT gene by GI, suggesting the significant contribution of Ag produced by other cell types to the magnitude of the humoral response.
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Adoptive transfer of migratory LC generates CTL
Migratory LC from gene gun-treated skin and gene gun-transfected XS106 cells were examined for their ability to initiate CTL responses. Transfection was achieved in both populations, as determined by RT-PCR signal analysis of the AAT gene (Fig. 3B). As described above, a single dose (2 x 105) of migratory LC or XS106 cells was transferred into naive mice by s.c. injection into the ear. Positive control mice received GI treatment with the AAT gene (GI-AAT). A group of mice were subjected to removal of the inoculated ear 1 h after gene gun treatment (VG experiments), serving as a parallel control (VG-AAT). After 3 wk, all mice were boosted by GI, their splenocytes harvested after 8 days, restimulated in vitro for 5 days, and assessed for CTL activities against AAT protein-pulsed or nonpulsed target cells by standard 51Cr-release assays. Fig. 5 shows that the level of CTL activity generated by LC (LC-AAT) was comparable to that obtained with transfected XS106 cells (XS106-AAT) and to that after standard GI (GI-AAT). This activity was Ag-specific because cytotoxicity was not observed against nonpulsed target cells. These data provide evidence that LC which initially migrate from the immunization site give rise to the majority of the CTL response generated by gene gun-mediated GI.
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The migratory LC collected from 2-day skin organ cultures may be loaded with Ag by either direct transfection or indirect uptake of Ag derived from neighboring transfected cells. To distinguish these two possibilities, we used an inducible GI vector system.
The two plasmid system consists of an activator and reporter plasmid. The activator plasmid encodes a chimeric transactivator that, upon binding the inducing drug RU486, activates transcription on a second reporter plasmid encoding either Luc or AAT. To test the effectiveness of this system in mouse skin organ culture, mouse ears were inoculated with the inducible Luc vector by gene gun and cultured in various doses of RU486. As shown in Fig. 6, we found that Luc gene expression occurred in a RU486 dose-dependent manner. Importantly, basal activity was undetectable in the absence of RU486. Another group of mice was pretreated with the drug RU486 an hour before gene gun inoculation in an attempt to maximize the induced transcription levels. We found that pretreatment of drug was sufficient to induce detectable expression of the Luc gene in the absence of further drug exposure during culture. This expression was further augmented when pretreated ear specimens were cultured in the presence of RU486, again, in a dose-dependent manner. Luc gene induction peaked at 100 ng/ml RU486 resulting in a 200-fold induction in this skin organ culture system and this concentration was used in combination with in vivo pretreatment in subsequent experiments. The peak level of Luc activity induced in vitro approached levels achieved by in vivo induction (as shown by a (+) symbol in Fig. 7) illustrating the effectiveness of the in vitro system.
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Using the inducible GI vector encoding the AAT Ag, three populations of migratory LC were compared for their ability to mount an immune response after adoptive transfer in naive mice. Exposure to the inducing drug RU486 occurred in three phases, in vivo pretreatment of the donor, during skin organ culture, and after transfer in recipient animals. Three migratory LC groups were derived from the following gene expression conditions: 1) "Continuous" expression, in which RU486 was preadministered to the donor GI-treated animals, during culture of the donor specimen, and after transfer of migratory LC in recipient animals. These conditions attempt to mimic those of standard GI, permitting both direct (by transfection) and indirect loading of Ag (produced by surrounding transfected skin cells) during LC migration. 2) "Induced" expression, in which donor ear specimens are derived from untreated animals, LC migrate into culture medium in the absence of RU486 and induced only after transfer to recipient mice treated with RU486. Without RU486 in culture, gene expression remains undetectable indicating that Ag is not produced under these conditions (Fig. 6). Therefore during migration, LC cannot be loaded with Ag. Only after transfer into mice treated with RU486 is gene expression induced. Thus, the Ag source in the Induced group can only be derived from transfected LC. 3) Uninduced LC are never exposed to RU486 induction in the donor, in culture, or in vivo upon adoptive transfer. Thus, immune response levels in the Uninduced group reflect background transcription of the transgene.
By comparing the magnitude of the immune response generated from Continuous vs Induced conditions, we assessed the contribution of indirect Ag loading in LC vs direct loading by transfected genes. As shown in Fig. 7, both Continuous and Induced groups generated significantly higher levels of anti-AAT Abs than the Uninduced group (p < 0.001). More importantly, we observed no significant difference in their responses when paired with each other, i.e., Continuous vs Induced groups. Furthermore, the Uninduced group did not generate significant levels of AAT-specific Ab when compared with the negative control Luc group, indicating that AAT-specific Abs could only be generated upon RU486 induction. Because no significant difference in the immune response was observed between Continuous and Induced groups, the results indicate that the majority of the immune response generated by inducible GI vectors was due to directly transfected LC and that uptake of exogenous Ag, under these conditions, played only a marginal role.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies by others have demonstrated small but significant numbers of transgene-expressing LC become detectable in the draining lymph node after gene gun treatment (11, 21). The functional contribution of these directly transfected LC on the immune response has been addressed in various ways. For example, some investigators used nonsecreted forms of Ags in GI so that the gene product is less likely to be transferred to neighboring LC (15, 21, 22, 23). In other studies, the role of cross-priming in GI was addressed by studying CTL priming in which expression of two genes, influenza nucleoprotein Ag and CD86, were required. They showed that bombardment with gold particles coated with both genes, and not a mixture of particles coated with individual genes, was necessary for developing gene gun-mediated responses, indicating that presentation of both gene products in the same cell could only be achieved by direct transfection (16). Together with our results the findings are all consistent with the hypothesis that uptake of exogenous Ag, in secreted or cell-associated form, is less efficient than direct transfection for LC Ag loading at the immunization site.
To address the role of migratory cells in mediating GI, Klinman et al. (24) studied the capacity of gene inoculated skin to transfer immunity to naive recipients. When naive mice were grafted with immunized skin, harvested from the donor as early as 1 day after GI, they failed to fully develop primary and memory immune responses. These results support our conclusion that migratory LC from the immunization site are responsible for initiating Ag-specific responses. Furthermore, Bouloc et al. (25), using a different GI technique of s.c. injection of DNA in saline, also showed that migratory LC expressed transgene RNA and conferred immunity in adoptive transfer experiments. We have extended their observations by examining the direct impact of gene gun treatment on LC migration and by testing the contribution of transfected LC, as well as the role of exogenous uptake for Ag loading to the immune response mediated by the first wave of migratory LC. We propose that cross-priming may play a more pronounced role in loading Ag into LC that repopulate the GI site. This interpretation is consistent with findings from Cho et al. (26), who addressed the role of cross-priming without direct transfection of LC, by demonstrating the ability to activate adoptively transferred Ag-specific CD8 T cells 7 and 21 days after GI, well after the first wave of LC migrated from the GI site. Although their data provide strong evidence that cross-priming plays a predominant role in generating humoral and cellular responses when using a keratinocyte promoter-driven GI vector, they did not directly examine the transfection of migratory cells from the GI site. Using DC-specific promoters to drive Ag expression, they were unable to demonstrate strong humoral responses and demonstrated a reduced capacity to activate CTL. Their data may differ from ours due to differences in the promoters used, as they demonstrated weak levels of Ag expression obtained with the keratinocyte and DC-specific promoters tested, and their use of a nonsecreted model Ag may have been less effective in promoting humoral responses as compared with the secreted AAT Ag used in our studies. Together, the previous findings by others and our results enforce the concept that presentation of Ag by directly transfected LC can be an important contributor in mediating GI.
Our results indicate that biolistic bombardment of skin by gene gun induces a synchronous, rapid, and enhanced emigration of LC from epidermis in skin organ cultures. The disappearance of LC after bombardment was also observed in vivo and was not dependent on the presence of DNA (Ref.27 and data not shown). The migration of untransfected and transfected LC from the site of inoculation is consistent with other reports showing that in addition to identifying rare transgene-expressing cells in draining lymph nodes, significant increases in total DC numbers were also observed (25). The mechanism by which LC are activated in the epidermis by gene gun requires further investigation. It is possible that microdamage of epidermal cells initiates stress signals, perhaps through apoptotic/necrotic mechanisms (28) and/or the elaboration of proinflammatory cytokines (29). We examined a number of DC-specific and leukocyte-specific markers to characterize the cells that migrate in response to gene gun. We found the both LC and T cells migrate, and that in general, LC markers were expressed at higher levels in gene gun-treated samples as compared with controls, indicating that LC were indeed activated. Unexpectedly, not all of the migratory LC from gene gun-treated skin exhibited typical phenotypic features of mature DC. One may interpret this to suggest that the signals generated by gene gun were sufficient to trigger migration but not full maturation of all LC. Immature DC have high levels of intracellular "empty" MHC class II molecules ready for loading processed Ag while mature DC traffic all their MHC II to the plasma membrane, and, therefore have no MHC molecules available for intracellular Ag loading (30). Thus, it may be advantageous that gene gun produces some DC that are not fully mature because GI-transfected migratory DC may have time to express, process, and load Ag-derived peptides onto MHC II before they fully mature in the lymph node.
In this report, we demonstrate the novel use of inducible GI vectors to address questions regarding the Ag-loading mechanisms involved in mediating GI from the first wave of LC that leave the GI site. Using this tool we showed that directly transfected LC have a predominant role in loading the first wave of LC that migrate from the GI site. Thus, future strategies for targeting gene expression exclusively to LC may offer more effective activation of desired immune responses. Collectively, our results with inducible vectors have important implications for the future development of engineered vaccines. Incorporating elements of inducible control in the design of vectors for GI or engineered DC-based vaccines may permit temporally precise and perhaps more predictable regulation of immunologic outcome in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Laura Timares at the current address: Department of Dermatology, University of Alabama, 1530 3rd Avenue S., Birmingham, AL 35294-0019. E-mail address: timares{at}uab.edu
3 Abbreviations used in this paper: GI, genetic immunization; DC, dendritic cell; LC, Langerhans cell; AAT, human -1-antitrypsin; GFP, green fluorescent protein; RT, reverse transcriptase; Luc, luciferase; VG, van Gogh.
Received for publication January 4, 2003. Accepted for publication March 20, 2003.
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B is an efficient human activator in the tetracycline-regulatable gene expression system. Gene 247:103.[Medline]
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) or nonpulsed target cells (
) by standard 51Cr-release assays. Mean and SEM values are shown for cytotoxicity at an E:T ratio of 80:1. *, p < 0.01; **, p < 0.001.
) RU486 in vivo treatment were genetically immunized by gene gun with the inducible vector system encoding the luciferase gene. Skin organ cultures were incubated in medium containing the indicated concentration of RU486. Luc activity was measured after 24 h. The (
) symbol indicates levels of Luc activity obtained from the ear specimen from in vivo RU486-treated mice harvested after 24 h and tested immediately. Similar results were obtained in two other experiments.