Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hung, C.-F.
Right arrow Articles by Wu, T-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hung, C.-F.
Right arrow Articles by Wu, T-C.
[Cancer Research 61, 1080-1088, February 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Enhancement of DNA Vaccine Potency by Linkage of Antigen Gene to a Gene Encoding the Extracellular Domain of Fms-like Tyrosine Kinase 3-Ligand1

Chien-Fu Hung2, Keng-Fu Hsu2, Wen-Fang Cheng, Chee-Yin Chai, Liangmei He, Morris Ling and T-C. Wu3

Departments of Pathology [C-F. H., K-F. H., W-F. C., C-Y. C., L. H., M. L., T-C. W.], Oncology [T-C. W.], Obstetrics and Gynecology [T-C. W.], and Molecular Microbiology and Immunology [T-C. W.], The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287; Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, Tainan, Taiwan [K-F. H.]; Department of Obstetrics and Gynecology, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan [W-F. C.]; and Department of Pathology, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan [C-Y. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, Flt3 (Fms-like tyrosine kinase 3)-ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). A recombinant chimera of the extracellular domain of Flt3-ligand (FL) linked to a model antigen may potentially target the antigen to DCs and their precursor cells. Using human papillomavirus-16 E7 as a model antigen, we evaluated the effect of linkage to FL on the potency of antigen-specific immunity generated by naked DNA vaccines administered intradermally via gene gun. We found that vaccines containing chimeric FL-E7 fusion genes significantly increased the frequency of E7-specific CD8+ T cells relative to vaccines containing the wild-type E7 gene. In vitro studies indicated that cells transfected with FL-E7 DNA presented E7 antigen through the MHC class I pathway more efficiently than wild-type E7 DNA. Furthermore, bone marrow-derived DCs pulsed with cell lysates containing FL-E7 fusion protein presented E7 antigen through the MHC class I pathway more efficiently than DCs pulsed with cell lysates containing wild-type E7 protein. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing metastatic tumors. The FL-E7 fusion vaccine mainly targeted CD8+ T cells, and antitumor effects were completely CD4 independent. These results indicate that fusion of a gene encoding the extracellular domain of FL to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antigen-specific cancer immunotherapy has emerged as a promising approach for controlling cancer because it is capable of developing specific immunity against neoplastic cells while not attacking normal cells. Increasing evidence suggests that professional APCs,4 particularly DCs, are the central players for mediating cancer immunotherapy. An effective vaccine most likely requires a strategy that targets antigen to professional APCs to activate antigen-specific T cells (reviewed in Ref. 1 ).

Recently, DNA vaccines have become an attractive approach for generating antigen-specific immunotherapy (reviewed in Refs. 2, 3, 4 ). One of the concerns about DNA vaccines is their potency, because they do not have the intrinsic ability to amplify in vivo as viral vaccines do. We reasoned that a DNA vaccine encoding a fusion antigen that is directed to cells which activate immune responses, such as DCs, may enhance vaccine potency. Previously, it has been demonstrated that the linkage of a GM-CSF gene to an antigen gene enhanced the potency of DNA vaccines against HIV (5) and hepatitis C (6) . It is believed that chimeric GM-CSF/antigen may act as an immunostimulatory signal to DCs, inducing differentiation from an immature DC form to a mature form (7) . Because DCs and their precursor cells express high levels of GM-CSF receptors, chimeric GM-CSF/antigen may target and concentrate the linked antigen to DCs and further improve DNA vaccine potency.

Another important molecule that also possesses a growth-stimulatory effect on DC precursors and has been shown to be capable of generating large numbers of DCs in vivo is FL (8 , 9) . FL has emerged as an important molecule for the development of tumor vaccines that augment the function and quantity of DCs in vivo. Flt3, a murine tyrosine kinase receptor, was first described in 1991 (10) and was found to be a member of the same family of receptors as c-kit and c-fms receptors, the type III receptor kinase family (reviewed in Ref. 11 ). In hematopoietic tissues, the expression of Flt3 is restricted to the CD34-positive progenitors. Flt3 has been used to identify and subsequently clone the corresponding ligand, FL (12 , 13) .

The predominant form of FL is synthesized as a transmembrane protein from which the soluble form is generated, presumably by proteolytic cleavage. The soluble form of FL (extracellular domain) has been shown to be functionally similar to FL (12) . These proteins function by binding to and activating unique tyrosine kinase receptors. Expression of the Flt3 receptor is primarily restricted, among hematopoietic cells, to the most primitive progenitor cells, including DC precursors. Several studies have shown that the soluble extracellular domain of FL generated strong antitumor effects against several murine model tumors including fibrosarcoma (14) , breast cancer (15 , 16) , liver cancer (17) , lung cancer (18) , melanoma, and lymphoma (19) . To date, FL has not been used in the form of chimeric DNA vaccines.

In our current study, we investigated whether linking a full-length E7 gene to a gene encoding the extracellular domain of FL would enhance the potency of DNA vaccines. We chose human HPV-16 E7 as a model antigen for vaccine development because HPVs, particularly HPV-16, are associated with most cervical cancers. The HPV oncogenic proteins, E6 and E7, are important in the induction and maintenance of cellular transformation and are coexpressed in most HPV-containing cervical cancers. Vaccines or immunotherapies targeting E7 and/or E6 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. We compared DNA vaccines containing wild-type HPV-16 E7 with DNA vaccines containing full-length E7 fused to FL for their generation of immune responses and their ability to protect animals against HPV-16 E7-expressing murine tumors (20) . Our data indicated that linkage of a gene encoding the extracellular domain of FL to E7 dramatically increases the expansion and activation of E7-specific CD8+ T cells, completely bypassing the CD4 arm. This strategy enhanced E7-specific CD8+ T-cell responses, resulting in potent antitumor immunity against established E7-expressing metastatic tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid DNA Constructs and Preparation.
We used pcDNA3 as an expression vector instead of a previously described pCMV-Neo-Bam vector (21 , 22) . We observed that the pCMV-Neo-Bam vector generated a strong antitumor effect but weak E7-specific T cell-mediated immune response in mice vaccinated with wild-type E7 DNA (21 , 22) . The degree of antitumor effect generated by DNA vaccines using pCMV-Neo-Bam vector did not appear to correlate well with E7-specific T cell-mediated immune activity. We chose to use the pcDNA3 expression vector, which has been used effectively to investigate the correlation between the E7-specifc T cell-mediated immune response with the antitumor effect generated by various DNA vaccines (23) . The generation of HPV-16 E7-expressing plasmid, pcDNA3-E7, has been described previously (23) . For the generation of plasmid encoding the extracellular domain of mouse FL, pcDNA3-FL, the DNA fragment encoding the signal peptide and extracellular domain of mouse FL was first amplified with PCR using conditions as described previously (23) with a mouse FL DNA template, sfHAV-EO410 (American Type Culture Collection, Manassas, VA) and a set of primers: 5'-gggtctagaatgacagtgctggcgccagc-3' and 5'-gggggatccctgcctgggccgaggctctgg-3'. The amplified product was then digested with XbaI and BamHI and further cloned into the XbaI and BamHI cloning sites of pcDNA3 vector (Invitrogen, Carlsbad, CA). For the generation of pcDNA3-FL-E7, the E7 DNA fragment was isolated from pcDNA3-E7 by digestion with BamHI and HindIII and gel recovered. The isolated E7 DNA fragment was further cloned into the BamHI and HindIII cloning sites of pcDNA3-FL. For the generation of pcDNA3-GFP, a DNA fragment encoding the GFP was first amplified with PCR using pEGFPN1 DNA (Clontech, Palo Alto, CA) and a set of primers: 5'-atcggatccatggtgagcaagggcgaggag-3'and 5'-gggaagctttacttgtacagctcgtccatg-3'. The amplified product was then digested with BamHI and HindIII and further cloned into the BamHI and HindIII cloning sites of pcDNA3 vector (Invitrogen). For the generation of pDNA3-E7-GFP, a DNA fragment encoding HPV-16 E7 first was amplified with PCR using pcDNA3-E7 as template and a set of primers: 5'-ggggaattcatgcatggagatacaccta-3' and 5'-ggtggatccttgagaacagatgg-3'. The amplified product was then digested with EcoRI and BamHI and further cloned into the EcoRI and BamHI cloning sites of pcDNA3-GFP vector. For the generation of pcDNA3-FL-E7-GFP, the DNA encoding the signal peptide and extracellular domain of FL was amplified with PCR using pcDNA3-FL as a DNA template and a set of primers: 5'-gggtctagaatgacagtgctggcgccagc-3' and 5'-cgagaattcctgcctgggccgaggctctg-3'. The amplified product was then digested with XbaI and EcoRI and further cloned into the XbaI and EcoRI cloning sites of pcDNA3-E7-GFP vector. The accuracy of these constructs was confirmed by DNA sequencing. pcDNA3 DNA with FL, E7, FL-E7, E7-GFP, or FL-E7-GFP gene insert and the "empty" plasmid, pcDNA3 vector, were transfected into subcloning-efficient DH5{alpha} cells (Life Technologies, Inc., Rockville, MD). The DNA was then amplified and purified as described previously (23) . The integrity of plasmid DNA and the absence of Escherichia coli DNA or RNA was checked in each preparation using 1% agarose gel electrophoresis. DNA concentration was determined by the absorbance measured at 260 nm. The presence of inserted E7 fragment was confirmed by restriction enzyme digestion and gel electrophoresis.

Cell Lines.
The production and maintenance of TC-1 cells has been described previously (20) . On the day of tumor challenge, TC-1 cells were harvested by trypsinization, washed twice with 1x HBSS, and finally resuspended in 1x HBSS to the designated concentration for injection. A human embryonic kidney 293 cell line expressing the Db and Kb (293 Db,Kb; Ref. 24 ) was a gift from Dr. J. C. Yang (National Cancer Institute, NIH, Bethesda, MD). It was grown in DMEM medium containing 10% heat-inactivated FCS, 0.3% glutamine, 0.01 M HEPES, 100 units/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml G418.

Confocal Fluorescence Microscopy.
293 Db,Kb cells transfected with pcDNA E7-GFP and pcDNA FL-E7-GFP DNA were cultured for 24–36 h and then cytospinned to glass slides. Cells were fixed with 4% paraformaldehyde in 1x PBS for 30 min at room temperature, permeabilized with 1x PBS containing 0.05% saponin and 1% BSA, and then incubated with mouse anti-calnexin MAb (Stressgen Biotechnologies, Victoria, British Columbia, Canada) at a concentration of 1 µg/ml for 30 min at room temperature. Unbound antibodies were removed by washing three times in 1x PBS. The cells were then incubated with Cy3-conjugated F(ab')2 fragment goat antimouse IgG (Jackson ImmunoReseach Laboratories) at the concentration of 10 µg/ml for 30 min. The slides were washed with 1x PBS containing and 1% BSA. The glass slides were mounted with anti-fading medium, Mowiol 4–88 (Calbiochem Inc., La Jolla, CA) and covered with coverslips. Slides skipping primary antibody were used as negative controls. Samples were examined on a confocal laser scanning microscopy.

Mice.
Female C57BL/6 mice, 6–8 weeks of age from the National Cancer Institute (Frederick, MD), were purchased and kept in the oncology animal facility of the Johns Hopkins Hospital (Baltimore, MD). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.

DNA Vaccination.
Preparation of DNA-coated gold particles and gene gun particle-mediated DNA vaccination was performed using a helium-driven gene gun (Bio-Rad, Hercules, CA) according to a protocol described previously (23) . Briefly, DNA-coated gold particles (1 µg DNA/bullet) were delivered to the shaved abdominal region of the mice using a helium-driven gene gun (Bio-Rad, Hercules, CA) with a discharge pressure of 400 p.s.i.

Intracytoplasmic Cytokine Staining and Flow Cytometry Analysis.
Splenocytes from naïve or vaccinated groups of mice were incubated either with the E7 peptide (amino acids 49–57) containing the MHC class I epitope (25) for detecting E7-specific CD8+ T cell precursors or the E7 peptide (amino acids 30–67) containing the MHC class II peptide (26) for detecting E7-specific CD4+ T helper cell precursors. The E7 peptide was added at a concentration of 2 µg/ml for 20 h. Golgistop (PharMingen, San Diego, CA) was added 6 h before harvesting the cells from the culture. Cells were then washed once in FACScan buffer and stained with phycoerythrin-conjugated monoclonal rat antimouse CD8 or CD4 antibody (PharMingen). Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer’s instructions (PharMingen). FITC-conjugated anti-IFN-{gamma} antibody and the immunoglobulin isotype control antibody (rat IgG1) were all purchased from PharMingen. Analysis was done on a Becton Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA).

ELISA.
The anti-HPV 16 E7 antibodies in the sera were determined by a direct ELISA as described previously (27) . Briefly, a 96-microwell plate was coated with 100 µl of 5 µg/ml bacteria-derived HPV-16 E7 proteins and incubated at 4°C overnight. The wells were then blocked with PBS containing 20% fetal bovine serum. Sera were prepared from the mice on day 14 after immunization, serially diluted in 1x PBS, added to the ELISA wells, and incubated at 37°C for 2 h. After washing with 1x PBS containing 0.05% Tween 20, the plate was incubated with 1:2000 dilution of a peroxidase-conjugated rabbit antimouse IgG antibody (Zymed, San Francisco, CA) at room temperature for 1 h. The plate was washed six times, developed with tetramethylbenzidine (Pierce Corp., Rockford, IL), and stopped with 1 M H2SO4. The ELISA plate was read with a standard ELISA reader at 450 nm.

In Vivo Tumor Protection Experiments.
For the tumor protection experiment, mice (five/group) were vaccinated via gene gun with 2 µg of FL DNA, E7 DNA, FL-E7 DNA, or FL mixed with E7 (FL+E7), or they were unvaccinated. One week later, the mice were boosted with the same regimen as the first vaccination. One week after the last vaccination, mice were s.c. challenged with 1 x l04 cells/mouse TC-1 tumor cells in the right leg and then monitored twice a week. Analysis was performed using SAS version 6.12 (SAS Institute Inc., Cary, NC). The percentage of tumor-free mice was analyzed according to Kaplan-Meier methods. Statistical significance was tested using log-rank statistics.

In Vivo Tumor Treatment Experiments.
The tumor cells and DNA vaccines were prepared as described above. Mice were i.v. challenged with 1 x 104 cells/mouse TC-1 tumor cells via tail vein on day 0. Three days after challenge with TC-1 tumor cells, mice were given 2 µg of FL DNA, E7 DNA, or FL-E7 DNA via gene gun or unvaccinated. One week later, these mice were boosted with the same regimen as the first vaccination. The mice were sacrificed on day 25. The number of pulmonary metastatic nodules of each mouse was evaluated and counted by experimenters blinded to sample identity. Statistical significance was tested using one-way ANOVA.

In Vivo Antibody Depletion Experiments.
In vivo antibody depletions have been described previously (20) . Briefly, mice were vaccinated with 2 µg FL-E7 DNA via gene gun, boosted 1 week later, and challenged with 5 x 104 cells/mouse TC-1 tumor cells. Depletions were started 1 week prior to tumor challenge. MAb GK1.5 (28) was used for CD4 depletion, MAb 2.43 (29) was used for CD8 depletion, and MAb PK136 (30) was used for NK1.1 depletion. Flow cytometry analysis revealed that >95% of the appropriate lymphocyte subsets were depleted with normal levels of other subsets. Depletion was terminated on day 40 after tumor challenge.

Generation of DCs.
DCs were generated by culture of bone marrow cells in the presence of GM-CSF as described previously (31) . Briefly, bone marrow was collected from the femurs and tibias of mice. Erythrocytes were lysed, and the remaining cells were passed through a nylon mesh to remove small pieces of bone and debris. The cells were collected, and 1 x 106 cells/ml were placed in 24-well plates in RMPI 1640, supplemented with 5% FCS, 2 mM ß-mercaptoethanol, 1% nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.), and 100 units/ml GM-CSF (PharMingen). Two-thirds of the medium was replaced every 2 days, and nonadherent cells were harvested on day 7. The collected cells were characterized by flow cytometry analysis for DC markers as described previously (32) .

Generation of E7-specific CD8+ T-Cell Lines.
Generation of E7-specific CD8+ cell lines has been described previously (32) . Briefly, female C57BL/6 (H-2b) mice were immunized by i.p. injection of vaccina-Sig/E7/LAMP-1. Splenocytes were harvested on day 8. For initial in vitro stimulation, splenocytes were pulsed with IL-2 at a concentration of 20 units/ml and 1 µM E7 peptide (amino acids 49–57) for 6 days. Propagation of the E7-specific CTL cell line was performed in 24-well plates by mixing (2 ml/well) 1 x 106 splenocytes containing E7-specific CTLs with 3 x 106 irradiated splenocytes and pulsing them with IL-2 at a concentration of 20 units/ml and 1 µM E7 peptide (amino acids 49–57). This procedure was repeated every 6 days. The specificity of the E7 CTL line was characterized by the CTL assay. Flow cytometry was performed to demonstrate the expression of the CD8 marker.

CTL Assay Using Transfected 293 Db,Kb Cells as Target Cells.
CTL assays were performed in 96-well round-bottomed plates as described by Corr et al. (33) . Cytolysis was determined by quantitative measurements of LDH as reported previously (33) . Transfected 293 Db,Kb cells were used as target cells, whereas E7-specific CD8+ T cells served as effector cells. 293 Db,Kb cells (5 x 106) were transfected with 20 µg of pcDNA3 (empty plasmid), E7, FL, or FL-E7 DNA vaccines via Lipofectamine 2000 (Life Technologies, Inc.), according to the manufacturer’s protocol. The 293 Db,Kb cells were collected 40–44 h after transfection. The levels of E7 protein expression as determined by ELISA were similar in E7- and FL-E7-transfected 293 Db,Kb. CTL assays were performed with effector cells and target cells (1 x 104 cells/well) mixed together at various ratios (1:1, 3:1, 9:1, and 27:1) in a final volume of 200 µl. After 5 h incubation at 37°C, 50 µl of the cultured medium were collected to assess the amount of LDH in the cultured medium using CytoTox assay kits (Promega Corp., Madison, WI) according to the manufacturer’s protocol. The percentage of lysis was calculated from the following equation: 100 x (A - B)/(C - D), where A is the reading of experimental-effector signal value, B is the effector spontaneous background signal value, C is maximum signal value from target cells, and D is the target spontaneous background signal value.

CTL Assay Using DCs Pulsed with Lysates of Transfected 293 Db,Kb Cells as Target Cells.
CTL assays using DCs pulsed with cell lysates as target cells were performed using a protocol similar to the protocol described by Uger and Barber (34) . Briefly, 5 x 106 293 Db,Kb cells were first transfected with 20 µg of pcDNA3 (empty plasmid), E7, FL, or FL-E7 DNA vaccines via Lipofectamine 2000 (Life Technologies, Inc.) according to the manufacturer’s protocol. The transfected 293 Db,Kb cells were collected 40–44 h after transfection and then treated with three cycles of freeze-thaw. The protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) using the vendor’s protocol. The quantity of E7 protein was determined using ELISA, and the cell lysates from E7- or FL-E7 DNA-transfected 293 Db,Kb cells were standardized for E7 protein concentration. The DCs were used as target cells and prepared by pulsing 1 million of DCs with different concentrations of cell lysates (50, 10, 2, and 0.4 µg/ml) in a final volume of 2 ml for 16–20 h. E7-specific CD8+ T cells were used as effector cells. CTL assays were performed at fixed E:T (9:1) ratio with 9 x 104 of E7-specific T cells mixed with 1 x 104 of prepared DCs in a final volume of 200 µl. Results from CTL assays were determined by quantitative measurements of LDH as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Linkage of the Extracellular Domain of FL to HPV-16 E7 Protein Reroutes E7 into the Endoplasmic Reticulum.
To determine the expression and localization of wild-type and modified HPV-16 E7 protein, we have added the gene encoding GFP to the 3' end of the E7 gene and chimeric FL-E7 genes as a tag. Transfection and subsequent examination with a fluorescence microscope were used to determine the expression and localization of wild-type and modified HPV-16 E7 protein. As shown in Fig. 1Citation , the level of protein expression was quite similar between cells transfected with E7-GFP or FL-E7-GFP. As expected, cells transfected with E7-GFP showed cytoplasmic/nuclear distribution (Fig. 1B)Citation . In comparison, cells transfected with the chimeric FL-E7-GFP construct displayed a network pattern consistent with ER localization (Fig. 1E)Citation . To further explore whether the FL-E7-GFP chimera had in fact been distributed to the ER, we performed immunofluorescent staining of cells transfected with either E7-GFP or FL-E7-GFP using an antibody against calnexin (Fig. 1, A and D)Citation , a well-characterized marker for the ER. As shown in Fig. 1, C and FCitation , colocalization of E7-GFP and the calnexin protein was observed only in cells transfected with FL-E7-GFP but not E7-GFP, indicating that at least some of the FL-E7 chimera was targeted to the compartments of the ER. These data indicated that the addition of the extracellular domain of FL to E7 may facilitate the entry of E7 into ER compartments.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1. Confocal fluorescence microscopic examination to demonstrate the expression and distribution of E7 and chimeric FL-E7 proteins. 293 Db,Kb cells were transfected with pcDNA3-E7-GFP (A–C) or pcDNA3-FL-E7-GFP DNA (D–F) using Lipofectamine. Immunofluorescent staining was performed as described in "Materials and Methods." For the detection of GFP protein, green fluorescence was noted (B and E). For the detection of endogenous calnexin protein, red fluorescence was observed (A and D). Controls omitting primary antibodies did not demonstrate specific red fluorescence (data not shown). Colocalization of GFP and calnexin was demonstrated by the yellow color in the combined image (C and F).

 
Vaccination with FL-E7 Fusion DNA Significantly Enhances E7-specific CD8+ T Cell-mediated Immune Responses.
CD8+ T lymphocytes are one of the most crucial effectors for inducing antitumor immunity. To determine the quantity of E7-specific CD8+ T-cell precursors generated by the FL-E7 DNA vaccine, we used intracellular cytokine staining, a sensitive functional assay for measuring IFN-{gamma} production at the single-cell level (35) . As shown in Fig. 2Citation , mice vaccinated with FL-E7 DNA generated the highest number of E7-specific IFN-{gamma}+ CD8+ T-cell precursors (94/3 x 105 splenocytes) using flow cytometry analysis, whereas mice vaccinated with E7 DNA generated fewer precursors (12/3 x 105 splenocytes). FL-E7 DNA immunization led to a nearly 8-fold increase in the number of E7-specific CD8+ T-cell precursors. These results also indicated that fusion of E7 to FL was required for enhancement of CD8+ T-cell activity, because FL mixed to E7 (FL+E7 DNA) did not generate enhancement of CD8+ T-cell activity.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2. Intracellular cytokine staining with flow cytometry analysis to determine E7-specific CD8+ T-cell precursors in C57BL/6 mice. Mice were immunized with FL DNA, E7 DNA, FL-E7 DNA, or FL mixed with E7 DNA (FL+E7) via gene gun or received no vaccination (Control). For vaccinated mice, 2 µg DNA/mouse were administered twice. Splenocytes were harvested 7 days after the last DNA vaccination. E7-specific CD8+ T cells are shown. A, splenocytes from vaccinated mice were cultured in vitro with E7 peptide (amino acids 49–57) overnight and were stained for both CD8 and intracellular IFN-{gamma}. The number of IFN-{gamma}-secreting, CD8+ T-cell precursors in mice immunized with various recombinant DNA vaccines was analyzed by flow cytometry. Mice vaccinated with FL-E7 DNA generated the highest IFN-{gamma}+ CD8+ double-positive T cells compared with other groups. B, the number of IFN-{gamma}-producing, E7-specific CD8+ T cells was determined using flow cytometry in the presence ({blacksquare}) or absence ({square}) of E7 peptide (amino acids 49–57). Data are expressed as the mean numbers of CD8+ IFN-{gamma}+ cells/3x105 splenocytes; bars, SE. The data from intracellular cytokine staining illustrated here are from one representative experiment of two performed.

 
Vaccination with FL-E7 Fusion DNA Does Not Generate Significant E7-specific CD4+ T Cell-mediated Immune Responses.
To examine the generation of E7-specific CD4+ T precursor cells and cytokine profiles (IFN-{gamma} or IL-4 secreting) by each of these vaccines, we performed double staining for CD4 surface marker and intracellular IFN-{gamma} or IL-4 on splenocytes obtained from immunized mice, followed by flow cytometry analysis. As shown in Fig. 3ACitation , mice vaccinated with FL-E7 DNA generated no significant increase in CD4+ IFN-{gamma}+ double-positive cells compared with mice vaccinated with FL DNA, wild-type E7 DNA, plasmid DNA, or naïve mice without vaccination. Splenocytes from Sig/E7/LAMP-1 DNA vaccinated mice (22) were used as positive controls. Similarly, no significant increase in CD4+ IL-4+ double-positive cells were identified in the mice vaccinated with FL-E7 compared with mice vaccinated with FL DNA, wild-type E7 DNA, plasmid DNA, or the naïve mice without vaccination (Fig. 3B)Citation . IL-4-secreting activated mouse splenocytes (MiCK-2; PharMingen) were used as positive controls to assure the success of intracellular IL-4 staining for this study.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. Flow cytometry analysis of IFN-{gamma}-secreting and IL-4-secreting E7-specific CD4+ cells in mice vaccinated with various recombinant DNA vaccines. Mice were immunized as described in Fig. 2Citation . A, splenocytes from vaccinated mice were cultured in vitro with E7 peptide (amino acids 30–67) overnight and were stained for both CD4 and intracellular IFN-{gamma}. The number of IFN-{gamma}-secreting CD4+ T cells was analyzed using flow cytometry. No significant difference in the frequency of E7-specific, IFN-{gamma}-secreting CD4+ cells was observed in mice immunized with various recombinant DNA vaccines. B, splenocytes from vaccinated mice were cultured in vitro with E7 peptide (amino acids 30–67) overnight and stained for both CD4 and intracellular IL-4. The percentage of IL-4-secreting CD4+ T cells was analyzed by flow cytometry. The IL-4-secreting, activated mouse splenocytes (MiCK-2) from PharMingen were used as positive controls to assure the success of intracytoplasmic IL-4 staining for this study. The specificity of IL-4 staining was demonstrated by the absence of CD4+ IL-4+ T cells when the IL-4 antibody was omitted. No significant difference in the frequency of IL-4-secreting E7-specific CD4+ cells was observed in mice immunized with various recombinant DNA vaccines. The intracellular cytokine staining illustrated here is from one representative experiment of two performed.

 
To determine the levels of E7-specific antibodies in the sera of the vaccinated mice, ELISA was performed 2 weeks after the last vaccination and read at 450 nm. No significant E7-specific antibody responses were detected in mice vaccinated with chimeric FL-E7 DNA, FL, or empty plasmid, or in naïve mice (data not shown).

Vaccination with Chimeric FL-E7 DNA Vaccine Enhances Protection of Mice against the Growth of TC-1 Tumors.
To determine whether vaccination with various DNA vaccine constructs protects mice against E7-expressing tumors, in vivo tumor protection experiments were performed. As shown in Fig. 4Citation , 100% of mice receiving FL-E7 DNA vaccination remained tumor-free 70 days after TC-1 challenge (log-rank, P < 0.001). In contrast, only 20% of mice receiving wild-type E7 remained tumor free after day 32, and all unvaccinated mice, or FL DNA-vaccinated mice, developed tumor growth within 20 days after tumor challenge. These results also indicated that fusion of E7 to FL was required for antitumor immunity, because only 20% of mice receiving FL mixed with E7 (FL+E7 DNA) remained tumor free after 32 days. Therefore, FL-E7 fusion DNA significantly enhanced the antitumor immunity against the growth of TC-1 tumors.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4. In vivo tumor protection experiments against the growth of TC-1 tumors. Mice were immunized with FL DNA, E7 DNA, FL-E7 DNA, or FL mixed with E7 DNA (FL+E7) via gene gun and boosted with the same regimen 1 week later. One week after the last vaccination, mice were challenged with 1 x 104 TC-1 cells/mouse s.c. Mice were monitored for evidence of tumor growth by palpation and inspection twice per week. One hundred % of mice receiving FL-E7 DNA vaccination remained tumor free 60 days after TC-1 challenge. The data collected from the in vivo tumor protection experiments illustrated here are from one representative experiment of two performed. •, FL-E7; {diamondsuit}, E7; {circ}, FL; {triangleup}, FL+E7; , Control.

 
Treatment with FL-E7 Fusion DNA Eradicates Established E7-expressing Tumors in the Lungs.
To determine the therapeutic potential of a chimeric FL-E7 DNA construct in treating TC-1 tumor metastases in the lungs, in vivo tumor treatment experiments were performed. As shown in Fig. 5ACitation , mice vaccinated with FL-E7 DNA demonstrated the lowest mean number of pulmonary nodules (5.8 ± 3.6) compared with mice vaccinated with wild-type E7 DNA (67.5 ± 3.5), FL mixed with E7 DNA (68 ± 15), or FL DNA (65 ± 5.0), or unvaccinated mice (50.66 ± 7.3; one-way ANOVA, P < 0.001). Data are expressed as mean number of pulmonary metastatic tumor nodules ± SE. Furthermore, mice vaccinated with FL-E7 DNA also had the lowest mean lung weight (g; 0.158 ± 0.025) compared with mice vaccinated with wild-type E7 DNA (0.462 ± 0.02), FL plus E7 DNA (0.469 ± 0.08), or FL DNA (0.6 ± 0.03), or unvaccinated mice (0.645 ± 0.08; one-way ANOVA, P < 0.001; Fig. 5BCitation ). Data are expressed as mean lung weight ± SE. Representative gross photographs of the lung tumors are shown in Fig. 6Citation .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. In vivo tumor treatment experiments against preexisting metastatic TC-1 tumor cells. The mice were i.v. challenged with 1 x 104 cells/mouse TC-1 tumor cells in the tail vein on day 0. Three days after challenge with TC-1 tumor cells, mice received 2 µg of FL DNA, E7 DNA, FL-E7 DNA, or FL mixed with E7 (FL + E7) via gene gun or unvaccinated. One week later, these mice were boosted with the same regimen as the first vaccination. The mice were sacrificed on day 25. The FL-E7 group has the least number of pulmonary metastatic nodules (A) and the lowest lung weight (B) as compared with the other vaccinated groups (one-way ANOVA, P < 0.001). The data obtained from these in vivo treatment experiments are from one representative experiment of two performed; bars, SE.

 


View larger version (49K):
[in this window]
[in a new window]
  		Fig. 6. Representative gross pictures of the lung tumors in each vaccinated group. After in vivo tumor treatment experiments against preexisting metastatic TC-1 tumor cells, there are multiple grossly visible lung tumors in unvaccinated control mice and mice vaccinated with FL, wild-type E7 DNA, or FL mixed with E7 DNA. The lung tumors in the FL-E7 vaccinated group cannot be seen at the magnification provided in this figure.

 
CD8+ T Cells but not CD4+ T Cells Are Essential for the Antitumor Effect Generated by DNA Vaccine with E7 Fused to FL.
To determine the subset of lymphocytes that are important for the rejection of E7-positive tumor cells, we performed in vivo antibody depletion experiments (20 , 36) . Depletion of lymphocyte subsets was assessed on the day of tumor injection and weekly thereafter by flow cytometry analysis of spleen cells. More than 99% depletion of the appropriate subset was achieved with normal levels of the other subsets (data not shown). As shown in Fig. 7, all mice without vaccination and all of the mice depleted of CD8+ T cells grew tumors within 14 days after tumor challenge. In contrast, all of the nondepleted mice and all of the mice depleted of CD4+ T cells remained tumor free 60 days after tumor challenge. Forty % of NK1.1-depleted mice grew tumors 6 weeks after tumor injections. Although NK cells appear to be important for the antitumor effect compared with mice with no depletion, this difference was not statistically significant (log-rank, P = 0.13). Our results suggest that CD8+ T cells are essential for E7-specific antitumor immunity generated by the FL-E7 DNA vaccine.

Enhanced Presentation of E7 through the MHC Class I Pathway in Cells Transfected with FL-E7 DNA.
From the immunological assays of vaccinated mice, we observed that mice vaccinated with FL-E7 generated the highest number of E7-specific CD8+ T-cell precursors (Fig. 2)Citation . To determine the mechanism that accounts for such a phenomenon, we first tested whether there was enhanced MHC class I presentation of E7 in cells expressing FL-E7 (in this case, human embryonic kidney 293 Db,Kb cells transfected with FL-E7). We used CTL assays with Db-restricted E7-specific CD8+ T cells as effector cells to determine whether target cells (293 Db,Kb cells) transfected with FL-E7 can be killed more efficiently than 293 Db,Kb cells transfected with wild-type E7. We chose 293 Db,Kb cells as target cells because 293 Db,Kb cells have been shown to have a stable high transfection efficiency (24) . In addition, the level of E7 expression in FL-E7 or E7 DNA transfected 293 Db,Kb cells was similar (data not shown). CTL assays were performed using 293 Db,Kb cells transfected with empty plasmid, FL, E7, or FL-E7 DNA or nontransfected 293 Db,Kb cells with various E:T ratios (1:1, 3:1, 9:1, and 27:1). As shown in Fig. 8Citation , 293 Db,Kb cells transfected with FL-E7 DNA generated significantly higher percentages of specific lysis compared with 293 Db,Kb cells transfected with wild-type E7 DNA. These results indicated that cells transfected with FL-E7 DNA presented E7 antigen through the MHC class I pathway more efficiently than cells transfected with wild-type E7 DNA.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 8. CTL assays to demonstrate enhanced presentation of E7 through the MHC class I pathway in cells transfected with FL-E7 DNA. 293 Db,Kb cells were transfected with various DNA vaccines with Lipofectamine and collected 40–44 h after transfection. Transfected 293 Db,Kb cells were used as target cells, whereas Db-restricted, E7-specific CD8+ T cells were used as effector cells. CTL assays with various E:T ratios were performed. Note: the 293 Db,Kb cells transfected with FL-E7 DNA generated significantly higher percentages of specific lysis as compared with 293 Db,Kb cells transfected with other DNA vaccines. CTL assays illustrated here are from one representative experiment of two performed; bars, SE.

 
Enhanced Presentation of E7 through the MHC Class I Pathway in DCs Pulsed with Chimeric FL-E7 Protein.
Another potential mechanism for the enhanced E7-specific CD8+ T-cell immune responses in vivo is the presentation of E7 through the MHC class I pathway by uptake of lysed cells expressing various E7 DNA constructs, also called "cross-priming." A cross priming experiment was performed to characterize the MHC class I presentation of E7 of DCs pulsed with cell lysates of 293 Db,Kb cells transfected with empty plasmid, FL, E7, or FL-E7 DNA. Lysates of transfected 293 Db,Kb cells were obtained from cycles of freeze-thaw. Bone marrow-derived DCs (1 x 106) were pulsed with a serial dilution of lysates derived from various transfected 293 Db,Kb cells (50, 10, 2, or 0.4 µg). DCs were used as target cells, whereas Db-restricted E7-specific CD8+ T cells served as effector cells. CTL assays were performed with a fixed E:T ratio (9:1). As shown in Fig. 9Citation , DCs pulsed with lysates from 293 Db,Kb cells transfected with FL-E7 DNA generated significantly higher percentages of specific lysis as compared with DCs pulsed with lysates from 293 Db,Kb cells transfected with the other DNA constructs and nontransfected DCs. These results suggested that DCs pulsed with FL-E7 fusion protein presented E7 antigen through the MHC class I pathway more efficiently than DCs pulsed with wild-type E7 protein. Thus, the fusion of FL to E7 may enhance E7-specific CD8+ T-cell immune responses via cross-priming effects.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 9. CTL assays to demonstrate enhanced MHC class I presentation of E7 in bone marrow-derived DCs pulsed with cell lysates containing chimeric FL-E7 protein. Bone marrow-derived DCs were pulsed with cell lysates from 293 Db,Kb cells transfected with various DNA vaccines in different concentrations (50, 10, 2, and 0.4 µg/ml) for 16–24 h. Db-restricted, E7-specific CD8+ T cells were used as effector cells. CTL assays were performed at fixed E:T (9:1) ratio with 9 x 104 of E7-specific T cells mixed with 1 x 104 of prepared DCs in a final volume of 200 µl. Results of CTL assays were assessed using quantitative measurements of LDH as described in "Materials and Methods." Note: DCs pulsed with lysates from cells transfected with FL-E7 DNA generated significantly higher percentages of specific lysis compared with DCs pulsed with lysates from cells transfected with other DNA vaccines. CTL assays illustrated here are from one representative experiment of two performed; bars, SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that linkage of the extracellular domain of FL to E7 can significantly enhance the potency of HPV-16 E7-expressing DNA vaccines. DNA vaccines encoding FL fused to HPV-16 E7 generated significant CD8+ T cell-dependent preventive effects against HPV-16 E7-expressing murine tumors. Furthermore, the chimeric FL-E7 DNA vaccine was capable of controlling lethal pulmonary metastatic tumors.

Our data demonstrated that the incorporation of FL can preferentially enhance CD8+ T-cell responses of E7 DNA vaccines in vaccinated mice. In contrast, E7-specific CD4+ T-cell responses were not significantly enhanced by the FL-E7 DNA vaccine. We found that the linkage of FL to E7 directly enhanced MHC class I presentation of E7 compared with wild-type E7 in transfected cells in vitro (Fig. 8)Citation . Because ballistic DNA delivery can introduce DNA directly into dermal professional APCs, the FL-E7 DNA-transfected APCs may directly enhance the presentation of E7 through MHC class I pathway to CD8+ T cells and contribute to the generation of E7-specific CD8+ T-cell precursors in vivo.

Although it is not clear how the linkage of FL to E7 can directly enhance MHC class I presentation of E7, one of the possible mechanisms for the enhancement of MHC class I presentation of E7 may be related to the chaperone effect of FL. FL expressed in cells may be distributed to the ER (37) . In our study, we have used fluorescence microscopic examination to investigate the distribution of E7 and FL-E7 proteins linked to GFP within transfected 293 Db,Kb cells. In cells transfected with FL-E7-GFP, most of the FL-E7-GFP protein showed colocalization with calnexin in the ER (Fig. 1)Citation , suggesting that linkage of FL to E7 may facilitate the entry of E7 into the ER. Several studies have demonstrated that ER targeting may lead to enhanced antigen-specific MHC class I-restricted CTL activity (38, 39, 40) .

Another mechanism that may contribute to enhanced E7-specific CD8+ T-cell immune responses in vivo is the so-called "cross-priming" effect, whereby lysis of cells expressing FL-E7 antigen can release exogenous protein to be taken up and processed by other APCs via the MHC class I-restricted pathway. Our data suggested that DCs pulsed with FL-E7 fusion protein are capable of presenting E7 antigen through the MHC class I pathway in a more efficient manner than DCs pulsed with wild-type E7 protein. (Fig. 9)Citation . However, the "cross-priming" of chimeric FL-E7 probably does not play a major role in gene gun-mediated FL-E7 DNA vaccination. It has been shown that direct priming, not cross-priming, of CD8+ T cells by gene-transfected DCs is the key event in gene gun-mediated DNA immunization (41 , 42) . However, we cannot completely rule out the possibility of cross-priming, because FL-E7 might be released from other cell types, such as keratinocytes (which were also transfected by gene gun vaccination), and then enter the DCs via the cross-priming mechanism.

In this study, we did not detect a significant increase in the number of DCs or NK cells in the spleens of mice vaccinated with FL-E7 DNA vaccines (data not shown), although FL has been shown previously to significantly expand DCs (17) and NK cells (43 , 44) . This may be related to the low quantity of FL-E7 released in blood circulation after DNA vaccination. We were not able to detect any FL-E7 protein in the sera derived from mice vaccinated with FL-E7 DNA (data not shown). This finding also raises an issue about the source of FL-E7 protein for cross-priming. One possibility is that FL-E7 protein from the lysis of transfected keratinocytes may be taken up by Langerhans’ cells and further processed in the draining lymph nodes without involving blood circulation.

It is interesting to note that the E7 DNA vaccine in the current study had a weaker antitumor effect compared with E7 DNA vaccine using a different mammalian expression vector (21 , 22) . In our previous study, we used a pCMV-Neo-Bam expression vector that contains the human cytomegalovirus promoter (21 , 22) . The E7 DNA vaccine using this vector generated a very impressive antitumor effect with a relative absence of E7-specific CD8+ T-cell immune response. In the current study, we observed a weak E7-specific CD8+ T-cell immune response as well as a weak antitumor effect in mice vaccinated with E7 DNA using a pcDNA3 expression vector. The discrepancy in the antitumor effect generated by the same gene in difference expression vectors may be explained by the fact that different vectors may have different levels of expression of the inserted gene. Furthermore, it is now clear that bacterial DNA can contain immunostimulatory elements such as CpG islands (45 , 46) , which have been shown to cause simultaneous maturation and activation of murine DCs (47) and act as an adjuvant for tumor antigen immunization (48) . We used pcDNA3 instead of pCMV-Neo-Bam in our current study because pCMV-Neo-Bam would likely generate a strong antitumor effect in mice vaccinated with either E7 and FL-E7 DNA, making it difficult to evaluate the correlation between E7-specific CD8+ T cell-mediated immune responses and the antitumor effect.

The FL-E7 DNA vaccine may raise certain safety issues that need to be addressed before it is used for widespread vaccination:

(a) There is the concern that DNA may integrate into the host genome, although it is estimated that the frequency of integration is much lower than that of spontaneous mutation and should not pose any real risk (49) .

(b) The second issue concerns potential risks associated with the presence of HPV-16 E7 protein in host cells. E7 is an oncoprotein that disrupts cell cycle regulation by binding to tumor suppressor pRB protein in nuclei (50) . Thus, the presence of E7 in host cells may lead to accumulation of genetic aberrations and eventual malignant transformation in the host cells. The oncogenicity of E7 can be eliminated by introducing mutations into E7 DNA so that the resulting E7 protein cannot bind with pRB (51) but still maintains most of its antigenicity.

(c) The third issue is the concern over the generation of autoimmunity that may be caused when FL leads to excessive expansion of DCs in vivo. However, we did not observe any significant increase in the number of DCs in the spleen or lymph nodes of mice vaccinated with FL or FL-E7 DNA vaccines. Furthermore, we performed pathological examination of the vital organs in all of the FL-E7-vaccinated mice, and we did not observe any significant pathology. These results indicated that FL-E7 can be used as a potent DNA vaccine with no detectable detrimental side effects.

In summary, our results indicated that fusion of the FL gene to the HPV-16 E7 gene generated potent E7-specific CD8+ T cell-mediated immune responses and antitumor effects against HPV-16 E7-expressing murine tumors. Our data suggest that linkage of the FL gene to an antigen gene may greatly enhance the potency of DNA vaccines and can potentially be applied to other cancer systems with known tumor-specific antigens.



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 7. In vivo antibody depletion experiments to determine the effect of lymphocyte subsets on the potency of FL-E7 DNA vaccine. Mice were immunized with 2 µg of FL-E7 DNA via gene gun and boosted with 2 µg of FL-E7 DNA 1 week later. One week after the last vaccination, mice were challenged with 1 x 104 TC-1 cells/mouse s.c. CD4, CD8, and NK1.1 depletions were initiated 1 week prior to tumor challenge and lasted 40 days after tumor challenge. Note: all of the unvaccinated mice and all of the mice depleted of CD8+ T cells grew tumors within 14 days after tumor challenge. The data of antibody depletion experiments illustrated here are from one representative experiment of two performed. {square}, no depletion; {diamondsuit}, CD8 depletion; •, CD4 depletion; {blacktriangleup}, NK depletion; , naive.

 

   ACKNOWLEDGMENTS
 
We thank Drs. Keerti V. Shah, Robert J. Kurman, Elizabeth Jaffee, and Drew M. Pardoll for insightful discussions. We also thank Drs. Richard Roden and Sara Pai for critical review of the manuscript. We greatly appreciate Lee Wu for excellent statistical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants NIH 5 PO1 34582–01, U19 CA72108–02, and RO1 CA72631-01; the Cancer Research Institute; the Richard W. TeLinde fund; and the Alexander and Margaret Stewart Trust grant. Back

2 The first two authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Department of Pathology, Johns Hopkins University School of Medicine, Ross Research Building, Room 659, Baltimore, MD 21205. Phone: (410) 614-3899; Fax: (410) 614-3548; E-mail: wutc{at}jhmi.edu Back

4 The abbreviations used are: APC, antigen-presenting cell; DC, dendritic cell; GM-CSF, granulocyte/macrophage-colony stimulating factor; FL, Flt3-ligand; Flt3, Fms-like tyrosine kinase 3; HPV, human papillomavirus; GFP, green fluorescent protein; MAb, monoclonal antibody; LDH, lactate dehydrogenase; ER, endoplasmic reticulum; IL, interleukin; NK, natural killer. Back

Received 3/21/00. Accepted 11/29/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chen C. H., Wu T. C. Experimental vaccine strategies for cancer immunotherapy.. J. Biomed. Sci., 5: 231-252, 1998.[Medline]
  2. Robinson H. L., Torres C. A. DNA vaccines.. Semin. Immunol., 9: 271-283, 1997.[Medline]
  3. Pardoll D. M., Beckerleg A. M. Exposing the immunology of naked DNA vaccines.. Immunity, 3: 165-169, 1995.[Medline]
  4. Donnelly J. J., Ulmer J. B., Shiver J. W., Liu M. A. DNA vaccines.. Annu. Rev. Immunol., 15: 617-648, 1997.[Medline]
  5. Lee A. H., Suh Y. S., Sung Y. C. DNA inoculations with HIV-1 recombinant genomes that express cytokine genes enhance HIV-1 specific immune responses.. Vaccine, 17: 473-479, 1999.[Medline]
  6. Lee S. W., Cho J. H., Sung Y. C. Optimal induction of hepatitis C virus envelope-specific immunity by bicistronic plasmid DNA inoculation with the granulocyte-macrophage colony-stimulating factor gene.. J. Virol., 72: 8430-8436, 1998.[Abstract/Free Full Text]
  7. Banchereau J., Steinman R. M. Dendritic cells and the control of immunity.. Nature (Lond.), 392: 245-252, 1998.[Medline]
  8. Maraskovsky E., Brasel K., Teepe M., Roux E. R., Lyman S. D., Shortman K., McKenna H. J. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.. J. Exp. Med., 184: 1953-1962, 1996.[Abstract/Free Full Text]
  9. Shurin M. R., Pandharipande P. P., Zorina T. D., Haluszczak C., Subbotin V. M., Hunter O., Brumfield A., Storkus W. J., Maraskovsky E., Lotze M. T. FLT3 ligand induces the generation of functionally active dendritic cells in mice.. Cell. Immunol., 179: 174-184, 1997.[Medline]
  10. Rosnet O., Marchetto S., deLapeyriere O., Birnbaum D. Murine Flt3, a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSF1R family.. Oncogene, 6: 1641-1650, 1991.[Medline]
  11. Lyman S. D. Biologic effects and potential clinical applications of Flt3 ligand.. Curr. Opin. Hematol., 5: 192-196, 1998.[Medline]
  12. Lyman, S. D., James, L., Vanden Bos, T., de Vries, P., Brasel, K., Gliniak, B., Hollingsworth, L. T., Picha, K. S., McKenna, H. J., Splett, R. R., et al. Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell, 75: 1157–1167, 1993.
  13. Hannum C., Culpepper J., Campbell D., McClanahan T., Zurawski S., Bazan J. F., Kastelein R., Hudak S., Wagner J., Mattson J., et al Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs.. Nature (Lond.), 368: 643-648, 1994.[Medline]
  14. Lynch D. H., Andreasen A., Maraskovsky E., Whitmore J., Miller R. E., Schuh J. C. Flt3 ligand induces tumor regression and antitumor immune responses in vivo.. Nat. Med., 3: 625-631, 1997.[Medline]
  15. Chen K., Braun S., Lyman S., Fan Y., Traycoff C. M., Wiebke E. A., Gaddy J., Sledge G., Broxmeyer H. E., Cornetta K. Antitumor activity and immunotherapeutic properties of Flt3-ligand in a murine breast cancer model.. Cancer Res., 57: 3511-3516, 1997.[Abstract/Free Full Text]
  16. Braun S. E., Chen K., Blazar B. R., Orchard P. J., Sledge G., Robertson M. J., Broxmeyer H. E., Cornetta K. Flt3 ligand antitumor activity in a murine breast cancer model: a comparison with granulocyte-macrophage colony-stimulating factor and a potential mechanism of action.. Hum. Gene Ther., 10: 2141-2151, 1999.[Medline]
  17. Peron J. M., Esche C., Subbotin V. M., Maliszewski C., Lotze M. T., Shurin M. R. FLT3-ligand administration inhibits liver metastases: role of NK cells.. J. Immunol., 161: 6164-6170, 1998.[Abstract/Free Full Text]
  18. Chakravarty P. K., Alfieri A., Thomas E. K., Beri V., Tanaka K. E., Vikram B., Guha C. Flt3-ligand administration after radiation therapy prolongs survival in a murine model of metastatic lung cancer.. Cancer Res., 59: 6028-6032, 1999.[Abstract/Free Full Text]
  19. Esche C., Subbotin V. M., Maliszewski C., Lotze M. T., Shurin M. R. FLT3 ligand administration inhibits tumor growth in murine melanoma and lymphoma.. Cancer Res., 58: 380-383, 1998.[Abstract/Free Full Text]
  20. Lin K-Y., Guarnieri F. G., Staveley-O’Carroll K. F., Levitsky H. I., August T., Pardoll D. M., Wu T-C. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen.. Cancer Res., 56: 21-26, 1996.[Abstract/Free Full Text]
  21. Chen C. H., Ji H., Suh K. W., Choti M. A., Pardoll D. M., Wu T. C. Gene gun-mediated, DNA vaccination induces antitumor immunity against human papillomavirus type 16 E7-expressing murine tumor metastases in the liver and lungs.. Gene Ther., 6: 1972-1981, 1999.[Medline]
  22. Ji H., Wang T-L., Chen C-H., Hung C-F., Pai S., Lin K-Y., Kurman R. J., Pardoll D. M., Wu T-C. Targeting HPV-16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine HPV-16 E7-expressing tumors.. Hum. Gene Ther., 10: 2727-2740, 1999.[Medline]
  23. Chen C-H., Wang T-L., Hung C-F., Yang Y., Young R. A., Pardoll D. M., Wu T-C. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene.. Cancer Res., 60: 1035-1042, 2000.[Abstract/Free Full Text]
  24. Bloom M. B., Perry-Lalley D., Robbins P. F., Li Y., el-Gamil M., Rosenberg S. A., Yang J. C. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.. J. Exp. Med., 185: 453-459, 1997.[Abstract/Free Full Text]
  25. Feltkamp M. C., Smits H. L., Vierboom M. P., Minnaar R. P., de Jongh B. M., Drijfhout J. W., ter Schegget J., Melief C. J., Kast W. M. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells.. Eur. J. Immunol., 23: 2242-2249, 1993.[Medline]
  26. Tindle R. W., Fernando G. J., Sterling J. C., Frazer I. H. A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes.. Proc. Natl. Acad. Sci. USA, 88: 5887-5891, 1991.[Abstract/Free Full Text]
  27. Wu T-C., Guarnieri F. G., Staveley-O’Carroll K. F., Viscidi R. P., Levitsky H. I., Hedrick L., Cho K. R., August T., Pardoll D. M. Engineering an intracellular pathway for MHC class II presentation of HPV-16 E7.. Proc. Natl. Acad. Sci. USA, 92: 11671-11675, 1995.[Abstract/Free Full Text]
  28. Dialynas D. P., Wilde D. B., Marrack P., Pierres A., Wall K. A., Havran W., Otten G., Loken M. R., Pierres M., Kappler J., et al Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen-reactivity.. Immunol. Rev., 74: 29-56, 1983.[Medline]
  29. Sarmiento M., Glasebrook A. L., Fitch F. W. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement.. J. Immunol., 125: 2665-2672, 1980.[Abstract]
  30. Koo G. C., Dumont F. J., Tutt M., Hackett J. , Jr., and Kumar, V.. The NK-1.1(-) mouse: a model to study differentiation of murine NK cells. J. Immunol., 137: 3742-3747, 1986.[Abstract]
  31. Fernandez N. C., Lozier A., Flament C., Ricciardi-Castagnoli P., Bellet D., Suter M., Perricaudet M., Tursz T., Maraskovsky E., Zitvogel L. Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate antitumor immune responses in vivo.. Nat. Med., 5: 405-411, 1999.[Medline]
  32. Wang T-L., Ling M., Shih I-M., Pham T., Pai S. I., Lu L., Kurman R. J., Pardoll D. M., Wu T-C. Intramuscular administration of E7-transfected dendritic cells generates the most potent E7-specific antitumor immunity.. Gene Ther., 7: 726-733, 2000.[Medline]
  33. Corr M., von Damm A., Lee D. J., Tighe H. In vivo priming by DNA injection occurs predominantly by antigen transfer.. J. Immunol., 163: 4721-4727, 1999.[Abstract/Free Full Text]
  34. Uger R. A., Barber B. H. Creating CTL targets with epitope-linked ß2-microglobulin constructs.. J. Immunol., 160: 1598-1605, 1998.[Abstract/Free Full Text]
  35. Murali-Krishna K., Altman J. D., Suresh M., Sourdive D. J., Zajac A. J., Miller J. D., Slansky J., Ahmed R. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.. Immunity, 8: 177-187, 1998.[Medline]
  36. Wu T-C., Huang A. Y. C., Jaffee E., Levitsky H., Pardoll D. M. A reassessment of the role of B7–1 in antitumor immunity.. J. Exp. Med., 182: 1415-1421, 1995.[Abstract/Free Full Text]
  37. Chklovskaia E., Jansen W., Nissen C., Lyman S. D., Rahner C., Landmann L., Wodnar-Filipowicz A. Mechanism of flt3 ligand expression in bone marrow failure: translocation from intracellular stores to the surface of T lymphocytes after chemotherapy-induced suppression of hematopoiesis.. Blood, 93: 2595-2604, 1999.[Abstract/Free Full Text]
  38. Shiver J. W., Davies M. E., Perry H. C., Freed D. C., Liu M. A. Humoral and cellular immunities elicited by HIV-1 vaccination.. J. Pharm. Sci., 85: 1317-1324, 1996.[Medline]
  39. Boyle J. S., Koniaras C., Lew A. M. Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocyte and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization.. Int. Immunol., 9: 1897-1906, 1997.[Abstract/Free Full Text]
  40. Hsu S. C., Obeid O. E., Collins M., Iqbal M., Chargelegue D., Steward M. W. Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-{gamma}.. Int. Immunol., 10: 1441-1447, 1998.[Abstract/Free Full Text]
  41. Porgador A., Irvine K. R., Iwasaki A., Barber B. H., Restifo N. P., Germain R. N. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.. J. Exp. Med., 188: 1075-1082, 1998.[Abstract/Free Full Text]
  42. Akbari O., Panjwani N., Garcia S., Tascon R., Lowrie D., Stockinger B. DNA vaccination: transfection and activation of dendritic cells as key events for immunity.. J. Exp. Med., 189: 169-178, 1999.[Abstract/Free Full Text]
  43. Williams N. S., Moore T. A., Schatzle J. D., Puzanov I. J., Sivakumar P. V., Zlotnik A., Bennett M., Kumar V. Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.. J. Exp. Med., 186: 1609-1614, 1997.[Abstract/Free Full Text]
  44. Shaw S. G., Maung A. A., Steptoe R. J., Thomson A. W., Vujanovic N. L. Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: implications for anticancer and antiviral therapy.. J. Immunol., 161: 2817-2824, 1998.[Abstract/Free Full Text]
  45. Sato Y., Roman M., Tighe H., Lee D., Corr M., Nguyen M. D., Silverman G. J., Lotz M., Carson D. A., Raz E. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.. Science (Washington DC), 273: 352-354, 1996.[Abstract]
  46. Klinman D. M., Yamshchikov G., Ishigatsubo Y. Contribution of CpG motifs to the immunogenicity of DNA vaccines.. J. Immunol., 158: 3635-3639, 1997.[Abstract]
  47. Sparwasser T., Koch E. S., Vabulas R. M., Heeg K., Lipford G. B., Ellwart J. W., Wagner H. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells.. Eur. J. Immunol., 28: 2045-2054, 1998.[Medline]
  48. Weiner G. J., Liu H. M., Wooldridge J. E., Dahle C. E., Krieg A. M. Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization.. Proc. Natl. Acad. Sci. USA, 94: 10833-10837, 1997.[Abstract/Free Full Text]
  49. Nichols W. W., Ledwith B. J., Manam S. V., Troilo P. J. Potential DNA vaccine integration into host cell genome.. Ann. NY Acad. Sci., 772: 30-39, 1995.[Abstract]
  50. Lukas J., Muller H., Bartkova J., Spitkovsky D., Kjerulff A. A., Jansen D. P., Strauss M., Bartek J. DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell’s requirement for cyclin D1 function in G1.. J. Cell Biol., 125: 625-638, 1994.[Abstract/Free Full Text]
  51. Heck D. V., Yee C. L., Howley P. M., Munger K. Efficiency of binding the retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses. Proc. Natl. Acad. Sci. USA, 89: 4442-4446, 1992.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
J. A. Triccas, E. Shklovskaya, J. Spratt, A. A. Ryan, U. Palendira, B. Fazekas de StGroth, and W. J. Britton
Effects of DNA- and Mycobacterium bovis BCG-Based Delivery of the Flt3 Ligand on Protective Immunity to Mycobacterium tuberculosis
Infect. Immun., November 1, 2007; 75(11): 5368 - 5375.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
F. Orlandi, F. M. Venanzi, A. Concetti, H. Yamauchi, S. Tiwari, L. Norton, J. D. Wolchok, A. N. Houghton, and P. D. Gregor
Antibody and CD8+ T Cell Responses against HER2/neu Required for Tumor Eradication after DNA Immunization with a Flt-3 Ligand Fusion Vaccine
Clin. Cancer Res., October 15, 2007; 13(20): 6195 - 6203.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C.-W. Liao, C.-A. Chen, C.-N. Lee, Y.-N. Su, M.-C. Chang, M.-H. Syu, C.-Y. Hsieh, and W.-F. Cheng
Fusion Protein Vaccine by Domains of Bacterial Exotoxin Linked with a Tumor Antigen Generates Potent Immunologic Responses and Antitumor Effects
Cancer Res., October 1, 2005; 65(19): 9089 - 9098.
[Abstract] [Full Text] [PDF]

Home page
<