Vaccination with the Prostate Cancer Over-Expressed Tumor Self-Protein TPD52 Elicits Protective Tumor Immunity and a Potentially Unique Subset of CD8+ T Cells

Research Article

Austin J Clin Immunol. 2014;1(2): 1007.

Vaccination with the Prostate Cancer Over-Expressed Tumor Self-Protein TPD52 Elicits Protective Tumor Immunity and a Potentially Unique Subset of CD8+ T Cells

Jennifer D Bright1, Joel F Aldrich1, Jennifer A Byrne2 and Robert K. Bright1,*

1Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center

2Children’s Cancer Research Unit, University of Sydney Discipline of Pediatrics and Child Health

*Corresponding author: Robert K. Bright, Department of Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, 3601 4th Street, MS 6591, Lubbock, TX 79430

Received: January 16, 2014; Accepted: February 10, 2014; Published: February 17, 2014

Abstract

Tumor protein D52 (D52) is expressed at low levels in normal cells, but over-expressed in prostate carcinomas and numerous other malignancies. Murine D52 (mD52) parallels the expression pattern of the human orthologue (hD52) and shares ~ 86% amino acid identity. Over-expression of mD52 in nontransformed murine fibroblasts induces anchorage independent growth and spontaneous metastasis. The TRAMP model was employed to study DNA-based D52 vaccines against prostate cancer. Immunizations consisted of mD52-DNA, hD52-DNA or a combination of both, followed by challenge with mD52 positive, TRAMP-C1 tumor cells. Greater protection (70%) was observed 10 months post challenge in mice immunized with hD52 DNA. Survivors of the initial tumor challenge rejected a second tumor challenge with mD52 positive, autochthonous TRAMP-C2 tumor cells given in the opposite flank more than four months after the first challenge. Analysis of the T cell function from survivors indicated that a Th1-type cellular immune response was involved in tumor rejection. A potentially unique subset of CD8+ IL-10+ T cells was also elicited and may play a role in inhibiting vaccine induced tumor immunity, suggesting that a deeper mechanistic understanding of these T cells in D52 vaccine-induced immunity may be important for developing a more potent cancer vaccine.

Keywords: Prostate; Vaccine; hD52; mD52; TPD52; Murine; TRAMP; CD8+ regulatory T cells.

Abbreviations

TPD52: Tumor protein D52; mD52: murine TPD52; hD52: human TPD52; TAA: tumor associated antigen; T regulatory cell, Treg

Introduction

A 2009 National Cancer Institute sponsored project to prioritize cancer vaccine target antigens for translational-research revealed that over expressed tumor self-proteins represent the largest number of untested antigens for vaccine development [1]. While it is arguable that antigens that are only found in tumors and not normal cells should be “ideal” targets for vaccination, most cancer antigens that have been isolated from tumor cells to date are self-proteins, specifically they are expressed at low levels in normal cells and over expressed in tumor cells, a characteristic that facilitated their discovery [2]. Until now only Her-2/neu could be classified as an over expressed tumor self-antigen that demonstrates a role in oncogenicity. This property has been proposed to be a desired and important characteristic for the next generation of cancer vaccine target antigens [1]. We recently described a novel over expressed tumor self-antigen, tumor protein D52 (D52). D52 represents a shared tumor antigen with a wide range of cancer associations to include but not limited to breast, prostate and ovarian cancers [3], and like Her-2/neu, D52 exhibits oncogenic properties [4,5].

Tumor protein D52 is a naturally expressed intracellular protein present at low but detectable levels in healthy cells and tissues where its normal function has yet to be defined. Increased expression of D52 has been demonstrated in association with prostate cancer as well as numerous other human malignancies [6-22]. The murine orthologue of D52 (mD52) is ~ 86% identical to human D52 (hD52) at the amino acid level [23]. Previous work from our laboratory demonstrated that over expression of mD52 in normal murine fibroblast cells induced anchorage independent growth in vitro and spontaneous lung metastasis in vivo [5]. We also demonstrated that reduction of hD52 expression via RNAi resulted in increased apoptosis in human breast cancer cells and hD52 over-expression correlated with decreased survival in human breast cancer patients [4]. Interestingly, shRNA reduction of mD52 expression abrogates spontaneous metastasis associated with murine 3T3.mD52 sarcoma cells [unpublished observation]. Thus, D52 is actively involved in transformation, leading to increased cell proliferation and metastasis. Involvement in oncogenesis suggests that these antigens may be critical for tumor survival, making the over expressed tumor self-protein D52 an excellent candidate for a cancer vaccine target.

Herein, we tested the hypothesis that the xenogeneic human orthologue of D52 (hD52) when administered i. m. as a simple DNAbased vaccine would elicit an anti-tumor immune response that is more potent than that elicited by the mD52 as assessed by protection from challenge with autochthonous TRAMP-C tumor cells which naturally contain elevated levels of mD52 protein [24].

Materials and Methods

Mice and tumor cell lines

Male 6- to 8-week old C57BL/6 mice were purchased from Jackson Labs (Bar Harbor, ME). All animals were cared for and treated according to Institutional Animal Care and Use Committee (IACUC) guidelines at Texas Tech University Health Sciences Center (Lubbock, TX). All experiments were conducted with IACUC approval. The tumorigenic, autochthonous C57BL/6 cell lines TRAMP-C1 and TRAMP-C2 [25] were used for tumor challenge and as targets for immunoassays. The tumorigenic SV40-transformed Balb/c murine kidney cell line designated mKSA was used as an mD52-positive MHC mis-matched control target for immunoassays [24,26,27].

Purification and validation of plasmid DNA used for immunization

Luria- Bertani (LB) broth supplemented with ampicillin (100ug/ ml) was inoculated with a starter culture of JM109 bacterial cells transformed with pcDNA, mD52pCDNA or hD52pcDNA plasmid and grown overnight with shaking (300 rpm) at 37oC. Bacterial cells were lysed, and plasmid DNA purified using Qiagen’s Endo Free Plasmid Purification kit (Valencia, CA) according to the manufacturer’s instructions. DNA concentrations were calculated using a bio-photometer (Eppendorf, Westbury, NY) [26]. Restriction enzyme digests were performed on all plasmids to confirm the presence of mD52 or hD52 cDNA inserts. Endpoint PCR was performed using primers specific for mD52 or hD52 for 30 cycles to confirm presence of the respective cDNA insert. Primer sequences: mD52cds-F-5’- TGC TGA AGA CAG AGC CGG, mD52cds-R-5’- ACG TCT TGC CAC CCT TTG, hD52-F-5’- GAT CTC GGG CTG GAG ACA TGG, hD52-R-5’- AAT TCG TGG GTA GCA GAA CAA AGG. Annealing temperatures used were 62oC and 60oC for mD52cds and hD52 primers, respectively. Primers for GAPDH were used as an internal control reference in PCR experiments [5]. To confirm mD52 and hD52 protein expression 3T3 cells were transfected with the vaccine plasmids containing cDNAs for mD52 or hD52, and whole cell protein lysates were prepared using methods previously described [4,28]. Protein expression was detected by Western analysis using an anti-TPD52 polyclonal antibody (generated by immunizing rabbits with N-terminal, carrier conjugated peptide GCAYKKTSETLSQAGQKAS; italics represents a region of TPD52 protein that is conserved between human and mouse) (Bio Synthesis, Inc, Lewisville, TX) [5].

Immunization and tumor cell challenge

Individual mice were immunized with 50 micrograms of D52- DNA administered i.m. in saline every 10 days for a total of 4 injections. Empty vector DNA (pCDNA 3.1 vector minus mD52 cDNA) served as a control immunization. Two weeks following the final immunization, mice in all groups were challenged with a tumorigenic dose (5x106) of autochthonous TRAMP-C1 tumor cells [25]. Mice that survived the primary challenge were re-challenged in the opposite flank with 1x106 TRAMP-C2 cells [25] approximately 150 days after the initial challenge. For some experiments, the TRAMP-C2 challenge dose was 5 X 105 cells, which was determined empirically to be 100% tumorigenic. Tumor size was determined by taking perpendicular measurements with calipers every 2 to 3 days and tumor volume (mm3) was calculated using the following formula: (a x b2) / 2, where b was the smaller of the two measurements [26,27].

To assess a role for CD4+ CD25+ or CD8+ CD122+ regulatory T cells in response to mD52 DNA vaccination, mice were injected i.p. with 300 μg of anti-CD25 mAb (PC-61.5.3), or anti-CD122 mAb (TM-beta 1) ), or both in 200 μl PBS on day 0, and again on day 28 at the time of the first and third mD52-DNA immunizations. At the time of tumor challenge (day 58), mice were injected i.p. with 600 μg of anti-CD25 mAb, or anti-CD122 mAb, or both in 200 μl PBS. For control (mock) depletions, mice were injected i.p. with isotype matched IgG on day 0, day 28 and at the time of tumor challenge with 300 μg, 300 μg and 600 μg, respectively.

T cell culture and ELISAs for cytokine production

T cells from immunized mice were stimulated in vitro by culturing Lympholyte-M® gradient separated spleen-derived lymphocytes with irradiated tumor cells (the same tumor cell line used for the in vivo challenge) in the presence of IL-2 (10 ng/ml), IL-7 (5 ng/ml), and IL-12 (5 ng/ml) at 37°C for 5-7 days. Culture supernatants used for cytokine analyses were harvested from 24 hr cultures of T cells (1x106 cells / ml in 200 μl of medium in 96 well plates) in medium alone, compared to T cells cultured with various tumor cell targets (1:1 ratio). Experimental targets were the TRAMP-C1 tumor cells (H-2b+, mD52+) and TRAMP-C2 tumor cells (H-2b++, mD52+). mKSA (H-2d+, mD52+) tumor cells, served as a control MHC mismatched, antigen positive target. Yac-1 cells served as an MHC-I negative control. To confirm MHC-I restricted tumor recognition, blocking assays were performed by incubating tumor cells with anti-H-2b or anti-H-2d (negative control) mAb, prior to incubation with T cells. Assessment of cytokine secretion by tumor-specific T cell cultures was accomplished by applying culture supernatants to commercially available sandwich ELISA’s for IFN-γ, IL-10, IL-4, and IL-17 detection (R&D Systems, Minneapolis,MN) as per the manufacturer’s instructions. Assays were analysed using a Victor3™ plate reader (Perkin Elmer, Boston, MA). We performed all assays with the manufacturer’s provided internal controls, from which standard curves were generated in order to determine concentration of cytokines produced in experimental sets for ELISA detection of IFN-γ, IL-10, IL-4, and IL-17 [26, 27].

Analysis of cytotoxic T lymphocyte (CTL)-mediated tumor cell lysis

T cells from spleens of immunized mice that survived tumor challenge were isolated and subjected to standard cytotoxic T lymphocyte (CTL)-mediated tumor cell lysis analysis. CTLs were generated by culturing spleen cells in the presence of irradiated tumor cells (using the same tumor cell line as was used for the in vivo challenge) in the presence of IL-2 (10 ng/ml), IL-7 (5 ng/ml), and IL- 12 (5 ng/ml) at 37°C for 5-7 days. Specificity was evaluated by mixing various numbers of CTLs with a constant number of target cells (5 x 103 cells per well) in 96 well round bottom plates. Specific lysis was determined using either a Europium time-resolved fluorescencebased method or LDH-release method, and measured using a Victor3™ plate reader (Perkin Elmer, Boston, MA) using previously described methods [27, 28, 29].

Flow cytometry

Lymphocytes from spleens cultured in vitro as described above were stained with monoclonal antibodies specific for CD3, CD4, CD19 and CD8. MHC class-I expression was assessed on tumor cell lines. Antibodies were purchased from BD-Bioscience (San Jose, CA). For determination of CD25+ Treg cell depletion, peripheral blood lymphocytes (PBLs) were collected 7 days following the 4th immunization via tail vein bleed, and lymphocytes were isolated using Lympholyte-M® density separation medium (Cedarlane Labs, Burlington, NC). Lymphocytes from animals in the same experimental group were pooled (n=10 per pooled sample) and stained with 1 μg each of anti-CD4-FITC and anti-CD25-PE or anti-CD122-PE mAbs per 1 x 106 cells. Antibodies were purchased from BD-Bioscience (San Jose, CA). Cells were fixed in 1% paraformaldehyde at 4oC for 1 hr and then analyzed by flow cytometry using a BD LSRII flow cytometer [26,27].

Real-time RT-PCR

Total RNA was extracted from pure T cell cultures that were generated by 7 day in vitro stimulation of lymphocytes from D52- DNA immunized mice with CD3, CD28 activation beads according to the manufacturer’s instructions (Life Tech, InVitrogen). For real-time RT-PCR, cDNA was generated using 1 μg of total RNA and oligo-dT primer and PCR reactions were performed using our previously published methods [5]. Primer sets for the targets depicted in figure 6b were purchased from Realtimeprimers.com (http://www.realtimeprimers.com/real-time-pcr-primer-sets-mouse-pcr-primersets.html). Reactions were performed using the Applied Bio systems Step One Plus Sequence Detection System and ABI SYBR green PCR core reagents kit, according to the manufacturer’s instructions (Applied Bio systems, Foster City, CA). PCR conditions for 40 cycles were: 60oC for 2 min, 95oC for 10 min, 95oC for 15 s, for all primers. Additional controls involved no template and no RT enzyme and were included in all real-time PCR reactions.

Statistical analysis

ELISA data were analyzed using one-way ANOVA with Bonferoni multiple comparison post test. A p value of less than 0.05 was determined to be statistically significant (Graph Pad Prism 5.0). Specific lysis data for CTL assays were analyzed using one-way ANOVA with Tukey-Kramer post test. A p value less than 0.05 was determined to be significant (Graph Pad Prism 5.0). Tumor challenge data were analyzed with a t-test to determine whether significant differences existed between mean tumor volume for D52-DNA immunized and control immunized mice [27].

Results

Intramuscular D52-DNA vaccination induces protective tumor immunity

Previously we reported on DNA-based vaccination against D52 using the TRAMP model of prostate cancer for which mD52-DNA was injected s.c. admixed with murine GM-CSF protein [26]. Though partial protection was observed following autochthonous TRAMP-C tumor challenge (40 % protected > 90 days), we postulated that vaccine approaches with xenogeneic hD52 might increase tumor rejection efficacy. To simplify the approach, we immunized mice i.m. with either hD52- DNA, mD52-DNA, hD52-DNA followed by mD52-DNA or mD52-DNA followed by hD52-DNA, and then challenged mice with a tumorigenic dose of TRAMP-C1 cells (Figure 1A). All four DNA vaccine approaches protected mice from tumor challenge. The majority of mice (70%) that received four injections of hD52-DNA remained free from TRAMP-C1 tumor growth for nearly eight months (Figure 1B), compared to 50% of mice that received four injections of mD52 prior to tumor challenge (Figure 1C). Similar tumor protection results were obtained for the primeboost approach, where mice received either two injections of hD52- DNA followed by two injections of mD52-DNA (60%) (Figure 1D) or two injections of mD52-DNA followed by two injections of hD52- DNA (70%) (Figure 1E). Interestingly, the average onset of tumor growth was delayed by nearly 21 days in mice that were immunized with hD52-DNA compared to the other vaccine groups (Figure 1CE). Overall the protective tumor immunity induced by i.m. D52-DNA vaccination was considerable and did not vary greatly whether the vaccine consisted of hD52-DNA, mD52-DNA or a combination of both. Control vaccines with empty vector DNA did not protect against tumor challenge (not shown) [26].