Photodynamic therapy (PDT) of tumours is based on administration of a photosensitiser followed by irradiation of the tumour with visible light leading to production of reactive oxygen species that cause direct tumour cell death and vascular damage. antigen. Induction of P1A with 5-aza-2′-deoxycytidine a methyltransferase inhibitor resulted in potentiated antitumour effects in mice with Lewis lung carcinoma and 4T1 mammary carcinoma when combined with PDT treatment. In CT26 colon carcinoma and EMT6 mammary carcinoma models the combination therapy resulted in complete responses and long-term survival. All long-term surviving mice were resistant to re-inoculation with the same tumour cells. Antitumour efficacy of the combination treatment was severely impaired by depletion of CD8+ cytotoxic T cells whereas adoptive transfer of CD8+ T cells from long-term surviving mice allowed for significant tumour growth delay in tumour-bearing mice. Taken together these findings show that PDT leads to strong specific antitumour immune responses and that epigenetic modification of tumour antigens levels may be a novel approach to further enhance the effectiveness of PDT. The present results provide a strong rationale for clinical development of this therapeutic approach. experiments were performed in accordance with the guidelines approved by the Ethics Committee of the Medical University of Warsaw. 2.3 Reagents Photofrin (Axcan Pharma Inc. Houdan France) was used as a photosensitiser. For studies Photofrin was dissolved in Dulbecco’s modified Eagle’s medium and for studies Photofrin was reconstituted in 5% glucose. 5-aza-dC was purchased in Sigma-Aldrich and dissolved in 0.9% NaCl. ROCK inhibitor Diluents were used as controls. 2.4 Reverse transcription polymerase chain reaction (RT-PCR) Cells were washed with phosphate buffered solution (PBS) and lysed in 0.5 ml of TRIzol reagent (Invitrogen) and the total RNA was isolated according to modified Chomczynski method. Concentration and purity of RNA were determined with a NanoDrop ND-2000c spectrophotometer (Thermo Scientific). For cDNA synthesis 1 μg of RNA was used with oligo(dT) primer and Avian Myeloblastosis Virus (AMV) reverse transcriptase (EURx). PCR was performed using Mastercycler personal (Eppendorf) and Color OptiTaq polymerase (EURx) or OneTaq? 2× Master ROCK inhibitor Mix (NEB). Products of PCR amplification were analysed by electrophoresis in ethidium bromide stained 1% agarose gel visualised under UV light and photographed using Alpha Imager (Cell eBioscence). The primer sequences were as follows: GAPDH forward – 5′-CCCTTCATTGACCTCAACT ACATGG-3′ and GAPDH reverse – 5′-CCTGCTTCA CCACCTTCTTGATGTC-3′; P1A forward – 5′-CGGA ATTCTGTGCCATGTCTGATAACAAGAAA-3′ and P1A reverse – 5′-CGTCTAGATTGCAACTGCATGC CTAAGGTGAG-3′. 2.5 Cell isolation and re-stimulation Spleens and tumour draining lymph nodes (LNs) (popliteal inguinal axillary brachial) were isolated from mice of each experimental group. To obtain single cell suspension spleens and LNs were forced through a 70 μm cell strainer. In order to lyse erythrocytes cells were incubated for 5 min in 37 °C in 150 mM NH4Cl 1 mM NaHCO3 pH 7.4 buffer. Next cells were washed and resuspended in RPMI 1640 medium with 10% foetal bovine serum. Cells were then counted stained with antibodies and analysed in flow cytometry. For stimulation 2 × 106 cells were seeded on 96-well plate and incubated in RPMI supplemented with 10% foetal bovine serum and antibiotic/antimycotic solution for 6 h at 37 °C with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) and 2 mg/ml ionomycin (Sigma-Aldrich). GolgiPlug (BD Biosciences) was added during the last 4 h of culture. After incubation time plate was placed in 4 °C overnight. 2.6 Flow cytometry 2.6 Rabbit Polyclonal to Src. In vitro staining After a 72 h incubation with 5-aza-dC tumour cells were trypsinised washed with PBS and incubated with anti-H2kd or anti-H2kb antibodies (mAbs) coupled with fluorescein isothiocyanate ROCK inhibitor (FITC) (BD Bioscience) in PBS containing 1% bovine serum albumin (BSA) for 30 min in 4 °C. Cells were analysed in FACScan (Becton Dickinson) using CellQuest Pro Software Version 5.2. 2.6 Ex vivo staining After isolation and re-stimulation with PMA and ionomycin cells were stained with anti-CD4 conjugated with PerCp-Cy5 and anti-CD8 coupled with FITC mAbs. Subsequently cells.