cross-presentation of the oncofetal tumor antigen 5t4...

Research Article

Cross-Presentation of the Oncofetal TumorAntigen 5T4 from Irradiated Prostate CancerCells—A Key Role for Heat-Shock Protein 70 andReceptor CD91Josephine Salimu1, Lisa K. Spary1, Saly Al-Taei2, Aled Clayton1, Malcolm D. Mason1,John Staffurth1, and Zsuzsanna Tabi1

Abstract

Immune responses contribute to the success of radiotherapyof solid tumors; however, the mechanism of triggering CD8þ

T-cell responses is poorly understood. Antigen cross-presen-tation from tumor cells by dendritic cells (DC) is a likelydominant mechanism to achieve CD8þ T-cell stimulation.We established a cross-presentation model in which DCspresent a naturally expressed oncofetal tumor antigen (5T4)from irradiated DU145 prostate cancer cells to 5T4-specificT cells. The aim was to establish which immunogenic signalsare important in radiation-induced cross-presentation. Radi-ation (12 Gy) caused G2–M cell-cycle arrest and cell death,increased cellular 5T4 levels, high-mobility protein group-B1(HMGB1) release, and surface calreticulin and heat-shockprotein-70 (Hsp70) expression in DU145 cells. DCs phago-cytosed irradiated tumor cells efficiently, followed by upregu-lation of CD86 on phagocytic DCs. CD8þ 5T4-specific T cells,

stimulated with these DCs, proliferated and produced IFNg .Inhibition of HMGB1 or the TRIF/MyD88 pathway only hada partial effect on T-cell stimulation. Unlike previous inves-tigators, we found no evidence that DCs carrying Asp299GlyToll-like receptor-4 (TLR4) single-nucleotide polymorphismhad impaired ability to cross-present tumor antigen. However,pretreatment of tumor cells with Hsp70 inhibitors resulted ina highly statistically significant and robust prevention ofantigen cross-presentation and CD86 upregulation on DCscocultured with irradiated tumor cells. Blocking the Hsp70receptor CD91 also abolished cross-presentation. Together,the results from our study demonstrate that irradiationinduces immunologically relevant changes in tumor cells,which can trigger CD8þ T-cell responses via a predominantlyHsp70-dependent antigen cross-presentation process. CancerImmunol Res; 3(6); 678–88. �2015 AACR.

IntroductionTraditional treatments of cancer, such as surgery, chemother-

apy, and radiotherapy, have been shown to trigger immuneresponses, which may contribute toward treatment outcome.Radiation is curative in up to 40% of patients with early-stage(localized) prostate cancer, but it is not yet clear what are thepredictors of complete responses. Radiotherapy in prostatecancer has been shown to be associated with increased fre-quencies of tumor antigen-specific T cells (1). The abscopaleffect of radiation (tumor regression at a distant site followinglocalized radiation) has been shown to be immune mediated

not only in mouse tumor models (2, 3) but also in patientswith metastatic melanoma and lung adenocarcinoma (4, 5).Furthermore, CD8þ T-cell infiltration in the irradiated tumortissue serves as a prognostic factor (4–7), indicating that radi-ation can switch the immunosuppressive tumor milieu to aproimmune environment.

For solid tumors, tumor antigen–specific CD8þ T-cellresponses can be induced either by tumor cells entering lymphnodes (8) or dendritic cells (DC) cross-presenting tumor antigenseither in lymph nodes or ectopic lymphoid tissues present insome tumors (9, 10). Efficient cross-presentation requirestumor cell damage or cell death and is associated with transloca-tion or release of damage-associated molecular patterns (DAMP).The precise nature of immunogenic cell death (ICD) is not welldefined but generally involves surface translocation of "eat me"signals, such as calreticulin (CRT), and stress-associated proteins,such as Hsp70. The release of chemoattractant molecules andHMGB1, representing DAMPs, also has been observed. However,there seems to be considerable plasticity in the combinationand extent of these changes. The type of trigger causing celldamage and cell death may influence the relative proportions ofkey ICD events (11). Our study focuses on ionizing radiation,which is known to cause primarily DNA damage, cell-cycle arrest,and cellular-damage responses. These changes can trigger eitherDNA repair or cellular senescence and also apoptotic, necrotic,

1Institute of Cancer and Genetics, School of Medicine, Cardiff Univer-sity, Cardiff, United Kingdom. 2Velindre Cancer Centre, Velindre NHSTrust, Cardiff, United Kingdom.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Zsuzsanna Tabi, Institute of Cancer and Genetics,Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff CF14 2TL, UnitedKingdom. Phone: 44-2920-196137; Fax: 44-2920-529625; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-14-0079

�2015 American Association for Cancer Research.

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or necroptotic cell death. The early release of IFNa/b by irradia-ted tumor cells can polarize antigen-presenting cells and aidtheir cross-presenting function (12). High-dose (10–100 Gy)in vitro irradiation of tumor cells enhances CRT translocationto the cell surface and dose-dependent release of HMGB1 andATP by breast, colon, and prostate cancer cell lines (13). Thesetypical ICDmarkersmay facilitate phagocytosis of damaged/deadcells and provide maturation signals for DCs (14).

The aim of our study was to determine the relative importanceof immunogenic signals in radiation-mediated tumor antigencross-presentation. As antigen cross-presentation studies oftenuse artificially overexpressed antigens, which may provide false-positive results, we established a model focusing on a naturallyoccurring oncofetal antigen, 5T4, which is expressed in mostsolid tumors (15). This cross-presentation model enabled us tostudy the effect of irradiated tumor cells on DC phenotypic andfunctional maturation, while the use of specific inhibitorsrevealed the main players of the cross-presentation process. Weshow here that in radiation-induced tumor antigen cross-pre-sentation, the Toll-like receptor 4 (TLR4) pathway is not themajor mechanism and the Asp299Gly TLR4 single-nucleotidepolymorphism (SNP) is not associated with any impairment ofthe process. Instead, we found that Hsp70 is crucially importantboth in activating DCs and triggering CD8þ T-cell responses toDCs cocultured with irradiated tumor cells. Our results highlightthe plasticity of tumor antigen cross-presentation and demon-strate the important immunologic role of Hsp70 followingtumor irradiation.

Materials and MethodsMedia and reagents

RPMI-1640 (Lonza) was supplemented with fetal bovineserum (FBS; PAA), AB-serum (Sigma) where indicated, 100 U/mLpenicillin, 100 mg/mL streptomycin, 2 mmol/L L-glutamine(Gibco), 25 mmol/L HEPES, and 1 mmol/L sodium pyruvate(Sigma). Lipopolysaccharide (LPS), oxaliplatin, and glycyrrhi-zin were obtained from Sigma, VER155008 and 2-phenylethy-nesulfonamide (PES) from Tocris Bioscience (R&D Systems),inhibitory peptide (and control) of MyD88 from ProImmune(Oxford), and TRIF from Invivogen.

Tumor cells and treatmentDU145 prostate cancer cells were obtained from the European

Collection of Animal Cell Cultures (ECACC) and maintained inculture with regular passaging for less than 6 months. Authenti-cation was carried out by the supplier using cytogenetic, isoen-zymatic, andDNAprofile analysis. TheHLA type ofDU145 cells isHLA-A03/A33/B50/B57 (Welsh Blood Transfusion Service, Car-diff, United Kingdom). The cells were Mycoplasma free, as testedmonthly using a MycoAlert Mycoplasma Detection Kit (Lonza).Irradiation was carried out using a 137Cs-source (with dosimetryquality assurance) at a rate of 0.627 Gy/min. Oxaliplatin (Sigma)was used at a dosage of 20 mmol/L.

Donors and DC preparationEthical approval was granted and informed consent was

obtained from healthy volunteers. HLA class-I typing was carriedout as above. Peripheral blood mononuclear cells (PBMC) fromvenous blood, collected in EDTA vacutainers, were prepared bydensity gradient centrifugation. CD14þ monocytes were isolated

by negative selection using the EasySepHumanMonocyte Enrich-ment Kit without CD16 Depletion (STEMCELL Technologies).Average purity of CD14þ cells was 70% to 80%. Cells wereincubated at 5�106 cells per well in 6-well trays in 5 mL/well of10% FBS-RPMI plus 500 ng/mL human recombinant GM-CSF(ProSpec) and 500 U/mL IL4 (Gentaur) for 5 to 6 days.

T-cell and B-cell linesA CD8þ T-cell line was developed from a HLA-A2þ healthy

donor by repeated stimulation of nonadherent PBMCs withautologous DCs loaded with 2 mg/mL 5T417-25 peptide (RLAR-LALVL; ProImmune), as described previously (16). T cells(1–2�106) were expanded with a mixture of 5 � 106 peptide-pulsed autologous B lymphoblastoid cells (BLCL) irradiatedwith 40 Gy; 5 � 107 allogeneic PBMCs from 2 to 3 donors,irradiated with 30 Gy; 50 U/mL IL2 and 1 mL/mL OKT3 hybrid-oma supernatant in 50 mL RPMI, supplemented as above, andwith 10% FBS and 1% AB-serum (16).

ImmunocytochemistryDU145 cells were seeded on coverslips and left untreated or

were irradiated with 12 Gy. After 72 hours, cells were fixed with a1:1 (v/v) mixture of ice-cold acetone:methanol for 5 minutes.After drying, cells were blocked in 1% BSA/PBS for 1 hour, thenstained with an anti-Hsp70 antibody (Enzo Life Sciences), and agoat anti-mouse Alexa Fluor 488 secondary (Life Technologies).Nuclei were stained with DAPI. Images were gathered on a ZeissObserver Z.1 microscope, fitted with an Apotome 2 module forstructured illumination, using a 63�/1.4 numerical aperture oilimmersion objective, and an Axiocam 506 monochrome camerasystem. Representative images from Z-axis sections were overlaidto generate maximum projection images.

Flow cytometryCells were labeled in flow cytometry buffer (PBS, 1 mmol/L

EDTA and 2% FBS) with fluorochrome-conjugated 5T4, CRT,HMGB1 (R&D Systems), Hsp70 (Enzo Life Sciences), CD91 (BDPharmingen), TLR4, HLA-DR, CD86, and CD83 (eBioscience)antibodies or unconjugated SREC-I (R&D Systems) followed by agoat anti-mouse Alexa Fluor 488 (Invitrogen) antibody andincubated on ice for 40 minutes. For intracellular labeling, thecells were fixed and permeabilized with eBioscience Fix/Permreagents before antibodies were added for 40 minutes at roomtemperature. For cytokine flow cytometry, T cells were fixed andpermeabilized as above andCD3, CD8, and IFNg antibodies wereadded together for 40 minutes. Flow cytometry was carried outusing a FACSCanto flow cytometer with FACSDiva software (BDBioscience).

For cell-cycle analysis, Guava Cell Cycle Reagent (Millipore)was used; cell death was assessed using the Annexin-V/PropidiumIodide (PI) Apoptosis Detection Kit (BD Bioscience).

For phagocytosis assays, DU145 cells at 72 hours after irradi-ation were labeled with 5 mmol/L CFSE (eBioscience) and werecocultured with DCs (5 � 104 cells/well; 1:1 ratio) in a 96-wellU-bottomed plate for 24 hours at 37�C. After incubation, the cellswere surface labeled with HLA-DR or CD86 antibodies.

Western blottingCell pellets were resuspended in 1-mL cold RIPA Lysis Buffer

(Santa Cruz Biotechnology) with freshly added protease and

Antigen Cross-Presentation from Irradiated Tumor Cells

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phosphatase inhibitors. Cells were incubated on ice for 30 min-utes, vortexing every 10 minutes, followed by centrifugation at14,000 � g for 15 minutes. Protein estimation from the super-natant was carried out using the BCA assay.

Five micrograms of protein was loaded and separated onNuPage 4% to 12% Bis–Tris gels under reducing conditions andtransferred onto polyvinylidene difluoride (PVDF) membranesusing the iBlot Gel Transfer Stack System (Life Technologies).The membrane was blocked with 5% nonfat dry milk andprobed with a sheep polyclonal IgG 5T4 antibody (R&D Sys-tems; AF4975) or a mouse monoclonal IgG1 anti-Hsp70 anti-body (C92F3A-5) and with a mouse monoclonal antibodyfor GAPDH (0411; both from Santa Cruz Biotechnology) for1 hour at room temperature followed by horseradish peroxi-dase–conjugated secondary antibody (Santa Cruz Biotechno-logy). Bands were developed by ECL on film (GE Healthcare).Relative density of the bands exposed was calculated using theImageJ Software.

HMGB1 ELISASupernatants from 0- or 12 Gy–irradiated DU145 cells grown

in T25 flasks (5 � 105 input cell number) were collected 0, 24,48, and 72 hours after irradiation and tested using an HMGB1ELISA kit (IBL International) according to the manufacturer'sprotocol.

Inhibition of TRIF, MyD88, HMGB1, Hsp70, and its receptorsThe MyD88 and TRIF inhibitory peptides that correspond

to the sequence of the BB-loop of MyD88 (RDVLPGT) andTRIF (FCEEFQVPGRGELH), respectively, serve as decoys bybinding to the TIR domains and interfering with TLR–adaptorinteractions (17, 18). The control peptides consist of the pro-tein transduction sequence alone, which renders the peptidescell-permeable. DCs were pretreated with 20 mmol/L ofMyD88 or 25 mmol/L of TRIF inhibitory or control peptide,respectively, for 6 hours before LPS stimulation (100 ng/mL)or with 25 mmol/L of both when adding DCs to DU145 cellsfor the cross-presentation assay.

Glycyrrhizin, an HMGB1-inhibitor, was added at 50 mmol/L atthe time of irradiation, while VER155008, an Hsp70-inhibitor,at 5 mmol/L to 0- and 12 Gy–irradiated DU145 cells at 0, 24, and48 hours of the 72-hour incubation, respectively. PES was addedto DU145 cells at 20 mmol/L at the time of irradiation. Hsp70receptor blocking was carried out by treating DCs with theSREC-I–specific purified goat IgG polyclonal antibody (R&DSystems) or CD91 mouse IgG1 monoclonal antibody (ThermoScientific) or relevant isotypes (R&D Systems) at 1 mg/mL for1 hour before adding DCs to DU145 cells.

Antigen cross-presentationDU145 cells were set up in two 96-well U-bottomed plates at 5

� 103 cells per well. One plate was irradiated with 12 Gy beforeincubation for 72 hours. DCswere then added to thewells at a 1:1DU145:DC ratio. After 48 hours, 5T4-specific T cells were added ata 1:1:5 (DU145:DC:T cell) ratio. Golgi Plug (0.5 mL/500mL) andGolgi Stop (0.35 mL/500 mL; Sigma) were added to the wells 1hour later and the cultures were incubated overnight. Cytokineflow cytometry was carried out to determine the percentage ofIFNgþCD8þ T cells.

T-cell proliferationDU145 cells were plated in two 24-well plates at 1 � 105cells

per well in 1.5 mL. One plate was irradiated with 12 Gy and 105

DCs in 0.5mLwas added to the wells. After 4 hours, CSFE-labeledT cells (5 � 105cells/well) were added and incubated for 5 days.Flow cytometry analysis of CFSE dilution in CD3þCD8þ cells wascarried out.

SNP analysisSNP analysis was performed by the Department of Medical

Genetics (Cardiff and Vale NHS Trust, University Hospital ofWales, Cardiff, United Kingdom). DNA amplification was carriedout from blood or established BLCLs by PCR followed by pyr-osequencing optimized for the Asp299Gly sequence of the TLR4polymorphism. Out of 67 samples tested, 4 donors were found tocarry the Asp299Gly SNP.

Statistical analysisStatistical analysis was carried out by applying the Student

t test, paired t test, and ANOVA with the Tukey post hoc test(GraphPad InStat 3.06). Statistically significant differences aremarked as �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

ResultsIrradiation induces immunologically relevant changes intumor cells

To establish the optimum minimal radiation dose causingsignificant changes in DU145 cells in vitro, dose-escalation andtime-kinetics experiments were performed. A significant propor-tion of irradiated cells was arrested in the G2–Mphase, detectablefirst at 24 hours after irradiation. There was a small but significantradiation dose-dependent increase at 48 hours in the proportionof cells in sub-G0 phase representing apoptotic cells with frag-mented DNA (Fig. 1A). The type of cell death, detected withAnnexin/PI-labeling, was mainly late apoptotic/necrotic and theproportion of cells with early apoptotic markers remained lowthroughout the 72-hour incubation (Fig. 1B). Other radiation-associated immunologically relevant changes were also observed.The total cellular HMGB1 content increased with early timekinetics (Fig. 1C, i), while significant amounts of HMGB1 werereleased from the cells, detectable from 48 hours after irradiationby ELISA (Fig. 1C, ii). Surface MHC class-I expression was notaltered by irradiation (not shown), but the cellular content of thetarget antigen, 5T4, was significantly elevated following 12-Gyradiation (Fig. 1D, i and ii) as detected by flow cytometry andconfirmed by Western blotting (Fig. 1E).

Irradiation of tumor cells induces Hsp70 cytoplasmictranslocation and surface expression

Radiation resulted in the significant upregulation of surfaceHsp70 on DU145 cells (Fig. 2A). Multicolor flow cytometryanalysis confirmed that upon irradiation in a large proportionof cells with cell surface Hsp70 expression CRT was also translo-cated to the cell surface; this double-positive subset was notobserved without irradiation (Fig. 2B, right vs. middle plot).Immunocytochemistry confirmed that while Hsp70 expressionwas predominantly nuclear in untreated DU145 cells, 72 hoursafter irradiation, cytosolic and cell surface expression becamedominant (Fig. 2C and Supplementary Fig. S1). Although a slightincrease in Hsp70 content was observed 3 hours after irradiation

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in DU145 cells by Western blotting (Fig. 2D), it was not assignificant as that observed 2 hours after 42�C heat treatment;by 72 hours after irradiation, no increase was detectable. Thissuggests that Hsp70's cellular localization may be important inantigen cross-presentation.

DC activation and tumor antigen cross-presentation followinguptake of DU145 cells

Next, we studied whether irradiated tumor cells are taken upby DCs and if they activate DCs and trigger their antigen-presenting function. Tumor cells were labeled with CFSE 72

hours after irradiation and added to DCs at a 1:1 ratio. Phago-cytosis was measured 24 hours later by determining the pro-portion of HLA-DRþCFSEþ cells. Although some uptake ofnonirradiated tumor cells was observed (Fig. 3A, i), the pro-portion of phagocytic DCs increased significantly uponencountering irradiated DU145 tumor cells (Fig. 3A, ii andiii). DC phenotype following phagocytosis of irradiated ornonirradiated tumor cells was also studied 24 hours afterDC:tumor cell coculture. CD86 expression was significantlyelevated on DC cultured with irradiated, but not with nonir-radiated, tumor cells (Fig. 3B). Furthermore, CD86 expression

Figure 1.Radiation causes cell-cycle arrest,cell death, and changes toimmunologically relevantmolecules inDU145 cells. A, cell-cycle analysis ofDU145 cells irradiated with increasingdose (x-axis) and incubated for 4, 24,or 48 hours, as indicated above thegraphs. B, percentage of DU145 cellsundergoing different types of celldeath, as indicated, following 12-Gyirradiation and cultures up to 72 hours(x-axis). C, i, HMGB1 expression infixed and permeabilizedDU145 cells atdifferent times after 12-Gy irradiation(x-axis) shown as mean fluorescenceintensity (mfi); ii, soluble HMGB1, asdetected by ELISA in the supernatantof DU145 cells as in (i). D, i, 5T4expression in fixed and permeabilizedDU145 cells 48 hours after irradiation;D, ii, representative FACS histograms.A–D, i, mean þ SEM of results fromtriplicate samples. E, Western blottingof 5T4 antigen in DU145 cell lysatewith or without irradiation. Raw data(left), adjusted density (right). Allexperiments were repeated two tothree times.






was significantly higher on those DCs that phagocytosed tumorcells, that is, that were positive for CFSE (Fig. 3C, cells in Q2of Fig 3A, ii) compared with those in Q1.

5T4 antigen cross-presentation from DU145 tumor cells byHLA-A2þDCs was measured by assessing proliferation and intra-cellular cytokine production of 5T4-peptide-specific T cells (16).Although some T-cell activation was triggered by DCs culturedwith nonirradiated DU145 cells, significantly more T-cell prolif-eration and IFNg production was observed when DCs werecocultured with irradiated DU145 cells (Fig. 3D and E). T-cellstimulation in the cross-presentation model was HLA class-I–restricted, as a class-I blocking antibody completely inhibitedT-cell stimulation (Fig. 3F, i). DU145 cells were not unique intheir ability to serve as antigen donors for 5T4-specific T-cell

activation, as 5T4-positive PC3 (prostate) and M38 (mesotheli-oma) cells behaved the same way (Fig. 3F, ii). LNCaP cells,expressing no or little 5T4, triggered no significant T-cellresponses. The experiments demonstrate that DCs are able topresent a naturally expressed tumor antigen to specific T cellsand that this process is significantly enhanced by tumor cellirradiation.

TLR4-MyD88 signaling is not the critical pathway in antigencross-presentation from irradiated tumor cells

Tumor antigen cross-presentation from tumor cells treatedwith chemotherapy, especially with anthracyclines, has beenstudied more extensively than that from irradiated tumor cells.However, it has been suggested that in both cases, antigen cross-presentation crucially depends on the TLR4–HMGB1 interactionand consequently patients with TLR4 polymorphism are un-able to mount immune responses to tumor antigens (19). Tostudy TLR4–HMGB1 signaling in antigen cross-presentation fromirradiated tumor cells, we applied Glycyrrhizin (GA), anHMGB1-inhibitor (20), to tumor cells before irradiation. HMGB1 inhibi-tion resulted in a small decrease in CD86 expression on DCsafter their coculture with tumor cells (Fig. 4A, i), and also a smallbut significant decrease in T-cell IFNg production (Fig. 4A, ii).HMGB1 can bind to multiple receptors, such as TLR2 and TLR4,so next we targeted the MyD88/TRIF signaling pathway withinhibitory peptides. We established that 20 to 25 mmol/L of theinhibitory peptides significantly reduced LPS-induced TNFa pro-duction by DCs compared with that of control peptides (Fig. 4B).Although neither TRIF nor the MyD88 inhibitory peptide alone(25 mmol/L each) had any effect on T-cell stimulation (notshown),when applied together, they resulted in a small inhibitoryeffect (Fig. 4C), indicating the importance of pathways other thanTLRs in antigen cross-presentation.

TLR4 polymorphism does not affect antigen cross-presentationfrom irradiated tumor cells

To elucidate whether TLR4 SNP results in impaired antigencross-presentation, as it has been shown to do with oxaliplatin-treated tumor cells (19), we conducted a series of experimentswith DCs generated from monocytes of donors carrying thenormal (Asp299; n ¼ 5) or the polymorphic Gly299 (n ¼ 4)TLR4 allele. All donors were HLA-A2þ. The general character-ization of monocytes and DCs revealed that TLR4 expressionlevels (Fig. 5A, i and ii) and LPS-induced TNFa production werecomparable between the two groups (Fig. 5A, iii). Phenotypicmaturation of DCs (CD86, HLA-DR, and CD83) carrying theSNP allele following coculture with irradiated tumor cells wasalso unimpaired (Fig. 5B). Most importantly, cross-presenta-tion of 5T4 from irradiated tumor cells was similarly efficientby DCs carrying the Asp299 or the Gly299 TLR4 allele, respec-tively (Fig. 5C). To see whether TLR4 SNP was affectingchemotherapy-induced but not radiation-induced tumor anti-gen cross-presentation, T-cell responses were also studied withoxaliplatin-treated tumor cells. T-cell responses were generallyweaker than those induced by DCs cocultured with irradiatedtumor cells, but significantly elevated responses were observedin one out of three normal subjects and 2 out of 3 TLR4 SNPdonors (Fig. 5C). Despite the low number of donors, the experi-ments clearly show that donors with TLR4 SNP are able to cross-present tumor antigen from either irradiated or chemotherapy-treated tumor cells.

Figure 2.Hsp70 translocation in irradiated tumor cells. A, Hsp70 surface expressionwas measured by flow cytometry on DU145 cells 24 or 72 hours afterirradiation. Mean þ SEM of Hsp70 mean fluorescence intensity (mfi; minusisotype control mfi values) from triplicate samples are shown. B,representative dot plots of DU145 cells 72 hours after irradiation, showingCRT (x-axis) and Hsp70 (y-axis) expression or isotype control (first panel).The numbers represent Hsp70 single-positive (top left) or Hsp70þ

CRTþ double-positive cells (top right). C, Hsp70-FITC (green) and DAPI(blue)-labeled DU145 cells 72 hours after 0-Gy (left) or 12-Gy (right)irradiation. Magnification, �63 (Axiovert-40, Zeiss). The white arrowsindicate surface Hsp70 expression. D, Western blotting of Hsp70 antigenin DU145 lysate, treatments are: (1) positive control: 42�C heat treatmentfor 2 hours followed by 1-hour incubation at 37�C; (2) 12-Gy irradiationfollowed by 72-hour incubation; (3) 12-Gy irradiation followed by 3-hourincubation; (4) no treatment. Blots are shown on left, adjusted densityon right.

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Hsp70 inhibition blocks antigen cross-presentationThe contribution of heat-shock proteins to antigen cross-pre-

sentation has been demonstrated in several models; we per-formed experiments to establish whether Hsp70 plays a role

Figure 4.Partial effect of HMGB1–TLR4 pathway inhibitors on DCmaturation and antigencross-presentation. A, DCs were cocultured with 0 Gy or 12 Gy–treated DU145cells in the presence or absence of 50 mmol/L glycyrrhizin (GA) for 72 hours.CD86 upregulation on DCs (i) and 5T4-specific T-cell stimulation (ii) wereanalyzed by flow cytometry. The columns show mean þ SEM of results fromtriplicate cultures. B, DCs were treated with LPS in the presence of inhibitorypeptides targeting MyD88 (20 mmol/L) or TRIF (25 mmol/L). Control peptides(cell-permeable domain of the inhibitory peptide) were used at the sameconcentrations. Mean þ SEM of the percentage of TNFa-producing DCsare shown, as detected by cytokine flow cytometry. C, DCs were cultured ina cross-presentation assay in the presence of MyD88 and TRIF inhibitorypeptides together or with control peptides (25 mmol/L each). Mean þ SEM ofpercentage of IFNgþ T cells from triplicate cultures are shown. The experimentswere repeated two to three times.

Figure 3.DC maturation and antigen cross-presentation by irradiated tumor cells. A,representative dot plot showing uptake of CFSE-prelabeled DU145 cells (x-axis) after 0-Gy (i) or 12-Gy (ii) irradiation by DCs (HLA-DRþ cells).Phagocytic DCs are in the top right quadrant (Q2). iii, summary of resultsfrom 5 donors; each symbol represents the mean percrentage of DCs in Q2from triplicate samples per donor. B, flow cytometry analysis of CD86expression after 24-hour coculture of DCs without (Nil) or with 0-Gy or12-Gy irradiated DU145 cells. Mean þ SEM of CD86 mean fluorescenceintensity (mfi) from triplicate cultures is shown. C, flow cytometry ofCD86 expression, analyzed on DCs gated as Q1 or Q2 DCs, respectively,after DCs coculture with DU145 cells. D, i, proliferation of CFSE-labeled 5T4-specific T cells 5 days after stimulation by autologous DCs coculturedwith DU145 cells, as indicated. Mean þ SEM of CFSE (mfi) of T cells fromtriplicate cultures are shown; ii, representative histograms of CFSE dilutionin T cells. The numbers represent the percentage of T cells that proliferated.E, 5T4-specific T cells were stimulated overnight with DCs (from 6donors), cocultured with DU145 cells. Each symbol represents the mean

percentage of IFNgþ T cells from triplicates from an individual donor. F, i,5T4 antigen cross-presentation is inhibited by HLA-class I blockingantibody; ii, 5T4þ (DU145, PC3, and M38) but not 5T4� (LNCaP) tumor cellsstimulate T cells in the cross-presentation assay. Meanþ SEM of percentageof IFNgþ T cells from triplicates are shown. NS, not statistically significant.






in cross-presentation of irradiated tumor cells. To test this, first weapplied to tumor cells before irradiation the small-moleculeinhibitor VER155008, which inhibits the activity of both theinduced and constitutive forms of Hsp70 (21, 22). Tumorcell numbers after 72 hours were only slightly lower whenVER155008 was applied to irradiated cells, presumably becausecell proliferation was already slowed down by the irradiation.However, as expected, the treatment inhibited the growth of

nonirradiated tumor cells by approximately 70% (Fig. 6A). Sim-ilarly, VER155008 significantly increased cell death of nonirradi-ated but not irradiated DU145 cells (Fig. 6B). The inhibitor didnot impair Hsp70 cell-surface expression on irradiated tumorcells (Fig. 6C). After establishing that VER155008 delivers theexpected effects to untreated tumor cells, we studied its effect onthe ability of irradiated tumor cells to upregulate CD86 on DCs.CD86 upregulation was partially inhibited (Fig. 6D) when DCsencountered irradiated tumor cells pretreated with VER155008.Finally, we applied VER155008 in the cross-presentation modelto test its effect on T-cell activation. The inhibitor significantlydecreased both the background cross-presentation of tumorantigen from nonirradiated tumor cells and the enhanced levelof cross-presentation observed from irradiated tumor cells(Fig. 6E), as detected by decreased T-cell IFNg production. Theresults were confirmed with DCs derived from 2 donors. Totest that the inhibition of cross-presentation with VER155008wasnot due to anoff-target effect,we appliedPES, anotherHsp70-inhibitor. PES binds only to stress-induced but not constitutiveHsp70 (23). Interestingly, PES only inhibited T-cell stimulationinduced by irradiated tumor cell-loaded DCs but not by theadditionof nonirradiated tumor cells (Fig. 6E). These experimentsindicate a crucial role for radiation-induced Hsp70 in tumorantigen cross-presentation.

Irradiated tumor cell-derived Hsp70 signals mainly via CD91on DCs

To further elucidate the importance of Hsp70 in the cross-presentation model, we tested the expression of potential recep-tors CD91 and SREC-I on day 5 DCs. Significant surface expres-sion of both receptors was observed (Fig. 7A). When neutralizingantibodies against these receptors were applied in the cross-presentation experiments, T-cell activation was completely inhib-ited in the presence of the anti-CD91- but not the SREC-I–specificneutralizing antibody (Fig. 7B). These results demonstrate thatCD91-mediated effects inDCs, such as tumor cell–derivedHsp70binding, are necessary for efficient antigen cross-presentationfrom irradiated tumor cells.

DiscussionAntigen cross-presentation has been indicated as an important

mechanism for generating CD8þ T-cell responses against solidtumors that do not migrate into lymph nodes or viruses that donot infect professional antigen-presenting cells. Although chemo-therapy-induced antigen cross-presentation has been studiedextensively, there is a paucity of information about ionizingradiation–mediated antigen cross-presentation. The abscopaleffect, observed in patients undergoing radiotherapy, has beendemonstrated to be immune-mediated and is likely to involveantigen cross-presentation from irradiated tumors (4, 5). Furtherstudies in this field would aid better understanding of howradiotherapy could be made more successful.

We studied the relative importance of immunogenic signals inantigen cross-presentation from irradiated human tumor cells.We established a model using a tumor-specific T-cell line as a de-tector of cross-presentation of a naturally expressed tumor anti-gen from irradiated, HLA-mismatched prostate cancer tumor cellsby DCs. The radiation dose (12 Gy) applied to tumor cells in theseexperiments is in line with the latest technical developments ofradiotherapy inprostate cancer andothermalignancies.High-dose

Figure 5.TLR4 polymorphism does not affect tumor antigen cross-presentation. A, i,TLR4 expression on monocytes from 5 donors with the Asp299 (299A)and 4 donors with the polymorphic Gly299 (299G) allele; ii, TLR4expression on day 5 DCs. iii, day 5 DCs were stimulated with LPS and TNFaproduction was measured by flow cytometry. Symbols representpercentage of positive cells from individual donors. The boxes show the 25%and 75% percentiles of the combined data, and the lines represent themedians. B, DC phenotyping from donors as in A. DCs were cocultured with0 Gy or 12 Gy–irradiated DU145 cells. Each line represents an individual donor.C, stimulation of 5T4-specific T cells with DCs, derived from 3 donors in eachgroup (as inA), loadedwith untreated, 12Gy–irradiated, or oxaliplatin-treatedDU145 cells, respectively. Mean þ SEM of percentage of IFNgþ T cells areshown from triplicate samples. NS, not statistically significant.

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brachytherapy and intensity-modulated radiotherapy offer treat-ments with fewer fractions but higher doses, delivered moreprecisely to the cancer. The cellular effect of radiation is complex,resulting in growth arrest, senescence, and different types of celldeath.We observed cell-cycle arrest in theG2–Mphase, as reportedby others (24), and a gradual increase of cell death with time

following irradiation. Cell death was predominantly of the lateapoptotic/necrotic type. The p53 gene is mutated in DU145 cells,which may affect radiation-mediated repair response and apopto-sis (25). As p53 mutations are frequent in prostate cancer, ourobservations are likely to be representative of the physiologicbehavior of the majority of prostate cancer cells.

Figure 6.Hsp70 inhibition abolishes antigencross-presentation. A, the effect ofVER155008 on DU145 cell numbersafter 72-hour culture. B, differenttypes of cell death as detected byAnnexin/PI staining in the absence orpresence of VER155008. C, surfaceexpression of Hsp70 (gray) versusisotype (black) in the absence orpresence of VER155008: (i) summaryfrom triplicates; (ii) representativehistograms. D, effect of VER155008-treated or untreated DU145 cells onCD86 expression of DCs following a24-hour coculture: (i) representativehistograms; (ii) summary fromtriplicates. E, stimulation of 5T4-specific T cells in a cross-presentationexperiment with DCs loaded withVER155008 or PES-treated oruntreated DU145 cells. Thisexperiment was carried out with DCsderived from 2 donors. A–E, mean þSEM of results from triplicate samplesare shown.






Radiation-induced upregulation of CRT from the endoplas-mic reticulum to the cell surface is one of the typical stressresponses with an important immunologic impact, such as thefacilitation of phagocytosis (26). The results show a partialcontribution by the TLR signaling pathway and HMGB1 toantigen cross-presentation. HMGB1 is both a nuclear factor anda secreted protein. In the nucleus, it acts as an architecturalchromatin-binding factor that influences DNA tertiary structure.When released from dying cells, it functions as a proinflamma-tory cytokine (27). However, its effects are pleiotropic and theydepend not only on its redox status but also on the particularreceptor it binds, such as RAGE, TLR2, or TLR4. Glycyrrhizin,which binds directly to HMGB1 and inhibits its chemokinefunction and autophagy induction (20), among other potentialeffects, was proved inhibitory in the cross-presentation model.HMGB1 has been shown to associate with TLR4 (28). TheAsp299Gly SNP of TLR4 causes structural changes of the TLR4extracellular domain, with a potential impact on LPS binding(29). Cross-presentation has been implied to be affected by thisSNP, similar to the effect observed in TLR4�/� knockout mice(19). However, LPS-induced cytokine production is not affectedby the TLR4 SNP even when present in a homozygous form(30). Our results are in agreement with this, as we observed noinfluence of Asp299Gly TLR4 SNP on LPS-induced TNFa pro-duction in DCs. However, we also found no effect by the TLR4SNP on radiation-induced antigen cross-presentation. Further-more, contrary to the observation by others (19), we also did notfind any evidence of impaired antigen cross-presentation fromoxaliplatin-treated tumor cells by DCs carrying the TLR4 SNP.The possible explanation behind this discrepancymay be relatedto donor variation as the previously published observation (19)was based on results from a single donor with TLR4 SNP.

Heat-shock proteins represent another group of damage-asso-ciatedmolecules, upregulated by irradiation and released into theextracellular space fromdying cells or secreted from live cells (31).In prostate cancer, Hsp70 has been shown to be protective, asits silencing enhanced tumor cell sensitivity to irradiation (32).

Hsp70 has well-described immunologic roles as well, as tumortissue–derived Hsp70 has been shown to be protective againsttumor challenge in mice (33). We observed a predominantlynuclear expression of Hsp70 in untreated DU145 cells, while inirradiated cells nuclear expression seemed lower and cytosolic andcell-surface expression increased significantly. This translocationobserved at 72 hours after irradiation may have been associatedwith enhanced repair activity in the irradiated cells. AlthoughHsp70 inhibition with VER155008, a specific Hsp70-familyinhibitor (21), did not influence tumor cell growth and celldeath following irradiation, it significantly inhibited the growthof nonirradiated tumor cells. VER155008 also did not influenceHsp70 cell-surface expression on irradiated DU145 cells; howev-er, it partially inhibited the ability of irradiated DU145 cellsto activate CD86 upregulation on DCs. The reason behind thisobservation is not clear. The inhibition of antigen cross-presen-tation was complete when VER155008 was added either toirradiated or control DU145 cells. Another Hsp70 family inhib-itor, PES (23), only affected the enhanced T-cell response observ-ed with irradiated but not that with untreated tumor cells.

Hsp70 can bind to TLR2 or TLR4, CD91, CD40, or toscavenger receptors such as SREC-I and LOX-1. Hsp70 bindingto TLR4 can upregulate HMGB1 in DCs (34), providing cross-talk between the heat shock and the HMGB1–TLR4 pathways.On the other hand, SREC-I and LOX-1 were shown to be bothresponsible for antigen cross-presentation in a murine system(35). To determine which receptor is important in this model,DC phenotyping and antibody-blocking experiments were car-ried out. These results are consistent with those from previousstudies (36, 37), showing that CD91 is the dominant receptorfor Hsp70 during the cross-presentation process.

We have not addressed directly whether Hsp70's role in thismodel liesmainly in antigen-chaperoning (33, 38, 39) or enhanc-ing autophagy (40) or triggering DC maturation (41, 42) withsubsequent increase in T-cell stimulation. All of these knownfunctions can be important and they likely play a synergistic role.Our model provides an opportunity to study the fine details of

Figure 7.Hsp70 receptor expression on DCsand the effect of receptor-blocking onantigen cross-presentation. A,representative data of CD91 or SREC-Iexpression on the surface of day 5 DCs(i) compared with isotype control.Numbers in histograms represent %positive DCs. (ii) Means þ SEM ofmean fluorescence intensity (mfi) ofantibody binding by DCs, as indicated,from triplicate samples. B, T-cellstimulation in a cross-presentationexperiment in the presence of CD91 orSREC-I neutralizing (N) antibodies orisotype controls (Iso), respectively.Mean þ SEM of percentage of IFNgþ

T cells are shown from triplicatesamples.

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heat-shock protein–mediated chaperoning of a naturally expres-sed antigen in a human tumor model. Such an analysis wouldrepresent an interesting follow-up to the work presented here.

Despite the long list of players necessary for "optimal" antigencross-presentation, the plasticity of the process has also beendemonstrated, as for example, highly polarized (type I) DCs canefficiently prime T cells evenwhen cocultured with apoptotic cells(43). Furthermore, DCs can acquire antigen from live cells forantigen cross-presentation in both tumor and viral settings(44–46). In the latter, while apoptosis is inhibited by the virus,Hsp70 expression is significantly upregulated (47). These exam-ples illustrate that if any key player of the antigen cross-presen-tation process is overexpressed or hyperactivated, it can generatea shortcut leading to antigen cross-presentation even if not all theelements, as discussed earlier, are present. Taken together, resultsfrom our study provide strong evidence that preexisting tumorantigen-specific T cells can be reactivated as a consequence ofirradiation of tumor cells. We also demonstrate that Hsp70plays a crucial role in antigen cross-presentation from irradiatedtumor cells. These observations have practical implications forthe design of future immuno-radiotherapy combinations.

Disclosure of Potential Conflicts of InterestJ. Staffurth has received speakers bureau honoraria from Jannsen and

Astellas, and is a consultant/advisory board member for Janssen. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Salimu, M.D. Mason, Z. TabiDevelopment of methodology: J. Salimu, Z. TabiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. SalimuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Salimu, L.K. SparyWriting, review, and/or revision of the manuscript: J. Salimu, L.K. Spary,S. Al-Taei, M.D. Mason, J. Staffurth, Z. TabiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. SalimuStudy supervision: A. Clayton, J. Staffurth, Z. Tabi

AcknowledgmentsThe authors thank Dr. Mario Labeta (Cardiff University, Cardiff, United

Kingdom) for helpful discussions, Drs. Rachel Butler and RanaHussein (Cardiffand Vale NHS Trust) for the TLR4 SNP analysis, Ms. Lynda Churchill fortechnical help, and all of our blood donors.

Grant SupportJ. Salimu is a recipient of a PhD scholarship from Cardiff University and

Cancer Research Wales; A. Clayton and Z. Tabi are supported by a CancerResearch Wales program grant.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 24, 2014; revised February 5, 2015; accepted February 5, 2015;published OnlineFirst February 12, 2015.

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2015;3:678-688. Published OnlineFirst February 12, 2015.Cancer Immunol Res Josephine Salimu, Lisa K. Spary, Saly Al-Taei, et al. Protein 70 and Receptor CD91

A Key Role for Heat-Shock−−Irradiated Prostate Cancer Cells Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from

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