Cross-presentation of the oncofoetal tumor antigen 5T4 from irradiated prostate cancer cells - a key role for Hsp70

Immune responses contribute to the success of radiation therapy of solid tumors; however, the mechanism of triggering CD8 T cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8 T cell stimulation. We established a crosspresentation model in prostate cancer in which DC present a naturally expressed oncofoetal tumor antigen (5T4) from irradiated DU145 tumor cells to 5T4-specific T cells. Ionising radiation (12 Gy) caused G2/M cell cycle arrest and cell death, increased cellular 5T4 and high-mobility protein group-B1 (HMGB1) levels and upregulated surface calreticulin and Hsp70 expression in DU145 cells. Co-culture of DC with irradiated tumor cells lead to efficient phagocytosis of tumor cells and upregulation of CD86 and HLA-DR on DC. CD8 5T4-specific T cells, stimulated with these DC, proliferated and produced IFNγ. Inhibition of HMGB1 decreased T cell stimulation but not DC activation, while TRIF/MyD88 inhibition only had a marginal effect on T cell stimulation. Unlike previous reports, we found no functional evidence that DC with Asp299Gly toll-like receptor-4 (TLR4) single nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, we observed a highly significant and robust prevention of antigen cross-presentation when tumor cells were pre-treated with the novel Hsp70 inhibitor, VER155008. The inhibitor also prevented CD86 upregulation on DC co-cultured with irradiated tumor cells. Together, our study demonstrates that radiation induces immunologically relevant changes in tumor cells, which can trigger CD8 T cell responses via a predominantly Hsp70-dependent antigen cross-presentation process. Antigen cross-presentation from irradiated tumor cells 3 Introduction Traditional treatments of cancer such as surgery, chemotherapy and radiotherapy (RT) have been shown to trigger immune responses, which may contribute towards treatment outcome. Radiation is curative in up to 40% of patients with early stage (localized) prostate cancer (PCa) but it is yet unclear what the predictors of complete responses are. RT in PCa has been shown to be associated with increased frequencies of tumor antigen-specific CD8 and CD4 T cells (1). The abscopal effect of radiation (tumor regression at a distant site following localized radiation) has been shown to be immune-mediated as demonstrated not only in mouse tumor models (2, 3) but also in patients with metastatic melanoma and lung adenocarcinoma (4, 5). Furthermore, CD8 T cell infiltration in the irradiated tumor tissue serves as a prognostic factor (4-8) indicating that radiation can switch the immunosuppressive tumor milieu to a pro-immune environment. For solid tumors, tumor antigen-specific CD8 T cell responses can be induced either by tumor cells entering lymph nodes and presenting antigens directly (9) or by denriti cells (DC) cross-presenting tumor antigens, either in the lymph node or in ectopic lymphoid tissues, as observed in melanoma (10, 11). Efficient crosspresentation requires tumor cell damage or cell death, associated with upregulation and/or release of danger signals or damage associated molecular patterns (DAMP). The precise nature of immunogenic cell death (ICD) is not well defined but generally involves surface translocation of “eat me” signals such as calreticulin (CRT) and stress-associated proteins such as Hsp70, release of chemoattractant molecules such as ATP and DAMP such as high-mobility protein group-B1 (HMGB1). However, there seems to be considerable plasticity in the combination and extent of these changes. The type of trigger causing cell damage and cell death may influence the relative proportions of key ICD events (12). Our study focuses on ionising radiation, which is known to cause DNA damage, cell cycle arrest and cellular damage response triggering either DNA repair or cellular senescence and apoptotic, necrotic or necroptotic cell death. Irradiated cells initiate post-phagocytic immune responses (13). The early release of IFNα/β by irradiated tumor cells can polarize antigen-presenting cells and aids their cross-presentation function (14). High dose (10-100 Gy) in vitro radiation of human tumor cells enhances CRT translocation to the cell surface and dose-dependent release of HMGB1 and ATP by breast, colon and prostate cancer cell lines (15). These typical ICD markers facilitate phagocytosis of damaged/dead cells and provide maturation signals for DC. Hsp70 translocation and release are also welldocumented consequences of radiation contributing to radiation resistance (16) with potential immunological effects (17). The aim of our study was to determine the mechanism of radiation-mediated antigen cross-presentation in PCa. As antigen cross-presentation studies often use artificially overexpressed antigens which may provide false positive results, we established a model focussing on a naturally expressed oncofoetal antigen, 5T4 (18), which is expressed in most solid tumors, including PCa. The cross-presentation model we established enabled us to study the effect of irradiated tumor cells on DC phenotypic and functional maturation and the use of specific inhibitors to reveal the mechanism of the cross-presentation process. We show here that unlike in crosspresentation induced by anthracyclines (19), in radiation-induced tumor antigen crosspresentation the toll-like receptor-4 (TLR4) agonist-triggered TRIF/MyD88 pathway (20) plays only a partial role and the Asp299Gly TLR4 single nucleotide polymorphism (SNP) is not associated with any impairment of CD8 T cell responses. Instead, we found that Hsp70 is crucially important both in activating DC and Antigen cross-presentation from irradiated tumor cells 4 triggering CD8 T cell responses via DC co-cultured with irradiated tumor cells. Our results highlight the plasticity of the tumor antigen cross-presentation process and demonstrate the important immunological role of Hsp70 following tumor radiation. Antigen cross-presentation from irradiated tumor cells 5 Materials and Methods Media and Reagents RPMI 1640 (Lonza, UK) was supplemented with low endotoxin fetal bovine serum (FBS, PAA Laboratories, Austria) 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (all from Gibco, UK), 25 mM Hepes buffer and 1 mM sodium pyruvate (both Sigma, UK). 1% AB-serum (Sigma) was added where indicated. LPS and Glycyrrhizin were obtained from Sigma, VER155008 from Tocris Bioscience (R&D Systems, UK), MyD88 inhibitory peptide (and control) from ProImmune (Oxford, UK) and TRIF inhibitory peptide (and control) from Invivogen (San Diego, CA). Tumor cells and irradiation DU145 human prostate cancer cells were obtained from ECACC and maintained in adherent cultures with regular passaging following trypsinization for less than 6 months. The cell line was authenticated by the supplier using cytogenetic, isoenzymatic and DNA profile analyzes. HLA typing results show that DU145 cells are HLA-A2 (and encode for HLA-A03, A33, B50, B57) (Welsh Blood Transfusion Service, Cardiff). The cells were mycoplasma-free, as tested monthly using a MycoAlert Mycoplasma Detection Kit (Lonza). Cells used for experiments were in exponential phase of growth. Irradiation was carried out using a Cs-source (with dosimetry quality assurance) at a rate of 0.627 Gy/min. Donors, dendritic cell preparation Ethical approval was granted for the study and informed consent was obtained from healthy volunteer donors. HLA Class I and II DNA typing was carried out as above. PBMC from venous blood collected in EDTA vacutainers was prepared by density gradient centrifugation. CD14 monocytes were isolated by negative selection using the EasySep Human Monocyte Enrichment Kit without CD16 Depletion (Stem Cell Technologies, Grenoble, France) according to the manufacturer’s protocol. The average purity of CD14 cells was 70-80%. The isolated cells were incubated at 5x10 cells/well in a 6 well tray and grown in 5ml/well in 10% FBS-RPMI in the presence of 500 ng/ml human recombinant (hr) GM-CSF (ProSpec, Israel) and 500 U/ml hr IL4 (Gentaur, Belgium) for 5-6 days. T cell and B cell line A CD8 T-cell line was developed from a HLA-A2 healthy donor by repeated stimulation of non-adherent PBMC with autologous DC, loaded with 2 μg/ml 5T417-25 peptide (RLARLALVL; 90.4% purity; ProImmune), as described previously (21). For the experiments, T-cells (1–2×10) were expanded using a mixture of 5×10 peptidepulsed autologous B lymphoblastoid cells (BLCL) irradiated with 40 Gy; 5×10 allogeneic PBMC mixed from 2 to 3 donors and irradiated with 30 Gy; 50 U/ml IL-2 and 1 μl/ml OKT3 hybridoma supernatant (MRC Cooperative, Cardiff University) in 50 ml RPMI supplemented as described above and with 10% FBS and 1% AB-serum in a T75 tissue culture flask (21). BLCL was developed from the same donor using EBV transformation of B cells from PBMC, following standard protocols. Immunocytochemistry DU145 cells were grown on coverslips and incubated for 72h after 0Gy or 12Gy radiation. They were fixed with acetone/methanol 1:1 (vol/vol) and labeled with Antigen cross-presentation from irradiated tumor cells 6 FITC-conjugated Hsp70 antibody (Enzo Life Sciences, Farmingdale, NY). The slides were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and visualized next day using an Axiovert 40 fluorescence microscope (Zeiss, Jena, Germany).