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Vaccine 26 (2008) 6422–6432

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

ost-apoptotic tumors are more palatable to dendritic cells and enhance theirntigen cross-presentation activity�

avide Brusaa, Stefano Garettoa, Giovanna Chiorinob, Maria Scatolinib, Elisa Migliorea,iovanni Camussi c, Lina Materaa,∗

Laboratory of Tumor Immunology, Department of Internal Medicine, University of Turin, Turin, ItalyCancer Genomics Lab Fondo Edo Tempia, Biella, ItalyDepartment of Internal Medicine, Research Centre for Experimental Medicine (CeRMS) and Centre for Molecular Biotechnology, University of Turin, Turin, Italy

r t i c l e i n f o

rticle history:eceived 16 June 2008eceived in revised form 21 August 2008ccepted 25 August 2008vailable online 9 October 2008

eywords:endritic cellsumor immunityaccinationntigen presentationpoptosis

a b s t r a c t

Critical issues for cytotoxic lymphocyte (CTL) cross-priming are (a) the maturation state of dendriticcells (DC), (b) the source of the tumor-associated antigens (TAA) and (c) the context in which they aredelivered to DCs. Drug-induced apoptosis has recently been implicated in CTL cross-priming. However,since drug-treatment produces in vivo more tumor cells than the DC default apoptotic clearance programcan cope with, they are expected to proceed to secondary necrosis and change their molecular pattern.Here we have addressed this issue on renal carcinoma cells (RCC) by using different apoptotic stimuli.UVC, but not �-irradiation or anthracyclins, induced after 4 h treatment of the RCC cell line K1 a com-bination of apoptotic (phosphatydilserine and calreticulin plasma membrane mobilization) and necrotic(membrane incompetence) features. Heat shock protein (Hsp)-70 and chromatin-bound high mobilitybox 1 HMGB1 protein, typical of necrosis, were released during the further 20 h and thus made accessibleto co-cultured monocyte-derived immature (i) DC. UVC-treated, secondary necrotic RCC cell lines were

cross-presented with higher efficiency by cytokine-matured (m) DC than their early apoptotic (i.e. �-irradiated) counterpart. Upstream events such as increased tumor uptake, activation of genes involved inthe antigen-processing machinery, and increased expression of costimulatory and maturation moleculeswere also observed after loading iDC with secondary necrotic, but not apoptotic, tumor cells. These dataoffer a description of the molecular and immunogenic characteristics of post-apoptotic tumors which canbe exploited to increase the efficiency of in vivo and ex vivo TAA delivery to the DC cross-presentation

giutt

pathway.

. Introduction

The aim of antigen-specific active antitumor immunotherapys to break tolerance against tumor-associated antigens (TAA) byresenting them in the context of costimulatory signals on antigen-

resenting cells. Dendritic cells (DC) capture tumor cells, processhem and present the relevant T-cell antigen epitope in the con-ext of both class II and class I MHC, thus cross-priming CD8 Tymphocytes (CTL) [1]. Capturing of particulate exogenous anti-

� Work supported by grants from Italian Ministries for the Universities, Regioneiemonte (Oncology project), CERMS/COES project funded by the ‘Compagnia di Sanaolo/FIRMS’, Compagnia di San Paolo (to L.M.), Fondazione Cassa di Risparmio diorino (to L.M.) and Fondazione Cariverona (to L.M). D.B. is a recipient of a fellowshiprom Compagnia di San Paolo.∗ Corresponding author. Tel.: +39 011 6961813; fax: +39 011 6634146.

E-mail address: lina.matera@unito.it (L. Matera).

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.08.063

© 2008 Elsevier Ltd. All rights reserved.

ens and antigen epitope presentation are specialized functions ofmmature (i) and mature (m) DC, respectively. mDC function entailsp-regulation of molecules including chemokine receptors and cos-imulatory molecules. Optimal cross-priming of tumor-specific CTLherefore relies on appropriate TAA delivery and DC maturation. iDCenerated from blood monocytes progress to mDC in the presencef cytokines which mimic the pro-inflammatory environment inhich optimal DC maturation occurs in vivo [2].

The context in which TAA are captured by iDC determines theutcome of the CTL response. Cell death occurs through apopto-is and/or necrosis, two mechanisms whose respective advantagesn generating efficient CTL cross-priming by loaded DC is uncer-ain. Necrotic death is accompanied by release of molecules that

avour both delivery of TAA to DC and creation of an inflam-

atory microenvironment which drives DC maturation [3,4].ffector molecules of necrosis-induced immunogenicity are theanger molecular pattern (DAMP) proteins heat shock protein0 (Hsp70) and high mobility group box 1 (HMGB1). Intracellu-

ine 26

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D. Brusa et al. / Vacc

ar Hsp70 is a highly conserved protein induced upon exposureo many forms of stress [5] that protects cells by antagoniz-ng apoptosis-inducing factors [6], whereas extracellular locatedr membrane-bound Hsp70 mediates immunological functions.sp70 released by necrotic cells induces maturation of DC and

haperones tumor antigen peptides to DC [7–9]. HMGB1, a non-istone protein loosely bound to chromatin, induces DC maturationnd migration [10,11]. Necrosis results in detachment of HMGB1rom chromatin and its extracellular translocation, whereas apop-osis stabilizes HMGB1 binding to DNA [12–15]. Apoptosis isiewed as a silent or tolerance-inducing cell death that producesither inhibition [16,17] or stimulation of DC-mediated antitumoresponse in vitro [18–23]. Recent data in vivo, however, have shownhat an anthracyclines-induced apoptotic tumor may prime themmune system for an antitumor response [24,25]. In those stud-es a prominent role in DC maturation and CTL cross-priming wasssociated with membrane translocation of calreticulin (CRT) fromhe ER [25].

Discrepancy in the data regarding the immunogenicity of apop-otic versus necrotic cells may stem from the difficulty of obtainingomogeneous apoptotic/necrotic cell populations in vivo and initro. During chemotherapy-induced tumor cell death, DCs areresented with more tumor cells than their default apoptotic clear-nce program can cope with. Most tumor cells, therefore, undergoecondary necrosis and change their molecular pattern [26]. Weave previously shown that secondary necrosis after anthracycline-

nduced apoptosis is endowed with high immunogenicity [27].e and others, in fact, have found that late but not early-stage

poptotic cells stimulate DCs [27–29]. We have now used a renalarcinoma cell (RCC) line to assess the effect of different stimuli,nd have focused on the biochemical and immunogenic proper-ies of early apoptotic versus secondary necrotic cell death. UVC,

known source of DNA damage, drove the short-term produc-ion of a population of tumor cells undergoing post-apoptoticecrosis, as shown by AnnV and CRT surface expression andxtracellular release of HMGB1. When co-incubated with DC,hese cells induced more efficient antigen cross-presentation than-rad-induced apoptotic cells. In addition to increased T-cell cross-riming, upstream events such as tumor uptake, transcriptionf antigen-processing molecules and expression of maturationarkers were also increased. UVC induction of cell death can be

xploited to increase the efficiency of in vivo and ex vivo TAA deliv-ry to the DC cross-presentation pathway.

. Materials and methods

.1. Reagents

Human recombinant granulocyte-macrophage colony stimulat-ng factor (GM-CSF) was purchased from Sigma–Aldrich (Milan,taly), IL-4, IL-1�, IL-6, TNF-� from Preprotech, IL-2 from ChironMilan, Italy) and PGE2 from Cayman Chemical Company (Inalco.p.a., Milan, Italy).

.2. Tumor cells

The clear cell RCC cell lines K1 [23] and RCC53 (a gift of Dr.olores J. Schendel Institute of Molecular Immunology, Munich,ermany) were grown as suspension cultures in RPMI 1640

Sigma–Aldrich), 10% heat-inactivated FCS (Life Technologies Ltd,aisley, Scotland, UK) and 1 �g/ml l-glutamine and subculturedvery 2–3 days. The adherent cells were detached by gently scrap-ng for flow cytometric analysis or by trypsin treatment for use inulture.

cWIMr

(2008) 6422–6432 6423

.3. Generation of iDC

iDC were generated as described [27]. Peripheral blood mononu-lear cells (PBMC) were isolated from heparinized blood oflood bank donors by standard density gradient centrifuga-ion (Histopaque-1077, Sigma–Aldrich), washed with RPMI 1640

edium supplemented with 5 mM EDTA (Sigma–Aldrich) and% heat inactivated FCS (Life Technologies) and suspended at× 106/ml in RPMI 1640 medium supplemented with 10% FCS.fter 2 h incubation at 37 ◦C in a humidified 5% CO2 incubator,on-adherent cells were removed and frozen for further use asutologous lymphocytes. Adherent cells were cultured in the aboveomplete medium supplemented with GM-CSF (1000 U/ml) andL-4 (1000 U/ml). Cytokines were replaced on day 3 and iDC wereollected on day 6.

.4. Flow cytometric analysis of DC membrane markers

Expression of relevant molecules on DC was analyzed withhe following mAbs: PE-conjugated anti-CD80 (Becton Dickin-on, Milan, Italy), anti-CD107a mAb (Becton Dickinson), anti-CCR7R&D Systems), anti-CD83 (Ancell Bayport, MN, USA), and FITC-onjugated anti-CD36 (e-Bioscience, San Diego, CA, USA) andPC-conjugated anti-CD80 (Genetex, San Antonio, TX, USA), using

he appropriate control isotypes. The purified mAb against theeceptor of advanced glycation end products (RAGE) (R&D Systemsinneapolis, MN, USA) was used at 10 �g/1 × 106 cells in 50 �l,

ollowed by PE goat anti-mouse (Abcam, Cambridge Science Park,ambridge, UK). Cells were washed, fixed in PBS 1% paraformalde-yde and analyzed using a FACSCalibur (Becton Dickinson). A totalf 10,000 events were analyzed for each sample.

.5. Killing of tumor cells

K1 cells received the following treatments: �-irradiation90 Gy), UVC (3.6 J/cm2) (UVC lamp PLS 9W/2P, W 2.3 Philips,

ilan, Italy), 60% distilled water (as a control for necrosis),erum-free RPMI (as a control for apoptosis), the anthracyclinesoxorubicin (25 �M) (Sigma–Aldrich) and mitoxantrone (1 �M)Sigma–Aldrich) or the topoisomerase inhibitor camptothecin8 nM) (Sigma–Aldrich).

.6. Analysis of tumor death parameters by flow cytometry

After 4, 24 or 48 h from the above treatments, K1 cells weretained with Annexin-V–FITC (AnnV) (Becton Dickinson) whichinds phosphatidylserine (PS) or with anti-CRT mAb 619 TO-11 (aift from S. Ferrone, Roswell Park Center University of Buffalo, NY,SA) or rabbit anti-human CRT (Stressgen, Temaricerca, Bologna,

taly) (followed by anti-mouse FITC and swine anti-human FITC,espectively), and with DNA-binding propidium iodide (PI) (Dako,ilan Italy). Cytometric analysis was performed as described above.

.7. Analysis of DAMP (Hsp70 and HMGB1) molecule release

The release of Hsp70 and HMGB1 was studied by immunoblotnalysis. At the indicated times cells were centrifuged (1200 rpm)nd the supernatants were collected and treated with RIPA buffernd protease inhibitors. Samples of equal volume (10 �l) wereeparated by SDS–PAGE (12% resolving gel) and blotted onto nitro-

ellulose. The filters were incubated with anti-Hsp70 mAb clone

27 SC-24 (Santa Cruz biotechnology, DBA Italia Segrate, Milan,taly), anti-Hsp70 polyclonal 4872 (Cell Signalling Celbio Pero,

ilan, Italy), rabbit polyclonal anti-HMGB1 (ab 18256; Abcam) orabbit polyclonal anti-tubulin N357 (GE Healthcare, Milan, Italy).

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he primary mAb/Abs was recognized by anti-mouse IgG HRPinked antibody (Biorad, Milan, Italy) and anti-rabbit IgG HRP linkedntibody (ab 7074; Cell Signalling), respectively. Proteins wereetected by ECL (Amersham, Milan, Italy). Images were capturedith CanoScan FB1210U Canon and processed by the Adobe Photo-

hop Microsoft Office Picture and Manager ScanGear CS-U.

.8. Uptake of dying tumors by iDC

The tumor cells were green-stained with PKH2 (Sigma–Aldrich)ccording to the manufacturer’s instructions, before receiving thebove killing treatments. After 4 h tumor cells were washed andncubated for 20 h at 37 ◦C with 1 × 105 iDC at a 2:1 ratio, andhe mixed culture was stained with the PE mAb CD80 (20 mint 4 ◦C). Phagocytosis of tumor cells by iDC was assessed by flowytometric analysis as the percentage of double-stained versus redCD80 PE) cells on a total of 10,000 events. In some experimentshe maturational changes of DC cultured with the unstained tumoras studied on CD80+ gated (APC anti-CD80) cells using the PEAbs CCR7 (R&D) and CD83 (Ancell). For microscopy analysis of

umor engulfment, DC and tumor cells cultured and stained asescribed above were mounted on a coverslip in Acqueous Gelounting (Sigma–Aldrich) and the fluorescence was assessed by

ITC and TRITC filters on a fluorescence microscope (Olympus BX41)quipped with a Leica DFC320 camera and Leica Qwin Software.mages were processed by Adobe Photoshop 5.0.

.9. Cross-priming of lymphocytes with tumor-loaded autologousC

Four hours after the indicated treatments, K1 cells were mixedith iDC at a ratio of 2:1 and the incubation proceeded for 20 h inM-CSF and IL-4-containing medium. iDC were then exposed for a

urther 48 h to a new medium containing a cocktail of the matura-ive cytokines TNF-� (10 ng/ml), PGE2 (1 �g/ml), IL-1� (1 ng/ml) eL-6 (10 ng/ml) and added to autologous T lymphocytes at a ratio of:10. rIL-2 (20 U/ml) was added to the cultures after 24 h and therocedure was repeated weekly, for up to 3 total stimulations. Gen-ration of specific antitumor response was evaluated 5 days afterhe last stimulation as described below.

.10. MHC class I-restricted IFN-� release ELISPOT assay

The frequency of IFN-�-secreting T lymphocytes in culturesas assessed in an ELISPOT assay, as previously described [27].itrocellulose membrane 96-well microtiter plates (Multiscreen,illipore, Vimodrome, Milan, Italy) were coated with anti-IFN-mAb (PharMingen). 105 T-cells (initial number in culture) were

eeded in triplicate wells containing medium alone, phorbol myris-ate acetate (50 ng/ml, Sigma–Aldrich) and ionomycin (1 �g/ml,igma–Aldrich), the unloaded, K1 lysate-loaded or MUC-1 STAP-VHNV peptide-loaded mDC as targets at a 1:2 ratio. Targetsere kept at 4 ◦C for 20 m with the anti-human MHC class Ilocking mAb W6/32 (Serotec, DBA Italia s.r.l., Milan, Italy) orn isotype-matched mAb (Becton Dickinson) (25 �g/ml for both)efore being used as targets. After 24 h, cells were lysed withistilled water and a biotinylated anti-IFN-� mAb (PharMingen)as added to the wells, followed by strept-avidin-horse radish

eroxidase-conjugated (PharMingen). The substrate solution (3-mino-9-ethylcarbazole) (Sigma–Aldrich) was then added andfter 30 min the reaction was stopped by rising with tap water.fter the plates had dried overnight, red spots (indicative of reactive

ymphocytes) were detected with the AID Elispot-Reader (Biolinemplimedical, Milan, Italy).

totm

(2008) 6422–6432

.11. Flow cytometry intracellular staining

Cells were fixed with 4% paraformaldehyde and permeabilizedith a buffer containing 0.1% of saponin (Sigma). IFN-� analysisas obtained with FITC-conjugated anti- IFN-� (Becton Dickinson)

fter gating on CD8+ cells. Degranulation by lymphocytes (as a signf target-induced activation) was detected with the anti-CD107aAb.

.12. Isolation of total RNA

Cells were homogenized by homogenizer and total RNA was iso-ated according to standard TriReagent protocol (Sigma–Aldrich, St.ouis MO).

.13. Microarray analysis

For each sample, mRNA was amplified starting from 1 �g ofotRNA for sample by Amino Allyl MessageAmp II aRNA Kit (Ambionnc. Austin TX, USA) to obtain amino allyl antisense RNA accordingo the method developed by Eberwine and coworkers [30]. Only oneound of amplification was performed, as for the manufacturer’srotocol and with minor modification. Briefly: mRNA was reverseranscribed in cDNA single strand; after the second strand synthe-is, cDNA was in vitro transcribed in aaRNA including an amino allyodified nucleotide (aaUTP). Both dsDNA and aaRNA underwent a

urification step-using column provided with the kit. Labeling waserformed using NHS ester Cy3 or Cy5 dies (GE Healthcare EuropeMBH, Uppsala, Sweden) able to react with the modified RNA. At

east 5 �g of mRNA, for each sample, were labeled and purified witholumns. Hybridization with dye-swap duplication was performedo compare samples versus reference. The same quantity of dif-erentially labeled samples and reference (0.75 �g for 44K arraysnd 0.825 �g for 4 × 44K arrays) was put together, fragmented andybridized to oligonucleotide glass array with sequences repre-enting over 37,000 well-characterized, full-length human genesHuman Oligo Whole Genome Microarray 44K and 4 × 44K, Agilentechnologies, Santa Clara, CA). All steps were performed followinghe “60-mer oligo microarray processing protocol” (Agilent Tech-ologies) for 44K arrays and “Two-Color Microarray-Based Genexpression Analysis” for 4 × 44K arrays. Then, slides were washedith the SSPE wash procedure, Acetonitrile and Drying Solution

Agilent Technologies) and scanned with the dual-laser microar-ay scanner Agilent G2505B (Agilent Technologies). totRNA andRNA quality was checked by RNA 6000 nano chip assays (Agi-

ent Technologies) and Agilent 2100 Bioanalyzer. Concentrationsnd labeling were also checked by NanoDrop ND-1000 Spectropho-ometer.

.14. Statistical analysis

Statistical significance of the treatments was calculated usinghe Student’s t-test for parametric data (percent double stainedells) and the Wilcoxon test for non-parametric data (ELISA andLISPOT assays).

. Results

.1. UVC-treatment is the best inducer of secondary necrotic death

The ability of different kinds of stimuli to induce apop-otic, necrotic or mixed markers was assessed during a 4–48 hbservation. Flow cytometry with AnnV and PI (Fig. 1) showedhat externalization of PS in the absence of membrane per-

eabilization (the AnnV+PI− phenotype indicative of early

D. Brusa et al. / Vaccine 26

Fig. 1. UVC treatment induces the largest number of cells expressing apoptotic andnecrotic markers. The renal carcinoma cell line K1 received the following treatments:incubation with serum-free RPMI (apoptosis), 70% distilled water (necrosis), 25 �Mdoxorubicin (DOXO), 1 �M mitoxantrone (MITOX) 8 nM camptothecin (CAMPTO),90 Gy (irr) and 3.6 J/cm2 UVC (UVC). Time kinetic analysis of surface exposure ofPS (AnnV+), membrane permeabilization (PI+), or both (AnnV+PI+) was performed.Membrane incompetence was detectable at 4 h only in cultures treated with UVC ordoxorubicin, with secondary necrosis (AnnV+PI+) being much more evident in theformer. Data are representative of three independent experiments.

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poptosis) was induced by the control apoptotic stimulus (i.e.nsulin–selenium–transferrin deprivation), mitoxantrone, camp-othecin and �-irradiation and was observed only at 4 h, declininghereafter. By contrast, membrane incompetence in the absence ofS externalization (the PI+AnnV− phenotype indicative of primaryecrosis) was detectable at 4 h only in cultures treated with dox-rubicin and at lower levels by UVC, and increased in all culturesfter 24 h. Importantly, UVC produced within 4 h a far higher pro-ortion of cells expressing both markers (the PI + AnnV + phenotype

ndicative of secondary necrosis), suggesting progression of apop-otic (AnnV+) cells to membrane incompetence (confirmed by theirailure to exclude trypan blue dye, not shown), In this context its necessary to point out that non-specific intracellular stainingue to membrane permeabilization, could have occurred beforeS externalization and been responsible for staining of PS on thenner plasma membrane. This was not the case, however, since PStaining (i.e. AnnV positivity) almost disappeared (2.33%) at 48 hhen the majority (97.12%) of cells were membrane-damaged (i.e.

I+) (Fig. 2A). The transient, and thus specific, nature of the AnnVontrasts with the stable expression of another marker of apoptosisCRT) (Fig. 2B), which is traslocated to the cell membrane from theR after apoptotic stimuli [25].

.2. UVC-treatment is the best inducer of CRT externalization

Assessment of CRT externalization on viable (PI−) cells (twoxperiments) showed higher percentage of CRT + PI− in the UVC-reated (23% of PI− cells) compared with �-irradiated (3.3%) orntreated (1.2%) live K1 cells (Fig. 3).

.3. UVC and �-irradiation as models for early and late apoptosissecondary necrosis)

On the basis of the above membrane features, UVC and �-radsere chosen as a model of post-apoptotic secondary necrotic death

nd of early apoptotic death, respectively. Individual data represen-ative of six experiments showing the AnnV/PI phenotype of UVC-nd �-rad-treated cells at 4 h, when they were used to feed iDC areiven in Fig. 4.

.4. Generation of CTL is increased by loading of iDC withVC-treated tumor

After 4 h (the time needed to induce secondary necrosis byVC as shown in Figs. 1 and 4), K1 or RCC53 cell lines were incu-ated with iDC. After 20 h incubation with the treated/untreatedumor cells, iDC were exposed to the maturation cytokine cock-ail to optimize their antigen-presenting activity and after 48 hsed as stimulators for the autologous lymphocytes. Stimulationas repeated twice. Five days after the third stimulation the

pecific antitumor response was assessed against mDC loadedith freeze/thawing K1 lysate, as antigen-expressing target. The

esponse from lymphocytes cultured with unloaded DC wasecreased when they were cultured with DC loaded with untreated1 (DC-K1UN) (Fig. 5A mean results of six experiments). However,VC treatment of K1 cells significantly reversed this inhibition. (DC-1UN versus DC-K1UVC p < 0.05). The increased response was MHClass I-restricted, since it was reduced by the MHC class I-blockingAb W6/32. No IFN-� release was observed in the presence of

he NK-sensitive K562 cells (not shown). Although CD4+ cells were

he predominant population in our cultures (50–65% range fromine experiments) flow cytometry showed that most of IFN-�xpressing lymphocytes were of the CD8+ phenotype and that auch higher percentage of CD8+ IFN-�+ cells was found in cul-

ures stimulated by DC-K1UVC (Fig. 5B, mean of two experiments).

6426 D. Brusa et al. / Vaccine 26 (2008) 6422–6432

Fig. 2. AnnV reacts with externalized PS. K1 cells were treated with UVC as described in legend to Fig. 1 and at different times from treatment were stained with AnnV andP sitivityp positt

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I or with the anti-CRT mAb 619 TO-11 and PI. The kinetics of AnnV/PI double poositivity. The transient positivity on gate PI+ for AnnV (A), as opposed to the stablehe inner leaflet of the disrupted (PI+) membrane.

ssessment of the cytolytic phenotype in a degranulation assaysing the anti-CD107a mAb also showed a significantly increase

n lymphocytes stimulated by DC-K1UVC compared to lymphocytestimulated by DC-K1UN (Fig. 5C mean ± S.D. of four experiments< 0.05). The HLA-A2+ CC53 line was treated in the same way toorroborate enhancement of CTL cross-priming by UVC-inducedecondary necrosis. To check for the specificity of the response,n some experiments the IFN-� release was tested against HLA-2+ donor autologous DC unloaded, loaded with the K1 lysater pulsed with a HLA-A2-restricted STAPPVHNV peptide of MUC-, which is expressed on most epithelial cancers [31]. Results ofig. 5D show that both K1 and RCC53 cells were more effectivehen UVC-treated. In addition, the difference in the response to

he different targets confirms that CTLs generated by the UVC-

reated RCCs maintain their specificity. The K1UVC-derived antigensrocessed and presented by the autologous DCs were recognizedy K1UVC-sensitised, but not by RCC53UVC-sensitised lymphocytes.ice versa, MUC-1 peptide-loaded autologous DCs were recognised

ig. 3. The percentage of CRT+ cells within the PI− population is increased after UVC.1 cells were untreated (UN), or treated with 90 Gy �-rads (irr) or 3.6 J/cm2 UVC (uvc)nd after 4 h were stained with PI and with the anti-CRT mAb 619 TO-11 or a rabbitnti-human CRT Ab (Stressgen). The percentage of CRT+ viable (i.e. PI− gated) cellsas strikingly increased after UVC. Results are the mean ± S.D. of two experimentsith the anti-CRT mAb 619 TO-11. Super imposable results were obtained in other

wo experiments using the rabbit anti-human CRT Ab (not shown).

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(central panel of Fig. 1) is compared here (up panel) with that of CRT/PI doubleivity for CRT (B) shows that the former is not due to mere staining of PS present on

y RCC53UVC-sensitised, but not by K1UVC-sensitised lymphocytes.hus, UVC secondary necrotic tumors convey a wide array of tumor-elated TAAs to the DC cross-presentation pathway.

.5. UVC induces the extracellular migration of Hsp70 andMGB1

Since UVC-treated K1 cells had apparently died of necrosis andhe DAMP molecules Hsp70 and HMGB1 (hallmarks of necrosis) arelso involved in DC activation, we investigated their release duringncubation of iDC with the untreated or UVC-treated K1 tumor. Ashown in Fig. 6 (representative results from four experiments) nei-her Hsp70 nor HMGB1 were detected in the supernatant of K1 afterh of UVC treatment when, according to our experimental design,

hey were exposed to iDC. They were however present at the end ofhe 20 h incubation (i.e. 24 h from treatment). A far higher amountf HMGB1 release was observed compared to Hsp70. It is worthoting that �-tubulin, a protein of the cytoskeleton, was not found

n the supernatant at late times after UVC treatment. Thus, despiteoss of membrane competence, UVC-treated cells still preserve theirrchitecture, as confirmed by microscope examination (not shown).

.6. UVC treatment increases tumor phagocytosis by iDC

Flow cytometry showed that UVC-treated K1 cells were engulfedith higher efficiency by iDC when compared with their untreated

ounterparts or with irradiated cells (50.4 ± 11.4% uptake for K1UVCompared with 22.3 ± 14.6% for K1UN and 20.7 ± 13.8% for K1irr).ig. 7A represents the dot blots from a representative experi-ent and Fig. 7B the mean ± S.D. of four experiments. Fluorescence

icroscopy confirmed increased K1UVC uptake by iDC (Fig. 7C), The

ercentage of iDC that expressed the PS receptor CD36 [18] wasnchanged after 20 h incubation with K1UVC (not shown), thus rul-

ng out its participation in the increased tumor uptake during the0 h incubation. The participation of HMGB1, present in the super-

D. Brusa et al. / Vaccine 26 (2008) 6422–6432 6427

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ig. 4. UVC and �-irradiation as representative stimuli of early and late apoptosis. Doells after �-rad and UVC treatments, respectively. UN: untreated.

atant of K1UVC, to increased uptake had also to be ruled out, sinceo inhibition was observed when iDC were pre-treated with theAb directed against the HMGB1 receptor RAGE (Fig. 7D).

.7. Effect of phagocytosis of UVC-treated tumors on DCaturation

In the resting/immature state DCs efficiently take up Ag,hereas after activation enhanced expression of MHC and cos-

imulatory molecules and secretion of proinflammatory cytokinesnables them to activate naive and memory T-cells and modulatehe immune response. The effect of loading DC with differen-

lvabe

ig. 5. UVC treatment increases the cross-priming of CTL precursors. iDC incubated for 20atured in the presence of a cytokine cocktail and used as stimulators for autologous ly

ssessed five days after the third stimulation. (A) IFN-� release was evaluated in a 24 hestriction of the response was assessed by pre-incubating the targets with the anti-MHCAb. Difference of the MHC I-restricted response in lymphocytes cultures stimulated by DCas statistically significant (p < 0.003, results from six experiments). Spots in the culture

howed that the highest number of CD8+anti-IFN-�+ cells developed in cultures stimulatenti-CD107a mAb, a lysosome-associated marker of degranulation activity, was also higheith DC-K1UN (p > 0.05) (mean ± S.D. of four experiments). (D) UVC-treatment of the HLA

ested in the IFN-� release Elispot assay. Specificity of CTL was shown by using as targets uepresentative data from two experiments.

s representative of six experiments show preponderance of Ann+PI− and of Ann+PI+

ially treated tumor was studied after 20 h incubation with theumor and a further 48 h incubation with the maturation cytokinesNF-�, PGE2, IL-1� and IL-6, prior to their use in cross-primingxperiments. The percentage of iDC expressing the costimula-ory molecule CD80, CD83 and the chemokine receptor CCR7 waslready increased after 20 h incubation with the UVC-treated tumornot shown) and the enhancement was confirmed when mDC

oaded with K1UN were compared to mDC loaded with K1UVC (45%ersus 75% p < 0.05 for CD80, 45% versus 100% p < 0.05 for CD83,nd 35% versus 90% p < 0.05 for CCR7) (Fig. 8A left column dotlots of a representative experiment and 8B mean ± S.D. of threexperiments).

h with untreated (UN), �-irradiated (irr) or UVC irradiated tumor cells were furthermphocytes. Stimulation was repeated twice and specific antitumor response wasELISPOT assay against K1 lysate-loaded mDC (DC + LysK1) as targets. The MHC Iclass I mAb W6/32 (25 �g/ml) or an equal amount of isotype-matched irrelevantloaded with K1UN versus lymphocytes cultures stimulated by DC loaded with K1UVC

s with PMA-ionomycin were 180 ± 15. (B) Flow cytometry of intracellular stainingd by DC-K1UVC (mean ± S.D. of two experiments). (C) Intracellular staining with ther in lymphocytes stimulated with DC-K1UVC compared to lymphocytes stimulated-A2+ RCC53 renal carcinoma cell line also induced increased CTL cross-priming asnloaded, K1 lysate-pulsed or MUC-1 STAPPVHNV peptide-pulsed autologous mDC.

6428 D. Brusa et al. / Vaccine 26 (2008) 6422–6432

F n blotv atmeno lts we

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ig. 6. DAMP molecules release occurs during the iDC-K1UVC coincubation. Westerirtually undetectable at the beginning of the iDC-tumor coincubation (4 h after tref at least three experiments using the anti-Hsp70 polyclonal Ab 4872. Similar resu

Since HMGB1 has a strong maturation effect on DC [10,11], itsnvolvement in K1UVC-induced DC maturation was assessed afterlocking the HMGB1 receptor RAGE with an antagonist mAb. Thexpression of maturational markers did not change when iDC werere-treated with the mAb (Fig. 8A right column dot blots of a rep-esentative experiment and 8B mean ± S.D. of three experiments),ndicating that the effect was not due to HMGB1.

.8. iDC loaded with UVC-treated K1 cells express a large numberf transcripts for immune-related genes

mRNA analysis was performed to secure gene-level confir-ation of the increased antigen-presenting activity of K1UVC-DC

Fig. 9). Genes IFNA2 IFNA5 IFNA21, encoding for inflammatoryype I interferon, but also IFNG or CSF2 encoding DC (IL12B (p40)r macrophage (GM-CSF)-specific cytokines were increased afterncubation with both the untreated and the UVC-treated tumor.he PSMB5 product is present in the proteasome and replaced inhe immunoproteasome by the product of PSMB8 (LMP7), inducedy IFN-�.

Genes IFNGR2, IL-1 A and TGFB2 were expressed only after iDCoading with untreated tumor as were genes PSMB5 and PSMB8. Anssential function of a modified proteasome, the immunoprotea-ome, is processing of class I MHC peptides [32]. The PSMB5 products present in the proteasome and is replaced in the immunoprotea-ome by the product of PSMB8 (LMP7), induced by IFN-�.

Ten genes whose products induce or are a marker of DC matu-ation (IFNGR1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA8, IFNB1, IFNW1,L-1 B and IL-12A (p35)) were differentially up-modulated onDC incubated with UVC-treated versus untreated K1 tumor cells.GFB3, TGFB2 and IL-4, which contrast DC maturation, were alsop-regulated. Genes encoding products of the antigen-processingathway [32] followed the same differential expression. The betaype 10 PSMB1 gene induced by IFN-�, whose gene product replacesatalytic subunit 2 (proteasome beta 7 subunit) in the immuno-roteasome, was up-regulated on iDC incubated with K1UVC. Soas TAP2, which encodes a subunit of the transporter associatedith antigen-processing (TAP) and TAPBP that encodes the bind-

ng protein tapasin. TAP is required for the transport of antigeniceptides across the endoplasmic reticulum membrane, whereasAPBP mediates interaction between newly assembled MHC class Iolecules and TAP. This interaction is essential for optimal peptide

oading on the MHC class I molecule.

pt[ae

analysis of the K1UVC supernatants shows that the release of Hsp70 and HMGB1 ist) but develops later on (i.e. after 24 h from treatment). Results are representative

re obtained with the mAb W27 SC-24 (not shown).

Transcriptional up-regulation of the two MHC class II genesHLA-DQA1 and HLA-DQA2) confirmed that UVC-treated tumorwitches iDC to mDC [33]. The contribution of K1UVC to the resultsas ruled out since it did not yield any RNA, probably due to UVC-

nduced degradation.

. Discussion

The immunogenic potential of dying tumor cells is receivingreat attention on account of both its importance in enhanc-ng T-cell directed immunotherapy and its indication of the bestmmunogenic source for ex vivo TAA DC loading [34,35]. Apopto-is is generally regarded as tolerance-inducing cell death [16,17],hereas necrotic death has long been considered highly immuno-

enic because of the danger signals it conveys [3,4,7–9]. However,poptotic death has also been reported to be immunogenic in vitrond in vivo [18–23]. Here we have outlined the molecular patternnduced in vitro by conventional chemotherapy, ionizing radiationsnd UVC and found that the last produced highly immunogenicecondary necrosis.

When compared with growth factor depletion, hypotonicedium, anthracyclins, the topoisomerase inhibitor campothecin

nd �-irradiation as death agents, 30 min exposure to 3.6 J/cm2

VC induced the highest co-expression of apoptotic and necroticolecules.Migration of PS on the outer membrane is an early event fol-

owing apoptotic signals. In our study it occurred when loss ofembrane competence by 80–90% of tumor cells) was observed.

his poses the question of whether co-existence of AnnV and PIositivity reflects post-apoptotic cell death or detection of unmo-ilized membrane PS as a result of membrane permeabilization36,37]. The first alternative seems more likely, since AnnV positiv-ty was a transient event, as opposed to CRT positivity, which wasetectable on PI+ cells at late times after UVC treatment.

It is worth noting that most UVC-treated live (PI−) cells stainedositive for CRT. Our observation is in keeping with the literatureata [25]. CRT is present in the ER where it participates in peptide-HC I assembly and is traslocated to the plasma membrane as a

re-apoptotic event preceding PS exposure [25]. Its expression onhe cell membrane determines the immunogenicity of cell death25]. However, calreticulin exposure alone is not sufficient to elicitn antitumor immune response, because live cells that expresscto-CRT are unable to induce DC maturation and antigen presenta-

D. Brusa et al. / Vaccine 26 (2008) 6422–6432 6429

Fig. 7. UVC-treated K1 cells are taken up with higher efficiency by iDC. Untreated (K1UN), �-irradiated K1 (irr) and UVC-treated (K1UVC) tumor cells were green labeled withPKH2, incubated with iDC and after 20 h stained with the PE-anti-CD80 mAb. (A) Flow cytometry of a representative experiment shows an increased number of phagocytosingiDC when K1 cells were treated with UVC. No changes were observed when iDC-tumors were cultured at 4 ◦C. (B) Fluorescence microscopy shows an increased number ofp the ut ) from(

teUsd

tcs

oK�

hagocytosing iDC (arrows) in the presence of the UVC-treated (right) compared toen fields from duplicate slides). (C) mean ± S.D. percent net uptake (37 − 4 ◦C valuesmean from two experiments).

ion and hence are non-immunogenic [25]. In our study membranexpression of CRT by PI− K1 cells was significantly increased byVC, but not by the other treatments, including �- irradiation, con-

onant with the notion that different apoptotic pathways induce

ifferent exposure of CRT [25].

UVC-induced death was much more immunogenic thanhat induced by irradiation, i.e. it induced the highest T-cellross-priming by tumor-loaded DC. Its efficacy in generating tumor-pecific CTL was confirmed with another RCC cell line. Investigation

mtpti

ntreated tumor (left). (X200 magnification: each image is representative of at leastfour experiments p < 0.05; (D) uptake of K1 cells in the presence of anti-RAGE mAb

f the mechanism underlying this effect showed that UVC-treated1 cells were engulfed far more efficiently than their untreated or-treated counterparts.

Increased phagocytosis of UVC-treated K1 renal tumor was

uch higher than that we obtained with a doxorubicin/epirubicin-

reated gastric carcinoma cell line [27,29]. In keeping with theresent data, CD36, the ligand for PS, which has a role inhe internalization of apoptotic tumors by DC [18], was notncreased.

6430 D. Brusa et al. / Vaccine 26 (2008) 6422–6432

Fig. 8. Phagocytosis of UVC-treated K1 cells induces maturational events in iDC. iDC were incubated for 20 h with untreated (A, full histogram) or UVC treated (A, greenand red histograms) K1 cells. After a further 48 h incubation with TNF-�, PGE2, IL-1� and IL-6 mDC were tested for expression of the maturation markers CD80, CD83 andCCR7. The putative involvement of HMGB1 on K1-induced DC maturation was assessed by pre-incubating iDC with the mAb directed against the HMGB-1 receptor RAGE (A,r untree isotypw beenp

etpbpccs�t

mnsbi

mhbT

rfwuppn

ight column) or with the control IgG (A, left column) before being loaded with thexperiments showing significant increase of CD80, CD83 and CCR7 markers on IgGith untreated K1 (IgG K1) and no difference between UVC-K1-loaded DC that hadre-treated with the anti-RAGE mAb (RAGE K1UVC).

iDC that had bona fide engulfed UVC-treated K1 cells had a higherxpression of genes encoding inflammatory cytokines (mostly ofhe IFN I family), as well as molecules involved in the antigen-rocessing machinery (immunoproteasome subunits, TAP and TAPinding protein) and in the antigen-presentation (MHC class II)rocess. Engulfment was also followed by up-regulation of theostimulatory (CD80) and maturative (CD83) molecules and thehemokine receptor CCR7 after 20 h incubation with the tumor (nothown), but also after subsequent maturation with IL-1�, IL-6, TNF-

and PGE2. This indicates that maturation of DC by UVC-treatedumor is induced by an independent factor/cytokine.

DAMP proteins are released by death cells as a consequence of

embrane permeabilization. In our study Hsp70 and HMGB1 were

ot detectable in the supernatant of K1 cells 4 h after UVC, despiteigns of membrane incompetence (i.e. PI and trypan bleu positivity),ut were released during the subsequent 20 h of incubation with

DC. Hsp70 chaperones antigenic peptides to DC and favours DC

m

hit

ated/UVC-treated cells. A: a representative experiment. B: mean ± S.D. from threee-pretreated DC loaded with UVC-K1 (IgG K1UVC) compared to the same DC loadedpre-treated with the IgG isotype (IgG K1UVC) and UVC-K1-loaded DC that had been

aturation [7–9]. Moreover, treatments which induce Hsp70 (i.e.eating) enhance cross-priming [38]. While this can be explainedy increased transfer of tumor peptides onto MHC class I, enhancedAA expression may also be involved [38].

HMGB1 is a potent DC maturating factor [10,11]. Its extracellularelease is a consequence of necrosis-driven exit of the acetylatedorm from the nucleus where it is loosely bound to chromatin,hereas this binding is increased in apoptotic cells even when theyndergo secondary necrosis [12–14]. This is in contrast with theost-apoptotic (AnnV+/PI+) predominant state of our K1UVC cellopulation. The recent finding that transfer of HMGB1 from theucleus to the cytosol is also promoted by alkylating agents [15]

ay resolve this discrepancy.Release of DAMP molecules during the iDC-tumor contact may

ave influenced tumor uptake, DC maturation or both. Failure tonhibit this activity by RAGE blockage does not formally rule outhe participation of HMGB1, because Toll Like Receptors (TLRs) are

D. Brusa et al. / Vaccine 26 (2008) 6422–6432 6431

Fig. 9. Engulfment of UVC-treated tumor induces expression of new genes by iDC. Some of the genes whose products are involved in DC maturation and in the antigen-p ted K1M y necrD d from

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rocessing pathways are up-regulated in iDC as a result of the uptake of the untreaHC class II molecules) are over expressed in iDC that have engulfed the secondarC from the immature to the mature phenotype. Analysis performed on cells poole

lso involved in the immunogenicity of HMGB1 [39,40]. However,ifferential usage of TLR2 and TLR4 in HMGB1 signalling in pri-ary cells and established lines has been described, thus adding

omplexity to studies of HMGB1 signalling [40].As an alternative hypothesis, the increased phagocytosis rate

induced by changes in the tumor membrane) may itself havepeeded up the acquisition of a mature DC phenoptype.

The molecular events associated with UVC treatment had pro-ound effects on the immunogenic behaviour of the tumor to theoint that inhibition of CTL generation was reversed. Since this inhi-ition is partially dependent on TGF-� release and independentf T regulatory (Treg) lymphocytes (manuscript in preparation),VC-induced recovery could be due to down-modulation of the

nhibitory factors TGF-� or to induction of stimulatory factors. Theecond possibility is more likely since down-regulation of TGF-�ccurs with both UVC and �-irradiation (manuscript in prepara-ion).

Investigation of the death parameters induced by UVC has pro-uced conflicting results. In contrast with our data, most studiesescribe only late (48–72 h) post-apoptotic necrosis [41–43], per-aps due to differences in UVC potency.

The relevance of UVC treatment to tumor immunogenicity haslso been addressed. In one study, UVC-treated tumor cells thatere allowed to progress to the post-apoptotic pathway until

hey were all permeable enhanced DC cross-presentation [26].ther groups have used mixed apoptotic/necrotic populations to

oad DC and have obtained antitumor protection in vivo [44] orevelopment of antigen-specific CTL in vitro [45]. In those studiespoptotic/necrotic cells were only defined as AnnV+/PI+, and AnnVinding may have been secondary as opposed to prior to membraneermeabilization.

Our UVC conditions resemble those of photodynamic ther-py (PDT). In vitro 100 J/cm2 PDT produced a mixed populationf apoptotic and necrotic cells with antitumor effectiveness inmurine colon carcinoma [46]. Our treatments have also com-on characteristics with hyperthermia, which increases TAA

ross-presentation [38], Hsp70 [38] and HMGB1 extracellular

ranslocation (manuscript in preparation).

In conclusion, we show that UVC treatment induces a post-poptotic immunogenic cell death. These findings are consonantith an immunoadjuvant role of tumor cell death induced by

hemotherapy/radiotherapy in vivo [34,35,39,47] and offer a means

[

[

RCC. However, a much higher umber of these genes (including those encoding forotic UVC-treated tumor. The overall changes are compatible with the transition of

five different donors.

f improving ex vivo tumor antigen loading in DC-based vaccinerotocols.

cknowledgments

We thank Prof. John Iliffe for revising the manuscript. We alsohank the AVIS blood bank for providing us with blood from normalonors.

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