Cell Cycle 9:15, 3072-3077; August 1, 2010; © 2010 Landes Bioscience

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*Correspondence to: Guido Kroemer; Email: kroemer@orange.frSubmitted: 05/14/10; Accepted: 05/23/10Previously published online: www.landesbioscience.com/journals/cc/article/12459DOI: 10.4161/cc.9.15.12459

Introduction

When confronted with dying or dead cells, the tasks of the inflammatory/immune system are to clear corpses, to stimulate the replacement of lost parenchymatous cells, to alert the host defense when cell death has been triggered by infectious patho-gens, and to eliminate cells that are on the verge of oncogenic transformation.2 The nature of the affected cell type, the history of prior attempts to cope with stress, as well as the biochemi-cal route leading to cell death, influence the dying cell’s surface composition, while they affect the release of ‘find-me’ signals (for the attraction of phagocytes),3-8 exposure of ‘eat-me’ signals (for corps engulfment),1,9-11 and disclosure of ‘hidden’ molecules.12-14 Particular combinations of these cell death-associated molecules (CDAMs) have a profound impact on the choice of engulfing cells, their activation/differentiation, as well as the handling of dead-cell-antigens.15-17 Thus, particular combination of CDAMs (the ‘key’) can be decoded by the microenvironment of dying cells to open different ‘locks’ that trigger adequate inflammatory/immune responses.18

In response to immunogenic cell death inducers, calreticulin (CRt) translocates from its orthotopic localization in the lumen of the endoplasmic reticulum (eR) to the surface of the plasma membrane where it serves as an engulfment signal for antigen-presenting cells.1 Here, we report that yet another eR protein, the lysyl-tRNA synthetase (KARS), was exposed on the surface of stressed cells, on which KARS co-localized with CRt in lipid rafts. Depletion of KARS with small interfering RNAs suppressed CRt exposure induced by anthracyclines or UVC light. In contrast to CRt, KARS was also found in the supernatant of stressed cells. Recombinant KARS protein was unable to influence the binding of recombinant CRt to the cell surface. Moreover, recombinant KARS protein was unable to stimulate macrophages in vitro. these results underscore the contribution of KARS to the emission of (one of) the principal signal(s) of immunogenic cell death, CRt exposure.

Lysyl tRNA synthetase is required for the translocation of calreticulin

to the cell surface in immunogenic deatholiver Kepp,1-3,† Abdelaziz Gdoura,1-3,† Isabelle Martins,1-3 theocharis panaretakis,1-4 Frederic Schlemmer,1-3 Antoine tesniere,1-3

Gian Maria Fimia,5 Fabiola Ciccosanti,5 Anne Burgevin,10,11 Mauro piacentini,5,6 paul eggleton,7 philip J. Young,7 Laurence Zitvogel,2,3,8,9 peter van endert10,11 and Guido Kroemer1-3,*

1INSeRM; U848; Villejuif, France; 2Institut Gustave Roussy; Villejuif, France; 3Université paris Sud-XI; Villejuif, France; 4Department of oncology/pathology; Karolinska Institutet; Sweden; 5National Institute for Infectious Diseases Lazzaro Spallanzani; Rome, Italy; 6Department of Biology; University of Rome tor Vergata; Via della Ricerca Scientifica;

7peninsula Medical School; University of exeter; exeter, UK; 8INSeRM; U1015; Villejuif, France; 9CICBt507; Villejuif, France; 10INSeRM; U1013; paris, France; 11Universite paris Descartes; Faculté de médecine René Descartes; paris, France

†these authors contributed equally to this work.

Key words: anthracyclines, apoptosis, calreticulin receptor, ER stress

Recently, we have identified one particular CDAM that has a major impact on the way cell death is interpreted by the immune system. In response to some anticancer therapeutics such as anthracyclines and ionizing irradiation, tumor cells can activate a complex pathway that leads to the translocation of cal-reticulin (CRT) from its orthotopic localization, the lumen of the endoplasmic reticulum (ER), to an ectopic site, the surface of the plasma membrane.1,19 There, CRT facilitates the engulfment of cell-associated antigens by dendritic cells (DC), which are optimal antigen presenters and required for launching cellular immune responses.1 The CRT exposure pathway that is activated before the cells acquire morphological or biochemical signs of apoptosis, involves the production of reactive oxygen species, an ER stress response linked to phosphorylation of the eukaryotic translation initiation factor eIF2a, the caspase-8-mediated cleav-age of the ER protein Bap31, anterograde transport of CRT from the ER to the Golgi apparatus and its active exocytosis towards the cell surface where it binds to a hitherto elusive CRT recep-tor.20 This CRT exposure pathway is phylogenetically ancient (it is conserved in yeast) and can be subverted by multiple viruses,21 suggesting that infectious pathogens may suppress this pathway

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cells that subvert or suppress immunogenic signaling through CRT exposure may be positively selected for, in vivo.

While performing a systematic search for plasma mem-brane proteins that appear on the surface of cells treated with

to elude an immune response. Along the same lines, it appears plausible that activation of the CRT exposure pathway, for instance in response to anthracycline-based anticancer therapies, can elicit a tumor-specific immune response and that malignant

Figure 1. Surface exposure and release of KARS in immunogenic cell death. (A and B) Ct26 cells were treated for 4 h with DX in the presence or absence of Z-VAD-fmk.1 Surface proteins have been biotinylated, purified and subsequently the plasma membrane proteome has been analyzed by 2D gel electrophoresis to identify changes upon the indicated treatments. In (A) squares indicate the position of two different spots, insets show gels at higher magnification, and circles indicate the DX-induced spots that subsequently have been identified by mass spectrometry as KARS. (B) the indicated peptide sequences (bold and underlined) were identified to correspond to the peptide sequence of KARS. (C) Cells were treated with 1 mM MtX or 100 J/m2 UVC and the surface exposed KARS was determined by FACS following immunostaining in viable, pI negative cells. Results are the means ± S.e.M. of triplicate samples. (D) In addition to surface exposure KARS was also found in the supernatant of cells treated with 1 mM MtX for the indicated time as assessed by immunoblotting of whole cell lysate (WCL) and supernatant (SN) fractions with a KARS specific antibody. Anti b-actin an-tibody was used to show equal loading of WCL and absence of any cellular contamination of the SN. (e) In contrast to LpS recKARS purified from insect cells failed to induce tNFa secretion in RAW 264.7 cells when incubated with the cells at 100 mM concentration for 18 h as assessed by eLISA. Results are the means ± S.e.M. of triplicate samples. the inset shows a Coomassie staining of recKARS.

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Figure 2. KARS dependent translocation of CRt. (A) eL4 cells were treated with 1 mM MtX for 4 h, incubated with recCRt for 30 min or were left untreated. Subsequently membrane exposed or bound CRt has been detected by immuno and chemical staining and confocal microscopy indicating differences in staining patterns of endogenous and ectopic surface-CRt. the cytoplasm and the nucleus were visualized by CMtMR and DApI counter-staining respectively. (B) endogenous CRt translocates to Ct-B stained patches on the plasma membrane of Ct26 cells upon treatment with 1 mM MtX for 4 h as visualized by immunostaining of CRt and Ct-B-FItC staining contrasted with nuclear DApI uptake. (C) CRt and KARS colocalize on the plasma membrane upon treatment with 1 mM MtX for 4 h as assessed by immunostaining of CRt and KARS with species specific different antibodies. (D and e) the siRNA mediated silencing of KARS reduced the immunodetectable amount of CRt on the surface of UVC irradiated Ct26 cells. Four different siRNA oligos have been tested for their capacity to induce an efficient knockdown by immunoblotting of KARS 48 h after transfection with specific antibody. the oligonucleotides that induced a significant reduction of KARS abolished CRt exposure upon UVC treatment as assessed by FACS. Results are the means ± S.e.M. of triplicate samples. (F and G) the effect could be validated by using Halotag-CRt stably expressing U2oS cells that have been transfected with control or KARS specific siRNA. the amount of Halotag that could be detected by FACS with membrane impermeable Halotag ligand upon UVC treatment, was significantly reduced upon siRNa mediated knockdown of KARS Results are the means ± S.e.M. of three independent experi-ments. the knockdown was validated by immunoblot with KARS specific antibodies. (H) For surface receptor competition studies U2oS cells were preincubated for 5 min with recKARS (isomolar, 5-fold or 10-fold excess) recCRt (isomolar, 5-fold or 10-fold excess), BSA or were left untreated. Follow-ing the cells were subjected to incubation with 12 mg/ml FItC labeled CRt for additional 30 min and the membrane bound CRt was evaluated by FACS on pI negative cells. Results are the means ± S.e.M. of triplicate samples.

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that can be detected by indirect immunofluorescence staining (Fig. 2E). Second, we used cells that were stably transfected with a HaloTag®-CRT fusion protein that does not stain with a membrane-impermeable fluorescent HaloTag® ligand unless they are driven to expose the HaloTag®-CRT chimera on the surface. Again, KARS depletion (Fig. 2F) strongly reduced HaloTag®-CRT exposure induced by UVC light (Fig. 2G). Next, we inves-tigated whether recombinant KARS would influence the binding of CRT to cells. Cells were left untreated or pre-incubated with bovine serum albumin (control), CRT or KARS and then treated with FITC labeled CRT protein, washed and subjected to the detection of surface-bound FITC-CRT. In conditions in which unlabeled CRT reduced the binding of labeled CRT (which indi-cates the presence of a saturable receptor), KARS was unable to influence CRT binding (Fig. 2H). Altogether, these results indi-cate that KARS is required for CRT exposure, yet likewise does not constitute the CRT receptor.

Materials and Methods

The rabbit polyclonal antibodies against human CRT and KARS were purchased from Abcam. The mouse monoclonal antibody specific for actin was from Chemicon. Propidium Iodide (PI) and chloromethyl tetramethylrhodamine (CMTMR) was purchased from Molecular Probes (Invitrogen) and HaloTag® dye was from Promega. The anthracylines mitoxantrone (MTX), doxorubicin (DX), FITC labeled choleratoxin (CT-B) and tumor necrosis factor alpha (TNFa) were obtained from Sigma-Aldrich. The pancaspase inhibitor carbobenzoxy-valyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) was purchased from Bachem.

Cells, transfection and treatments. Human osteosarcoma (U2OS) cells expressing HaloTag®-CRT and human cervix car-cinoma (HeLa) cells were cultured in DMEM and mouse colon carcinoma (CT26), mouse leukemic monocyte/macrophage cells (RAW 264.7) and EL4 cells were cultured in RPMI. All media were supplemented by heat-inactivated FBS, 10 mM HEPES, 100 U/ml penicillin and 100 mg/ml streptomycin. CT26 cells were transfected with siRNA heteroduplexes specific for mouse KARS siGenome Set of 4 (Dharmacon) and U2OS cells with siRNA specific for human KARS (5'-GCU CUA AGA UCA UCA CAU A-3') (Proligo), at a final concentration of 100 nM using HiPerFect (Qiagen) according to the manufacturer’s instructions. Unrelated siRNA provided by Dharmacon was used as control. 48 h posttransfection the knockdown was validated by immunoblotting. For the induction of immunogenic cell death the cells were incubated in the presence of 1 mM MTX or 25 mM DX for 4 h in the presence or absence of 50 mM Z-VAD-fmk or were irradiated with UVC light (100 J/m2).

Purification of plasma membrane proteins. Biotinylation and recovery of cell surface proteins was performed using a method described before.22,23 In short: 20 x 106 CT26 cells were washed three times with cold PBS. Membrane proteins were biotinylated for 30 min at 4°C with 1.25 mg/ml NHSSS-biotin (Pierce) in biotinylation buffer (10 mM triethanolamine, 2 mM CaCl

2, 150

mM NaCl, pH 7.5) with gentle agitation. The cells were rinsed with PBS containing 100 mM glycine and unreacted biotin was

anthracyclines, we discovered that the ER protein lysyl-tRNA synthetase (KARS) translocates to the surface of anthracyclin-treated tumor cells. Here, we show that KARS is indispensable for the voyage of CRT to the cell surface.

Results and Discussion

Anthracyclin-induced translocation of KARS to the cell surface. We biotinylated the surface-exposed proteins from non-permeabilized HeLa cells that were left untreated (controls) or treated with the CRT exposure-inducing anthracycline doxo-rubicin (DX) alone or in combination with the synthetic caspase-inhibitor Z-VAD-fmk (DXZ), which inhibits CRT exposure. The biotinylated proteins were then purified on a streptavidin column and separated by two-dimensional gel electrophoresis. Careful inspection of the gels revealed the presence of two adja-cent spots that were upregulated by one order of magnitude in doxorubicin-treated cells as compared to untreated controls and by a factor of ~3 as compared to cells treated with doxorubicin plus Z-VAD-fmk (Fig. 1A). Mass spectrometry identified at least nine peptides within these spots that are derived from the mature KARS protein (Fig. 1B). Although treatment with anthracy-clines such as doxorubicin (not shown) or mitoxantrone (MTX) did not cause an overexpression of KARS at the whole-cell level, it did cause translocation of immuno-detectable KARS to the cell surface (Fig. 1C) and secretion of KARS into the supernatant (Fig. 1D). This was an early process that was detectable within 4 to 8 hours, that is well before mitoxantrone induces significant apoptosis in the tumor cells (Fig. 1C and D). An unconfirmed study reported that recombinant KARS protein (produced in bacteria) stimulates tumor necrosis factor-a (TNFa) produc-tion by macrophages.26 However, recombinant KARS protein that was generated in insect cells using a baculovirus expression system failed to elicit TNFa secretion by the same macrophage cell line (Fig. 1E). In conclusion, it appears that cells treated with anthracyclines transport KARS to their surface and secrete it.

Involvement of KARS in the CRT exposure pathway. After anthracycline treatment, tumor cells expose endogenous CRT on small patches of their plasma membrane, as determined by indirect immunofluorescence staining of CRT. This patchy dis-tribution contrasts with that obtained by absorbing recombi-nant CRT to healthy cells, yielding a near-to-continuous ring of CRT that decorates the entire plasma membrane (Fig. 2A). Endogenous CRT present on the surface of mitoxantrone-treated cells localized in discrete patches that correspond to rafts and can be labeled with a FITC-choleratoxin B conjugate (Fig. 2B). Endogenous KARS that translocates to the plasma membrane of mitoxantrone-treated cells co-localized with endogenous CRT and hence also distributed into lipid rafts (Fig. 2C). Next, we explored the possibility that KARS would be required for CRT exposure. Knockdown of KARS with several non-overlapping small interfering RNAs (siRNAs) (Fig. 2D) reduced the fre-quency of cells that expose CRT in response to anthracyclines (not shown) or UVC light. This result was obtained using two different technologies for the quantitation of CRT expo-sure. First, knockdown of KARS reduced the presence of CRT

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(Southern Biotechnologies Associates) and detected by ECL (Pierce). Anti-actin antibody was used as a control to determine equal loading of cell lysates.

Analysis of surface exposed CRT and surface exposed HT CRT. For the detection of extracellular HaloTag® moieties, cells were treated and incubated as described above before detach-ment. Subsequently the cells were incubated for 30 min with HaloTag® Alexa Fluor® 488 Ligand, diluted in DMEM medium containing 10% of fetal bovine serum according to the manu-facturer’s intruction. The cells were washed twice with PBS and incubated for additional 30 min in DMEM medium. Thereafter, cells were rinsed twice in PBS and incubated with 1 mg/ml PI (Invitrogen) for necrosis detection. For CRT immune staining, cells, treated as described above were washed twice with cold PBS and fixed in 0.25% w/v paraformaldehyde in PBS for 5 min on ice. Following repeated washing steps with cold PBS, cells were incubated for 30 min with the primary antibody, diluted in cold blocking buffer (2% fetal bovine serum in PBS), followed by washing and incubation with the Alexa488-conjugated monoclo-nal secondary antibody in a blocking buffer for additional 30 min. Samples were analyzed by means of a FACScan (Becton-Dickinson) for cell surface CRT- HaloTag® or endogenous cell surface CRT respectively. Isotype-matched IgG antibodies were used as controls, and the fluorescent intensity of stained cells was gated on PI-negative cells.

FITC-CRT binding studies. To study a competing effect of KARS and CRT concerning surface receptor binding 1 x 105 cells have been collected in cold PBS and were pretreated for 5 min with recKARS, recCRT at the indicated concentrations or left untreated. The cells were washed with PBS and were exposed to FITC labeled CRT in PBS for 30 min in cold PBS. Upon addi-tional washing the cells were co-stained with PI and analyzed in a FACS Calibur (Becton-Dickinson) for cell surface bound FITC-CRT. BSA was used as control, and for the analysis the fluores-cent intensity of stained cells was gated on PI-negative cells.

Immunofluorescence staining and concofocal microscopy. For the detection of surface exposed CRT or KARS, cells grown on coverslips were washed twice with cold PBS and fixed in 0.25% w/v PFA in PBS for 5 min on ice. For visualization of lipid rafts the cells were incubated with FITC labeled CT-B (final concentration of 2.5 mg/ml) for 30 min at room temperature. Subsequently the cells were washed with cold PBS and primary antibody, diluted in cold blocking buffer, was added for 30 min. Following several washing steps with cold PBS, the cells were incubated for addi-tional 30 min with the appropriate secondary antibody diluted in cold blocking buffer. Cells were then washed with PBS and mounted on slides with DAPI containing mounting medium (vectashield). The samples were analyzed in a Leica TCS SPE con-focal microscope (Leica Microsystems, Wetzlar, Germany).

Macrophage stimulation assay. 2 x 105 RAW264.7 cells were cultured in 24-well plates for 5 h and then incubated for addi-tional 18 h with 100 nM recKARS (purified from insect cells), 100 ng/ml LPS or with PBS. After centrifugation at 1,000 g for 5 min the secreted TNFa was detected by using a TNFa (Mono/Mono) ELISA kit following the manufacturer’s instructions (Pharmingen).

quenched for 20 min. The cells were then rinsed twice with PBS and pelleted before solubilization for 45 min in lysis buffer (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5) containing protease inhibitors. The samples were incubated overnight with packed streptavidin-agarose beads to recover the biotinylated proteins.

Two-dimensional gel electrophoresis and proteomic identification. This method was carried out as described before.20 In short: purified proteins were precipitated and subsequently resuspended in urea buffer. For the first dimension of protein separation, isoelectric focusing was performed using immobi-lized nonlinear pH gradient strips (pH 3 to 10; GE Healthcare). 100 mg of protein was loaded and then run. Prior to the second-dimension electrophoresis, IPG gel strips were equilibrated and subsequently derivatized. Strips were transferred to 10% (wt/vol) polyacrylamide gels and the second-dimension gels were run before staining with Sypro Ruby (BioRad) and visualization by means of a Typhoon 9,200 scanner (GE Healthcare). The Investigator HT analyser (Genomic Solutions Inc.) was used for matching and analysis of visualized protein spots among dif-ferential gels. Differentially expressed spots were excised from the gels with an automatic spot picker (Investigator ProPic, Genomic Solutions Inc.) destained and washed with acetoni-trile. The dried gel pieces were reswollen and digested with tryp-sin. MALDI-MS and MALDI-MS/MS were performed on an Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF ion optics. Spectra were acquired and calibrated. Mass spectra were obtained from each sample spot by 30 sub-spectra accu-mulation in a 750 to 4,000 mass range. MS and MS/MS data were interpreted using the GPS Explorer software (Version 2.1, Applied Biosystems). Peptide mass fingerprints obtained from MS analysis were used for protein identification in Swiss Prot non-redundant database.

Purification of human recombinant KARS and CRT. RecCRT was produced as described before.24 A full length cDNA encod-ing murine KARS was obtained from Geneservice (Cambridge, U.K.), amplified using primers adding a 5' EcoRV site and a 3' NheI site, sequenced to confirm absence of errors, and cloned into a pVL1393-based plasmid already containing a sequence encoding a C-terminal poly-histidine tag. The resulting plasmid was used to produce a recombinant baculovirus using standard procedures. Recombinant KARS was purified from lysates of infected insect cells prepared in 1% CHAPS by incubation with a Ni-Nta resin (ProBond, Invitrogen) and dialyzed against 25 mM Hepes pH 7.4, 200 mM NaCl and 10% glycerol before use. Purity of both proteins was checked by Coomassie staining and immunoblot. RecCRT for FITC labeling was produced as described before.25 Subsequently it was labeled using the FITC labeling kit from Pierce following the manufacturer’s instruction.

Immunoblots. Cells were washed and lysed in lysis buffer (50 mM Tris HCl pH 6.8, 10% glycerol and 2% SDS) supple-mented with protease inhibitors (Roche). Protein concentration was determined by Biorad protein assay (Biorad) and 30 mg of protein was separated on Nu-Page precast gels (Invitrogen). The primary antibody detecting KARS was revealed with the appropriate horseradish peroxidase-labeled secondary antibody

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19. Obeid M, Panaretakis T, Joza N, Tufi R, Tesniere A, van Endert P, et al. Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC light-induced apoptosis. Cell Death Differ 2007; 14:1848-50.

20. Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC, et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J 2009; 28:578-90.

21. Kepp O, Senovilla L, Galluzzi L, Panaretakis T, Tesniere A, Schlemmer F, et al. Viral subversion of immuno-genic cell death. Cell Cycle 2009; 8:860-9.

22. Gottardi CJ, Dunbar LA, Caplan MJ. Biotinylation and assessment of membrane polarity: caveats and method-ological concerns. Am J Physiol 1995; 268:285-95.

23. Hanwell D, Ishikawa T, Saleki R, Rotin D. Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells. J Biol Chem 2002; 277:9772-9.

24. Culina S, Lauvau G, Gubler B, van Endert PM. Calreticulin promotes folding of functional human leu-kocyte antigen class I molecules in vitro. J Biol Chem 2004; 279:54210-5.

25. Young P, Szestakowska D, Morse R, Winyard P, Whatmore J, Reid K, et al. Purification, Isolation and Characterization of Native and Recombinant Calreticulin. Calcium Binding Proteins 2006; 1:160-16.

26. Park SG, Kim HJ, Min YH, Choi EC, Shin YK, Park BJ, et al. Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response. Proc Natl Acad Sci USA 2005; 102:6356-61.

27. Panaretakis T, Joza N, Modjtahedi N, Tesniere A, Vitale I, Durchschlag M, et al. The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death. Cell Death Differ 2008; 15:1499-509.

9. Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001; 411:207-11.

10. Srivastava P. Roles of heat-shock proteins in innate and adaptive immunity. Nat Rev Immunol 2002; 2:185-94.

11. Savill J, Dransfield I, Gregory C, Haslett C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2002; 2:965-75.

12. Aymeric L, Apetoh L, Ghiringhelli F, Tesniere A, Martins I, Kroemer G, et al. Tumor cell death and ATP release prime dendritic cells and efficient anticancer immunity. Cancer Res 70:855-8.

13. Sancho D, Joffre OP, Keller AM, Rogers NC, Martinez D, Hernanz-Falcon P, et al. Identification of a dendritic cell receptor that couples sensing of necrosis to immu-nity. Nature 2009; 458:899-903.

14. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent con-tribution of the immune system to anticancer chemo-therapy and radiotherapy. Nat Med 2007; 13:1050-9.

15. Tesniere A, Apetoh L, Ghiringhelli F, Joza N, Panaretakis T, Kepp O, et al. Immunogenic cancer cell death: a key-lock paradigm. Curr Opin Immunol 2008; 20:504-11.

16. Green DR, Ferguson T, Zitvogel L, Kroemer G. Immunogenic and tolerogenic cell death. Nat Rev Immunol 2009; 9:353-63.

17. Krysko DV, D’Herde K, Vandenabeele P. Clearance of apoptotic and necrotic cells and its immunological consequences. Apoptosis 2006; 11:1709-26.

18. Zitvogel L, Kepp O, Kroemer G. Decoding cell death signals in inflammation and immunity. Cell 2010; 140:798-804.

Concluding Remarks

In this report, we demonstrate that KARS translocates to the surface of cells that have been treated with immunogenic cell death inducers and that KARS is actually required for the sur-face exposure of CRT. Both KARS and CRT co-localize at the surface of stressed cells within or in close proximity of microdo-mains that incorporate choleratoxin B and hence constitute bona fide lipid rafts. These results suggest to speculate that KARS and CRT may co-translocate in the same molecular complex to the cell surface. CRT and the disulfide isomerase ERp57 have previ-ously been shown to interact within the ER and to co-translocate to the cell surface of anthracycline-treated cells.27 Knockout or knockdown of ERp57 suppressed CRT exposure while deletion or depletion of CRT abolished ERp57 translocation to the plasma membrane, indicating that the two molecules co-translocate in an obligatory fashion. Moreover, CRT that was mutated on two amino acids that are required for its interaction with ERp57 was unable to restore the ERp57 exposure induced by anthracyclines, supporting the notion that the physical interaction between ERp57 and CRT is crucial for their joint translocation to the cell surface.27 The data presented here suggest that ERp57 and CRT travel together in a larger molecular complex that includes other ER proteins including KARS. At difference with CRT

(which we were unable to detect in the soluble fraction), however, KARS was secreted into the cell free supernatant. Whether this reflects a dissociation of the multiprotein complex once it is pres-ent on the cell surface or additional mechanisms of KARS release has not yet been determined.

Soluble KARS has previously been reported to stimulate macrophages in vitro, suggesting that KARS might have immu-nostimulatory properties.26 We have been unable to recapitulate these results using a preparation of recombinant KARS protein that was generated in the absence of any possible contamination with bacterial lipopolysaccharide. As a result, the exact— inflam-matory or antiphlogistic, immunostimulatory or immunosup-pressive—function of extracellular KARS remains enigmatic.

Acknowledgements

G.K. is supported by the Ligue Nationale contre le Cancer (Equipes labellisée), Agence Nationale pour la Recherche (ANR), European Commission (Apo-Sys, ChemoRes, ApopTrain), Fondation pour la Recherche Médicale (FRM), Institut National du Cancer (INCa) and Cancéropôle Ile-de-France. O.K. receives a post-doctoral fellowship from INSERM and F.S. is supported by FRM. T.P. is supported by Cancerfonden, Barncancerfonden, the Swedish Royal Academy of Sciences and the Åke Wiberg Stiftelse. I.M. is supported by La Ligue Nationale Contre le Cancer.

References1. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM,

Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13:54-61.

2. Fadeel B. Programmed cell clearance. Cell Mol Life Sci 2003; 60:2575-85.

3. Ghiringhelli F, Apetoh L, Tesniere A, Aymeric L, Ma Y, Ortiz C, et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med 2009; 15:1170-8.

4. Le LQ, Kabarowski JH, Weng Z, Satterthwaite AB, Harvill ET, Jensen ER, et al. Mice lacking the orphan G protein-coupled receptor G2A develop a late-onset autoimmune syndrome. Immunity 2001; 14:561-71.

5. Gude DR, Alvarez SE, Paugh SW, Mitra P, Yu J, Griffiths R, et al. Apoptosis induces expression of sphingosine kinase 1 to release sphingosine-1-phos-phate as a “come-and-get-me” signal. FASEB J 2008; 22:2629-38.

6. Knies UE, Behrensdorf HA, Mitchell CA, Deutsch U, Risau W, Drexler HC, et al. Regulation of endothelial monocyte-activating polypeptide II release by apopto-sis. Proc Natl Acad Sci USA 1998; 95:12322-7.

7. Nishimura T, Horino K, Nishiura H, Shibuya Y, Hiraoka T, Tanase S, et al. Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemo-tactic S19 ribosomal protein dimer. J Biochem 2001; 129:445-54.

8. Segundo C, Medina F, Rodriguez C, Martinez-Palencia R, Leyva-Cobian F, Brieva JA. Surface molecule loss and bleb formation by human germinal center B cells undergoing apoptosis: role of apoptotic blebs in mono-cyte chemotaxis. Blood 1999; 94:1012-20.