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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Gene Ther. Author manuscript; available in PMC 2010 June 21.
Published in final edited form as:
PMCID: PMC2888272
NIHMSID: NIHMS199702

Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

Abstract

Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal timeframe. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation.

Keywords: HPV, DNA vaccine, calreticulin (CRT), E7

Introduction

DNA vaccines have emerged as an encouraging approach for antigen-specific immunotherapy due to their excellent safety profile, stability, and ease of production. Intradermal administration of DNA vaccines via a gene gun serves as an efficient method to deliver DNA directly into the professional antigen-presenting cells in the skin, the dendritic cells (DCs). These DCs express the DNA and mature and migrate to the draining lymph nodes, where they activate naive T cells to differentiate into activated helper or killer T cells in vivo.1,2 Thus, intradermal administration via a gene gun has enabled us to enhance DNA vaccine potency by using strategies to modify the properties of dendritic cells (for review, see 3,4).

One strategy to enhance DNA vaccine potency is to improve antigen expression, processing, and presentation in DCs using intracellular targeting strategies (for review, see 3,4). Several of our previous studies have employed DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) 510 and have shown that these vaccines are capable of enhancing the E7-specific CD8+ T cell immune responses in vaccinated mice. Furthermore, mice vaccinated with the DNA vaccines encoding CRT/E7 have been shown to generate stronger antitumor effects against E7-expressing tumors when compared with mice vaccinated with wild-type E7 DNA.

In general, current DNA vaccination strategies employ regimens of multiple intradermal or intramuscular administrations, with about 2 week intervals, and require at least 1 month to achieve immunity.11,12 This may be the cause for the relatively slow development of T cell immune responses after DNA vaccination in contrast to the rapid immune responses induced by physiological antigen encounter, such as viral infection. It has been shown that a cluster (short-interval) DNA vaccination method generates potent humoral as well as T cell-mediated immune responses within a minimal time frame.13

Thus, we aim to employ this novel cluster vaccination regimen in order to generate potent immune responses following DNA vaccination. In the current study, we hypothesized that the cluster intradermal CRT/E7 DNA vaccination would generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors, TC-1, in tumor-bearing mice. Furthermore, we reasoned that effective vaccination would result in an increase in apoptotic tumor cell death. Thus, we have investigated the effect of cluster intradermal CRT/E7 DNA vaccination in TC-1 tumor-bearing mice. Our data serve as an important foundation for future clinical translation.

Results

Cluster Intradermal CRT/E7 DNA Vaccination Rapidly Induces E7-Specific CD8+ T Cells Compared to Long Interval DNA Vaccination

In order to determine if cluster intradermal CRT/E7 DNA vaccination would rapidly induce E7-specific immune responses in vaccinated mice, we performed intracellular cytokine staining followed by flow cytometry analysis. C57BL/6 mice (5 per group) were vaccinated twice with pNGVL4a-CRT/E7(detox) DNA vaccine at either 4 days interval (short interval/cluster vaccination) or 7 days interval (long interval). The vaccination regimen is depicted in Figure 1A. Splenocytes were harvested on day 8 and day 14 after the initial vaccination and characterized for E7-specific CD8+ T cells using intracellular IFNγ staining followed by flow cytometry. As shown in Figure 1B, the cluster intradermal DNA vaccination schedule generated robust E7-specific CD8+ T-cell immune responses as measured on day 8 and day 14 (p<0.05). We also followed the immune responses for 3 weeks after the initial vaccination. We observed that the level of E7-specific CD8+ T cell immune responses were quite comparable between cluster and long interval DNA vaccination regimens (See Supplementary Figure 1). Thus, our data suggests that cluster intradermal DNA vaccination is capable of rapidly generating E7-specific CD8+ T cell immune responses in vaccinated mice compared to DNA vaccination using the long interval vaccination regimen.

Figure 1
Characterization of E7-specific CD8+ T cell immune responses in mice vaccinated with pNGVL4a-CRT/E7 (detox) DNA vaccine

Cluster Intradermal CRT/E7 DNA Vaccination is Capable of Generating Significant Therapeutic Antitumor Effects Against E7-Expressing Tumors

The ability of cluster DNA vaccination regimen to rapidly induce robust E7-specific T cell immune responses in vaccinated mice raised the possibility that this regimen may lead to better therapeutic antitumor effects against E7-expressing tumors compared to DNA vaccination using the long interval regimen. One explanation for such may be that the tumor load would be relatively less at the completion of cluster DNA vaccination. We therefore performed in vivo tumor treatment experiments using E7-expressing TC-1 tumors. C57BL/6 mice (5 per group) were inoculated with TC-1 tumor cells subcutaneously. The TC-1 tumor-bearing mice were then treated with the pNGVL4a-CRT/E7 (detox) DNA vaccine using either the cluster or long interval vaccination regimen. The vaccination regimen is depicted in Figure 2A. As shown in Figure 2B, we found that the tumor-bearing mice treated with the pNGVL4a-CRT/E7 (detox) DNA vaccine using the short interval cluster vaccination schedule demonstrated significantly better anti-tumor therapeutic effects, compared to the mice vaccinated using the long interval vaccination schedule (p<0.05). Thus, our data suggests that cluster intradermal DNA vaccination is capable of generating potent therapeutic antitumor effects against E7-expressing tumors.

Figure 2
In vivo antitumor effects generated by treatment with pNGVL4a-CRT/E7 (detox) DNA vaccine

Cluster Intradermal CRT/E7 DNA Vaccination in the Presence of Tumor Generates Significantly Higher E7-specific CD8+ T Cell Immune Response Compared to Vaccination after Tumor Resection

We further determined if the presence of tumor would influence the E7-specific CD8+ T cell immune responses generated by cluster pNGVL4a-CRT/E7(detox) DNA vaccination. C57BL/6 mice (5 per group) were inoculated subcutaneously with TC-1 tumor cells. Ten days later, these mice then received cluster intradermal vaccination with pNGVL4a-CRT/E7(detox) DNA vaccine (4aCRT/E7(detox) + TC-1). Another group of tumor bearing mice received tumor resection on day 10. Two days later, these mice were vaccinated with the same dose and regimen (4aCRT/E7(detox) - TC-1). As shown in Figure 3A, we observed a significantly higher number of E7-specific CD8+ T cells in the mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine in the presence of tumor compared to those in mice vaccinated with the same dose and regimen after tumor resection (p<0.05). A graphical representation of the number of E7-specific CD8+ T cells is depicted in Figure 3B. We also characterized the E7-specific T cells in TC-1 tumor-bearing mice without pNGVL4a-CRT/E7(detox) DNA vaccination. We found that only low levels of E7-specific T cells were detected in TC-1 tumor-bearing mice without the DNA vaccination compared to TC-1 tumor-bearing mice treated with pNGVL4a-CRT/E7(detox) DNA vaccine (see Supplementary Figure 2). Thus, our data indicate that cluster intradermal CRT/E7(detox) DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in vaccinated mice compared to vaccination after tumor resection.

Figure 3
Intracellular cytokine staining followed by flow cytometry analysis to characterize E7-specific CD8+ cell precursors in mice vaccinated with pNGVL4a-CRT/E7(detox)DNA vaccine before and after tumor resection

Cluster Intradermal Vaccination with CRT/E7(detox) in the Presence of Tumor Generated Significant E7-specific CD8+ T cell Immune Response in Tumor Infiltrating Lymphocytes and in Draining Lymph Nodes of Vaccinated Mice

In order to determine if cluster intradermal vaccination with CRT/E7(detox) in TC-1 tumor bearing mice will lead to an increase in the number of E7-specific CD8+ T cell immune responses in the tumor infiltrating lymphocytes and in draining lymph nodes compared to TC-1 tumor bearing mice vaccinated with CRT DNA, we performed intracellular cytokine staining followed by flow cytometry analysis. C57BL/6 mice (5 per group) were inoculated subcutaneously with TC-1 tumor cells. Ten days after tumor inoculation, the mice were vaccinated either with pNGVL4a-CRT/E7(detox) or pNGVL4a-CRT DNA vaccine. We observed a significantly higher number of E7-specific CD8+ T cells in tumor infiltrating lymphocytes (Figure 4A) as well as in the draining lymph nodes (Figure 4C) of the TC-1 tumor bearing mice vaccinated with pNGVL4aCRT/E7(detox) DNA vaccine compared to those in TC-1 tumor bearing mice vaccinated with control pNGVL4a-CRT DNA (p<0.05). A graphical representation of the number of E7-specific CD8+ T cells in tumor infiltrating lymphocytes as well as in the draining lymph nodes is depicted in Figure 4B and 4D respectively. Thus, our data indicate that cluster intradermal vaccination with pNGVL4a-CRT/E7(detox) in the presence of tumor generated significant E7-specific CD8+ T cell immune response in tumor infiltrating lymphocytes and in draining lymph nodes.

Figure 4Figure 4
Intracellular cytokine staining followed by flow cytometry analysis to characterize the E7-specific CD8+ cell precursors in the tumor and draining lymph nodes of tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox)DNA vaccine

Cluster Intradermal Vaccination with CRT/E7(detox) in the Presence of Tumor Generated Significantly Lower Levels of CD4+Foxp3+ T Regulatory cells and Myeloid Suppressor Cells in Peripheral Blood and in Tumor Infiltrating Lymphocytes Compared to Vaccination with CRT DNA

Effective vaccination has been shown to lead to a reduction of CD4+Foxp3+ T regulatory cells.14,15 It has been generally accepted that the high-levels of expression of Foxp3 is the most distinctive marker for the T regulatory lineage, particularly in the murine system (for review, see 16,17). Furthermore, myeloid suppressor cells have been shown to be important in the tumor microenvironment to modulate the T cell responses.18,19 Thus, in order to determine if cluster intradermal vaccination with pNGVL4a-CRT/E7(detox) in TC-1 tumor bearing mice would lead to a decrease in the number of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells in peripheral blood and in tumor infiltrating lymphocytes, we performed flow cytometry analysis. C57BL/6 mice (5 per group) were inoculated subcutaneously with TC-1 tumor cells. Ten days after tumor inoculation, mice were vaccinated intradermally via gene gun either with pNGVL4a-CRT/E7(detox) DNA vaccine or pNGVL4a-CRT DNA alone. We observed a significantly lower number of CD4+Foxp3+ T regulatory cells (p<0.05) (Figure 5A) and Gr-1+CD11b+ myeloid suppressor cells (p<0.05) (Figure 5C) in the peripheral blood of tumor bearing mice vaccinated with CRT/E7(detox) DNA vaccine compared to mice vaccinated with CRT DNA. A graphical representation of the number of CD4+Foxp3+ T regulatory cells and Gr-1+CD11b+ myeloid suppressor cells in the peripheral blood is depicted in Figure 5B and 5D respectively.

Figure 5
Flow cytometry analysis of CD4+Foxp3+ regulatory T cells and Gr-1+CD11b+ myeloid suppressor cells in peripheral blood of TC-1 tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine

We further characterized the number of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells in tumor infiltrating lymphocytes in tumor bearing mice vaccinated intradermally via gene gun either with CRT/E7(detox) DNA vaccine or CRT DNA. We observed a significantly lower number of CD4+Foxp3+ T regulatory cells (p<0.05) (Figure 6A) and CD124+CD11b+ myeloid suppressor cells (p<0.05) (Figure 6C) in the tumor infiltrating lymphocytes of tumor bearing mice vaccinated with CRT/E7(detox) DNA vaccine compared to mice vaccinated with CRT DNA. A graphical representation of the number of CD4+Foxp3+ T regulatory cells and CD124+CD11b+ myeloid suppressor cells in the tumor infiltrating lymphocytes is depicted in Figure 6B and 6D respectively. Taken together, our data indicate that cluster intradermal vaccination with CRT/E7(detox) in the presence of tumor generated significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells in peripheral blood and in tumor infiltrating lymphocytes in vaccinated mice. The significant reduction of the CD4+Foxp3+ T regulatory cells and myeloid suppressor cells in vaccinated mice are likely due to a smaller tumor load in the tumor-bearing mice treated with the DNA vaccine.20,21

Figure 6
Flow cytometry analysis of CD4+Foxp3+ regulatory T cells and CD124+CD11b+ myeloid suppressor cells in tumor infiltrating lymphocytes of TC-1 tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine

Cluster Intradermal Vaccination with CRT/E7(detox) in the Presence of Tumor Leads to Significantly Enhanced Apoptotic Tumor Cell Death in Vaccinated Mice

In order to determine the apoptotic tumor cell death induced by the cluster intradermal CRT/E7(detox) DNA vaccination, we performed a TUNEL assay on paraffin-embedded TC-1 tumor tissue sections. C57BL/6 mice (5 per group) were inoculated subcutaneously with TC-1 tumor cells. Ten days after tumor inoculation, mice were treated intradermally via gene gun either with CRT/E7(detox) DNA vaccine or CRT DNA alone. As shown in Figure 7A, we found that the tumor sections from mice treated with pNGVL4a-CRT/E7 (detox) DNA vaccine showed the highest numbers of positively stained apoptotic cells compared to tumor sections derived from untreated mice or mice treated with CRT DNA alone (p<0.001). A graphical representation of the number of apoptotic cells/ 200X HPF is depicted in Figure 7B. Thus, our data indicates that CRT/E7(detox) DNA vaccination leads to enhanced apoptotic tumor cell death which correlates with the enhanced E7-specific CD8+ T cell immune response in vaccinated mice.

Figure 7
TUNEL assays to determine the apoptotic tumor cell death induced by the cluster intradermal CRT/E7(detox) DNA vaccination

Discussion

In the current study, we have investigated the effect of cluster intradermal CRT/E7 DNA vaccination in E7 expressing TC-1 tumor-bearing mice. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells in peripheral blood and in tumor infiltrating lymphocytes and ultimately led to apoptotic tumor cell death. Thus, our data serve as an important foundation for future clinical translation. The success of the cluster vaccination strategy using intradermal administration via gene gun warrants the consideration of the cluster vaccination strategy in other DNA vaccination methods such as intramuscular administration of DNA vaccine with or without electroporation.

In the current study we observed that vaccination of TC-1 tumor-bearing mice treated with the pNGVL4a-CRT/E7(detox) DNA vaccine generated significantly higher numbers of IFNγ-secreting, E7-specific CD8+ T cells compared to tumor-free mice treated with the same DNA vaccine. Several reasons may account for the observed phenomenon. First, we recently observed that TC-1 tumor-bearing mice have higher frequencies of IFNγ-secreting, E7-specific CD8+ T cells compared to mice without TC-1 tumors (see Supplementary Figure 3). Therefore, the higher frequency of the E7-specific CD8+ T cells in TC-1 tumor-bearing mice (compared to tumor-free mice) may contribute to the observed higher numbers of E7-specific CD8+ T cell immune response following the pNGVL4a-CRT/E7(detox) DNA vaccination. Second, in the current study, we observed that TC-1 tumor-bearing mice treated with the pNGVL4a-CRT/E7(detox) DNA vaccine generated significantly higher numbers of tumor-apoptotic cell death compared to TC-1 tumor-bearing mice without the DNA treatment (see Figure 7). We have recently observed that increased apopototic tumor cell death of E7-expressing TC-1 tumors could result in increased frequency of E7-specific CD8+ T cells.22 Thus, the potential increase in apopototic cell death of E7-expressing TC-1 tumors following the initial DNA vaccine treatment may further contribute to the observed higher numbers of E7-specific CD8+ T cell immune response following the repeated pNGVL4a-CRT/E7(detox) DNA vaccination. These factors may contribute to the mechanism by which the vaccine induces higher number of T cells in the presence of E7-expressing tumor.

We also observed significant reduction in the numbers of CD4+Foxp3+ Treg cells and myeloid suppressor cells in peripheral blood as well as in tumor infiltrating lymphocytes in mice vaccinated with the pNGVL4a-CRT/E7(detox) DNA vaccine (see Figure 5 and Figure 6). It has been reported that the numbers of CD4+ Treg cells and myeloid suppressor cells closely correlate with the size of the tumor.20,21 Thus, the significant reduction of the CD4+Foxp3+ Treg and myeloid suppressor cells in vaccinated mice are likely due to a smaller tumor load in the tumor-bearing mice treated with the DNA vaccine. However, we cannot exclude the possibility of the direct influence of the DNA vaccine on the reduction of the numbers of CD4+Foxp3+ Treg and myeloid suppressor cells.

For clinical translation of the therapeutic HPV DNA vaccines, it is important to consider issues relating to safety. Concerns are raised regarding the potential for oncogenicity associated with administration of E7 as DNA vaccines into the body. Thus, it is important to use attenuated (detox) versions of E7 that has been mutated. It has been demonstrated that a mutation at E7 positions 24 and/or 26 will disrupt the Rb binding site of E7, abolishing the capacity of E7 to transform cells.23,24 In addition, clinical translation may preclude the use of the pcDNA3 vector, because it contains an ampicillin resistance gene. It will important to use an appropriate DNA vector such as the pNGVL4a vector, which was obtained from the NIH National Gene Vector Laboratory for DNA vaccine development. This vector is a second-generation plasmid derived from pNGVL-3, which has been previously used for human clinical trials.25 The pNGVL4a vector lacks the ampicillin resistance gene and thus would be suitable for clinical translation of the current DNA vaccines.

In fact, a phase I clinical trial is currently being planned using the cluster intradermal DNA vaccination regimen in stage IB1 cervical cancer patients (Drs. Ronald Alvarez and Cornelia Trimble, personal communication). In this trial, we aim to investigate whether the cluster intradermal CRT/E7 DNA vaccination is safe and able to generate E7-specific CD8+ T cell immune responses in patients with stage 1B1 cervical cancer. We have chosen stage IB1 patients because the standard care of stage IB1 cervical cancer patients allows us to resect the tumor and its draining lymph nodes, thus permitting us to evaluate the tumor microenvironment. The clustered vaccination regimen allows us to complete the vaccinations before tumor resection, thus making it possible to assess the changes in tumor microenvironment with or without DNA vaccination without compromising the standard care of the patient. The pNGVL4a-CRT/E7(detox) DNA vaccine has been formulated in a proprietary ND-10 gene gun device, that has been approved for clinical trials.

In summary, the current cluster intradermal DNA vaccine regimen is highly effective in inducing a rapid and potent E7-specific CD8+ T cell immune response, resulting in antitumor effects and apoptotic cell death in E7-expressing tumors. Thus, the current study has immense potential for clinical translation in the near future.

Materials and methods

Mice, cell lines, peptides and antibodies

C57BL/6 mice (6- to 8-week-old) were purchased from the National Cancer Institute (Frederick, MD). All animals were maintained under specific-pathogen free conditions, and all procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.

E7 expressing TC-1 cells were generated as previously described 26 and were maintained in RPMI medium supplemented with 2mM glutamine, 1mM sodium pyruvate, and 20mM HEPES, 5×10−5 M β-mercaptoethanol, 100 IU/ml penicillin, 100µg/ml streptomycin and 10% fetal bovine serum.

The H-2Db restricted HPV-16 E7 peptide, RAHYNIVTF (E7 aa49–57) 27 was synthesized by Macromolecular Resources (Denver, CO) at a purity of ≥70%. Antibodies against mouse CD4 (FITC-conjugated, clone RM4-5), Foxp3 (PE-conjugated, FJK-16s) were purchased from eBiosciences (San Diego, CA), CD11b (FITC-conjugated, clone M1/70), Ly6-G (Gr-1, FITC or APC-conjugated, clone RB6-8C5). Antibodies against mouse CD8 (PE-conjugated, clone 53.6.7), IFN-γ (FITC-conjugated, clone XMG1.2), CD124 (PE-conjugated, clone mIL4R-M1) were purchased from BD Pharmingen (San Diego, CA).

In vivo tumor treatment experiments

For in vivo tumor treatment experiments, C57BL/6 mice (five per group) were inoculated subcutaneously with 1×l05 TC-1 tumor cells per mouse on the left flank. Mice were monitored for tumor growth by measuring diameters with calipers twice a week.

Surgical resection of TC-1 tumors

TC-1 tumors were surgically excised on day 10 after tumor inoculation when the tumor size reached approximately 5–6 mm in diameter. Mice were anesthetized with isoflurane and tumors were excised and incisions were closed with 4-0 nylon. Mice with recurrent primary tumors after surgery (less than 10%) were excluded from the study.

DNA vaccination

Generation of pNGVL4a-CRT and pNGVL4a-CRT/E7(detox) has been described previously.10 E7(detox) represents an E7 gene with mutations at E7 positions 24 and 26 which disrupt the Rb binding site of E7. The mutation at these locations abolishes the capacity of E7 to transform cells.23,24 Clinical grade pNGVL4a-CRT/E7(detox) plasmids were prepared by NIH RAID. For the gene gun-mediated intradermal vaccination, DNA-coated gold particles (1 µg DNA/bullet, 2 bullets per vaccination) were delivered to the shaved abdominal region of C57BL/6 mice using a helium-driven gene gun device from BioRad (BioRad, Hercules, CA, USA), or ND-10 gene gun device from PowderMed/Pfizer with a discharge pressure of 400 p.s.i., according to a previously described protocol.28 Naïve C57BL/6 mice or TC-1 tumor-bearing mice (d10 after tumor inoculation), or TC-1 tumor postsurgical mice (2 days after resection) were vaccinated twice with 2 µg/mouse of pNGVL4a-CRT/E7(detox) DNA vaccine at either 4 days interval (short interval/cluster vaccination) or 7 days interval (long interval) as depicted in Figure 1A.

Preparation of single-cell suspensions from TC-1 tumors

One week after last vaccination, TC-1 tumors were surgically excised under sterile conditions and placed in RPMI 1640 medium containing 100 U/ml penicillin, 100µg/ml streptomycin on ice, and washed with PBS. A fraction of the tumors were either snap-frozen in Tissue-Tek O.C.T. compound immediately (Sakura Finetek, Torrence, CA) or fixed with 10% Formalin. The remaining solid tumors were then minced into 1–2-mm pieces and immersed in serum-free RPMI-1640 medium containing 1 mg/ml collagenase D, and 0.25 mg/ml DNase I (both are from Roche), 100U/ml penicillin, 100 µg/ml streptomycin, and incubated at room temperature overnight with gentle agitation. It was then filtered through a 70-µm nylon filter mesh to remove undigested tissue fragments. The resultant single tumor cell suspensions and tumor infiltrating lymphocytes were washed twice in HBSS (400 × g for 10 min), and viable cells were determined using trypan blue dye exclusion.

Intracellular cytokine staining and flow cytometry analysis

Fresh single-cell suspensions were prepared and 1×106 cells were used for flow cytometry analysis. For cell surface markers staining, cells were stained with indicated conjugated antibodies at 4°C for 30 minutes. The cells were washed twice with FACS wash buffer (PBS containing 0.5% BSA). For Foxp3 staining, cells stained for surface markers were fixed and permeabilized. After wash, the cells were then stained with anti-Foxp3 antibody. The cells were then washed twice before analysis. For the characterization of the CD4+Foxp3+ Treg cells in the tumor infiltrating lymphocytes, the gated lymphocytes were used for analysis.

For intracellular IFN-γstaining, 5×106 pooled splenocytes from each vaccination group were incubated with 1 µg/ml of the HPV-16 E7 aa49-57 peptide in the presence of GolgiPlug (BD Pharmingen, San Diego, CA) at 37°C overnight. Cells were then washed once with FACS wash buffer and stained with phycoerythrin-conjugated monoclonal rat antimouse CD8a. Cells were subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer’s instruction (BD Pharmingen, San Diego, CA). Intracellular IFN-γwas stained with FITC-conjugated rat antimouse IFN-γ. Flow cytometry analysis was performed using FACSCalibur with CELLQuest software (BD biosciences, Mountain View, CA).

Apoptotic assays

Terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) assay was done on paraffin-embedded tumor tissues using In Situ Cell Death Detection Kit, POD (Roche, Indianapolis, IN). Tissue sectioning was done by the Reference Histology Laboratory of Johns Hopkins University. The assay was done according to the manufacturer's instruction. Briefly, tissue sections were deparaffinized, permeabilitied with the proteinase K (Roche) at 37°C for 15min, then the slides were incubated in 3% hydrogen peroxide in PBS at room temperature for 10min. The sections were sequentially incubated in the labeling mixture contained terminal nucleotidyl transferase (TdT) and digoxigenin-labeled dUTP for 60min at 37°C. Then, the converter-POD (a peroxidase-conjugated antidigoxigenin antibody) was added to the tissue sections and the product was visualized by staining with the substrate DAB (3,3’-diaminobenzidine tetrahydrochloride) (St. Louis, MO, Sigma), followed by counterstaining with Hematoxylin (Dako, Carpinteria, CA). Tissue sections processed without TdT enzyme were used as the negative control. Only nuclear staining was considered as positive. The data was generated using 200X HPF from 10 different fields for each tumor. Three tumors were used for each group.

Statistical analysis

All numerical data are expressed as means ±s.d.. Comparisons between experimental and control groups were made using Student’s t test. All p values less than 0.05 were considered statistically significant.

Supplementary Material

Acknowledgements

This work was supported by the Flight Attendant Medical Research Institute and National Cancer Institute SPORE in Cervical Cancer P50 CA098252 and the 1 RO1 CA114425-01.

References

1. Porgador A, Irvine KR, Iwasaki A, Barber BH, Restifo NP, Germain RN. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization. J Exp Med. 1998;188:1075–1082. [PMC free article] [PubMed]
2. Condon C, Watkins SC, Celluzzi CM, Thompson K, Falo LD., Jr DNA-based immunization by in vivo transfection of dendritic cells. Nat Med. 1996;2:1122–1128. [PubMed]
3. Hung CF, Yang M, Wu TC. Modifying professional antigen-presenting cells to enhance DNA vaccine potency. Methods Mol Med. 2006;127:199–220. [PubMed]
4. Tsen SW, Paik AM, Hung CF, Wu TC. Enhancing DNA Vaccine Potency by Modifying the Properties of Antigen-Presenting Cells. Expert Review of Vaccines. 2007;6:227–239. [PubMed]
5. Cheng WF, Hung CF, Chai CY, Hsu KF, He L, Ling M, et al. Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen. J Clin Invest. 2001;108:669–678. [PMC free article] [PubMed]
6. Kim TW, Lee JH, Hung CF, Peng S, Roden R, Wang MC, et al. Generation and characterization of DNA vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus. J Virol. 2004;78:4638–4645. [PMC free article] [PubMed]
7. Peng S, Ji H, Trimble C, He L, Tsai YC, Yeatermeyer J, et al. Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6. J Virol. 2004;78:8468–8476. [PMC free article] [PubMed]
8. Peng S, Tomson TT, Trimble C, He L, Hung CF, Wu TC. A combination of DNA vaccines targeting human papillomavirus type 16 E6 and E7 generates potent antitumor effects. Gene Ther. 2006;13:257–265. [PMC free article] [PubMed]
9. Peng S, Trimble C, Ji H, He L, Tsai YC, Macaes B, et al. Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies. J Biomed Sci. 2005;12:689–700. [PubMed]
10. Kim JW, Hung CF, Juang J, He L, Kim TW, Armstrong DK, et al. Comparison of HPV DNA vaccines employing intracellular targeting strategies. Gene Ther. 2004;11:1011–1018. [PubMed]
11. Gurunathan S, Klinman DM, Seder RA. DNA vaccines: immunology, application, and optimization. Annu Rev Immunol. 2000;18:927–974. [PubMed]
12. Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. DNA vaccines. Annu Rev Immunol. 1997;15:617–648. [PubMed]
13. Bins AD, Jorritsma A, Wolkers MC, Hung CF, Wu TC, Schumacher TN, et al. A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression. Nat Med. 2005;11:899–904. [PubMed]
14. Nitcheu-Tefit J, Dai MS, Critchley-Thorne RJ, Ramirez-Jimenez F, Xu M, Conchon S, et al. Listeriolysin O expressed in a bacterial vaccine suppresses CD4+CD25high regulatory T cell function in vivo. J Immunol. 2007;179:1532–1541. [PubMed]
15. Powell DJ, Jr, Felipe-Silva A, Merino MJ, Ahmadzadeh M, Allen T, Levy C, et al. Administration of a CD25-directed immunotoxin, LMB-2, to patients with metastatic melanoma induces a selective partial reduction in regulatory T cells in vivo. J Immunol. 2007;179:4919–4928. [PMC free article] [PubMed]
16. Belkaid Y. Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol. 2007;7:875–888. [PubMed]
17. Colombo MP, Piconese S. Regulatory-T-cell inhibition versus depletion: the right choice in cancer immunotherapy. Nat Rev Cancer. 2007;7:880–887. [PubMed]
18. Bunt SK, Yang L, Sinha P, Clements VK, Leips J, Ostrand-Rosenberg S. Reduced inflammation in the tumor microenvironment delays the accumulation of myeloid-derived suppressor cells and limits tumor progression. Cancer Res. 2007;67:10019–10026. [PubMed]
19. Nagaraj S, Gupta K, Pisarev V, Kinarsky L, Sherman S, Kang L, et al. Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med. 2007;13:828–835. [PMC free article] [PubMed]
20. Yu P, Lee Y, Liu W, Krausz T, Chong A, Schreiber H, et al. Intratumor depletion of CD4+ cells unmasks tumor immunogenicity leading to the rejection of late-stage tumors. J Exp Med. 2005;201:779–791. [PMC free article] [PubMed]
21. Sinha P, Clements VK, Bunt SK, Albelda SM, Ostrand-Rosenberg S. Cross-talk between myeloid-derived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. J Immunol. 2007;179:977–983. [PubMed]
22. Kang TH, Lee JH, Song CK, Han HD, Shin BC, Pai SI, et al. Epigallocatechin-3-gallate enhances CD8+ T cell-mediated antitumor immunity induced by DNA vaccination. Cancer Res. 2007;67:802–811. [PubMed]
23. Edmonds C, Vousden KH. A point mutational analysis of human papillomavirus type 16 E7 protein. J Virol. 1989;63:2650–2656. [PMC free article] [PubMed]
24. Munger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, Grace M, et al. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene. 2001;20:7888–7898. [PubMed]
25. Trimble C, Lin CT, Hung CF, Pai S, Juang J, He L, et al. Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun, biojector, and syringe. Vaccine. 2003;21:4036–4042. [PubMed]
26. Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, et al. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 1996;56:21–26. [PubMed]
27. Feltkamp MC, Smits HL, Vierboom MP, Minnaar RP, de JB, Drijfhout JW, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol. 1993;23:2242–2249. [PubMed]
28. Chen CH, Wang TL, Hung CF, Yang Y, Young RA, Pardoll DM, et al. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Cancer Res. 2000;60:1035–1042. [PubMed]