Sylvain Ladoire

Dalil Hannani

Marie Vétizou

Clara Locher

Laetitia Aymeric

Lionel Apetoh

Oliver Kepp

Guido Kroemer

François Ghiringhelli

Laurence Zitvogel

Marie Vetizou

¸ois Franc

Ghiringhelli

Cell-Death-Associated Molecular Patterns As Determinants of Cancer Immunogenicity

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction: Immunogenic Cancer Cell Death in Cancer?

S ince most human malignancies result from chronic inflammatory processes, oncogenic transformation was not considered intrinsically immunogenic. However, recent lines of evidence indicate that this assumption needs to be revisited. First, the composition, function, and gene expression profile of the immune infiltrates in the tumor has a major impact on the fate of cancer patients [START_REF] Disis | Immune regulation of cancer[END_REF][START_REF] Finn | Cancer immunology[END_REF][START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF]107). Second, meta-analyses of multiple clinical studies indicate that severe lymphopenia ( < 1000 cells/ll) negatively affects the chemotherapeutic response of multiple distinct solid cancers (122). Third, some oncogenic mutations may be capable of eliciting major histocompatibility complex (MHC) restricted T-cell immune responses [START_REF] Ait-Tahar | CD4 T-helper responses to the anaplastic lymphoma kinase (ALK) protein in patients with ALK-positive anaplastic large-cell lymphoma[END_REF][START_REF] Ait-Tahar | B and CTL responses to the ALK protein in patients with ALK-positive ALCL[END_REF][START_REF] Disis | Immune regulation of cancer[END_REF][START_REF] Kobayashi | Defining promiscuous MHC class II helper T-cell epitopes for the HER2/neu tumor antigen[END_REF][START_REF] Kroemer | Can the exome and the immunome converge on the design of efficient cancer vaccines?[END_REF]. Finally, infiltration by CD8 + T cells also has a positive predictive value, for instance, in breast cancers (BC) treated by neoadjuvant chemotherapy [START_REF] Denkert | Tumor-associated lym-phocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer[END_REF].

For many decades, most anticancer therapies, especially radiotherapy and chemotherapy, were considered direct tumoricidal compounds, killing cancer cells or inducing a permanent arrest in their cell cycle. Chemotherapies target without any distinction tumor cells and rapidly proliferating host's immune cells, leading to the assumption that cytotoxic chemotherapeutics would induce immunosuppressive rather than immunostimulatory off-target effects (167,171). This belief has largely discouraged oncologists from performing extensive studies on the contribution of the immune system in anticancer therapies. Indeed, drug discovery programs have neglected the possibility that host immune responses may account for -at least part of -the efficacy of cytotoxic drugs. For example, the guidelines for drug screening and validation provided by the National Cancer Institute recommended the (xeno-) grafting of human cancer cell lines into immunodeficient hosts (168). Moreover, arguing against a dominant role of the immune system in the antitumor effects mediated by select chemotherapies, the fact that most treatment modalities induce ''apoptosis,'' a cell demise is considered ''immunologically silent,'' if not ''tolerogenic'' [START_REF] Kroemer | Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009[END_REF][START_REF] Matzinger | The danger model: a renewed sense of self[END_REF]131).

Despite these early observations, recent experimental evidence from several groups challenged this notion that necrosis would be immunogenic while apoptosis would not [START_REF] Green | Immunogenic and tolerogenic cell death[END_REF], indicating that the nature of cell death defined by distinct molecular, biochemical, and metabolic hallmarks would convey the mandatory links between dying cells, innate and cognate immunity. Hence, while many chemotherapeutic agents (e.g., etoposide, mitomycin C, and cisplatin) trigger a cell death modality that is not immunogenic, distinct cytotoxic compounds such as doxorubicin, oxaliplatin, cyclophosphamide, or ionizing radiation could favor the activation of immune effectors cells while inducing apoptotic tumor cell death [START_REF] Casares | Caspasedependent immunogenicity of doxorubicin-induced tumor cell death[END_REF]102). This means that (i) cancer cells dying after anthracyclines, oxaliplatin, or irradiation can mount an anti-cancer immune response in the absence of adjuvants and immunize the host against a subsequent tumor rechallenge [START_REF] Casares | Caspasedependent immunogenicity of doxorubicin-induced tumor cell death[END_REF]102); (ii) that established tumors which regressed postchemotherapy are massively invaded by cytotoxic T cells; and (iii) that chemotherapy-induced control of transplantable tumors depends on T lymphocytes. Indeed, we reported that these anti-cancer treatments were more efficient in immunocompetent than in immunocompromised animals (101, 102, 147).

We showed a hierarchy of cytokine-associated events that can be summarized as follows. First, the triggering of the autophagy machinery in chemotherapy-exposed tumor cells promotes adenosine tri-phosphate (ATP) release, which is indispensable for the recruitment of inflammatory monocytes-derived dendritic cells (in a P2Y2-dependent manner) [START_REF] Michaud | Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice[END_REF]. The ATP/P 2 RX 7 /Nlrp3/Caspase-1 axis in local dendritic cells (DCs) exposed to dying tumor cells promotes IL-1b release by DCs and activation of c4/c6 cdT cells that secrete IL-17 and IL-22 three days after chemotherapeutic treatment. Those cdT17 cells markedly correlate with the brisk recruitment and accumulation of interferon-c (IFNc)-producing CD8 + CTLs in tumor beds by day 8 postsystemic chemotherapy. Hence, using neutralizing antibodies and loss-offunction mice targeting IL-17/IL-17RA, IL-1b/IL-1R1, and IFNc/IFNRII, we showed that these three cytokines are crucial for the immunogenicity of cell death and anticancer efficacy mediated by chemotherapy.

These data generated in transplantable tumor models as well as in methylcholanthrene-induced sarcoma have been extended to humans. Anthracycline-killed tumor cells also appear particularly immunogenic [START_REF] Fucikova | Human tumor cells killed by anthracyclines induce a tumor-specific immune response[END_REF]. After anthracylinebased chemotherapy, the intratumoral leukocyte infiltrate changes its composition in cancer patients. Accordingly, after exposure to neoadjuvant chemotherapy, one third of stage II/ III BCs appear highly infiltrated by cytotoxic T cells with a concomitant reduction of B lymphocytes and Th2-CD4 + T cells (127). Moreover, the ratio of macrophages to CD8 + T cells in primary BCs not only predicts overall survival, but also predicts a pathologic response to chemotherapy in chemo-naı ¨ve patients [START_REF] Denardo | Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy[END_REF]. An increased abundance of T lymphocytes, as well as an ameliorated ratio of CD8 + T cells over Foxp3 + regulatory cells postchemotherapy, also predicted favorable therapeutic responses to combination therapy with anthracyclines/taxanes in human BC, and so does invasion by CD8 + T cells in colorectal cancer liver metastases [START_REF] Denardo | Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy[END_REF][START_REF] Halama | Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy[END_REF][START_REF] Ladoire | In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival[END_REF]161). This review will summarize the mandatory cell-deathassociated molecular patterns (CDAMP) for immunogenic cell death (ICD) to occur and will discuss the clinical implementations of this concept.

Early Signaling During ICD: Role of Calreticuline in Antigen Uptake

The uptake of dead cell-derived antigens by DCs constitutes an early step in the sequence of events that leads to anticancer immune responses. One of the first and cardinal molecular events during ICD is the rapid exposure of molecules on the outer leaflet of the plasma membrane that act as ''eat-me'' signals for antigen-presenting cells such as DCs [START_REF] Gardai | Recognition ligands on apoptotic cells: a perspective[END_REF]. Interestingly, the analysis of surface proteome changes in anthracycline-treated tumor cells revealed that ICD is associated with the ectopic exposure of the endoplasmic reticulum (ER) chaperone calreticuline (CRT) (102, 109). In homeostatic conditions, CRT is mainly located in the lumen of the ER, in which it functions as a molecular chaperone and modulates calcium signaling and homeostasis (65). In response to anthracyclines or ionizing radiation, CRT exposure occurs before the first morphological signs of apoptosis and before the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Another prerequisite for surface CRT exposure is the activation of caspases; the Z-VAD fmk caspase inhibitor abolishing anthracycline-mediated CRT exposure to the cell surface [START_REF] Casares | Caspasedependent immunogenicity of doxorubicin-induced tumor cell death[END_REF]. This process is accompanied by other hallmarks of the ER stress response such as phosphorylation of the eukaryotic initiation factor 2a (Fig. 1) and co-trafficking with CRT of another ER-resident protein, the disulfide isomerase ERp57. ERp57 knockdown tumor cells fail to elicit immune responses and to respond to chemotherapy. This can be overcome by exogenous recombinant CRT protein in immunocompetent but not nu/nu mice, indicating that tumors with an intrinsic defect in the CRT-translocation machinery become unable to elicit an efficient anticancer immune response, contributing to tumor regression (109). The presence of CRT on the surface of tumor cells that succumb to distinct anticancer agents correlates with their uptake by DCs (102). Ecto-CRT appears to be indispensable for phagocytosis and ICD in that neutralizing CRT-specific antibodies or silencing of CRT by RNA interference abolished phagocyte engulfment of dying bodies and the immunogenicity of tumor cell death (102).

While we described an ER stress-response-dependent CRT exposure in the context of anthracyclines, cyclphosphamide, oxaliplatin, and irradiation, other reports confirmed the cardinal function of CRT in the recognition of dying cells and the elimination of nascent tumors.

CRT translocation as triggered by hypericin-based photodynamic therapy was independent of ERp57, increased cytosolic Ca 2 + concentrations, eIF2a phosphorylation, caspase-8 activation, and lipid rafts [START_REF] Garg | A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death[END_REF][START_REF] Krysko | Immunogenic cell death and DAMPs in cancer therapy[END_REF]. Conversely, hypericinbased photodynamic therapy promoted ICD via a molecular cascade that relies on reactive oxygen species (ROS) overgeneration, PERK, BAX, and BAK, PI3K activity, and the ERto-Golgi transport apparatus [START_REF] Garg | A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death[END_REF][START_REF] Krysko | Immunogenic cell death and DAMPs in cancer therapy[END_REF].

Interestingly, Beneteau et al. just reported that inhibition of glycolysis converted etoposide-mediated non-ICD into an efficient vaccine procedure in mice. They demonstrated that this effect was at least partially mediated through ERp57/ CRT exposure on the plasma membrane [START_REF] Beneteau | Combination of glycolysis inhibition with chemotherapy results in an antitumor immune response[END_REF].

Chao et al. demonstrated that CRT is a dominant prophagocytic signal in several human cancers (such as leukemia, neuroblastoma, bladder cancers, and lymphoma) binding to CD91 on macrophages and counterbalancing the CD47/SIR-Pa-mediated anti-phagocytic signal. Interfering in the delicate balance between pro-and anti-phagocytic signals by selectively targeting tumor cells by anti-CD47 antibody effectively prevents immune evasion [START_REF] Chao | Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47[END_REF].

Hence, the ER stress response constitutes a central hub in the signaling cascades, leading to the emission of ICDpromoting signals. The accumulation of misfolded proteins, the inhibition of protein glycosylation, and the deregulation of redox and Ca 2 + homeostasis can disrupt the delicate homeostasis of the ER-Golgi network, resulting in the activation of adaptive cell responses (120). More work is required to establish which transcription factors of the unfolded protein response are mandatory for the ICD and how the ER stress coopts the pattern recognition receptor (TLR4 in this case) signaling pathway to ignite innate immunity [START_REF] Martinon | TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages[END_REF].

Late Signaling During ICD: HMGB1 and Cross-Presentation of Tumor Antigens

The CRT-driven uptake of tumor antigens by DCs is per se insufficient to elicit an antitumor immune response, as these tumor antigens should be also efficiently processed by DCs FIG. 1. Molecular mechanisms dictating early exposure of ecto-CRT. Immunogenic cell death (ICD) requires an ''endoplasmic reticulum (ER) stress'' for which the signaling axis phospho extracellular-signal-regulated kinase (PERK)-eukaryotic initiation factor 2a (eIF2a) is mandatory for anthracyclines and oxaliplatin-mediated calreticuline calreticulin (CRT) exposure. The ICD also requires an intact apoptotic signal that involves caspase-8, the pro-apoptotic B cell lymphoma 2 family members BAX and BAK, and the ER protein BAP31. The inhibition of glycolysis could favor ecto-CRT exposure, rendering apoptosis inducers (but not intrinsically ER stressors) as immunogenic treatments. Strong ecto-CRT exposition overcomes the negative and exposed at their surface within MHC complexes for the cross-priming of CD4 + and CD8 + T lymphocytes. This implies that, during ICD, other signaling pathways involved in antigen processing, presentation, and polarization are required after antigen uptake. We will now deal in detail with how high mobility group box 1 protein (HMGB1) and ATP can also determine the immunological fate of cell death.

HMGB1 in cancer

The HMGB1 is ubiquitously present in the nucleus of almost all mammalian cells, in which it stabilizes nucleosomes and regulates the transcription of many genes, promoting access to DNA to transcriptional factors (111, 143). Within the nucleus, it binds to the minor groove of DNA and enables interaction between nucleic acids and a variety of molecules, including p53, NF-kB, and steroid hormone receptors. Besides its nuclear role, HMGB1 also functions as an extracellular cytokine-like molecule during various physiological or pathological processes, such as inflammation, cell differentiation, cell migration, or tumor metastasis [START_REF] Xda | High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy[END_REF][START_REF] Ellerman | Masquerader: high mobility group box-1 and cancer[END_REF][START_REF] Lotze | High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal[END_REF][START_REF] Muller | New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal[END_REF]. Indeed, HMGB1 is secreted by macrophages [START_REF] Bonaldi | Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion[END_REF]158), mature DCs [START_REF] Dumitriu | Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products[END_REF], and natural killer (NK) cells (139) in response to injury, infection, or other inflammatory stimuli. When released outside the cell, HMGB1 can bind with a high affinity to several receptors, including the receptor for advanced glycation end products (RAGE), TLR-2, TLR-4, TLR-9, and, as a negative signaling molecule, CD24, mediating the response to infection and injury, thereby igniting inflammation [START_REF] Xda | High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy[END_REF][START_REF] Lotze | High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal[END_REF]132,148,158). For a long time, HMGB1 has been thought to exert a proinflammatory function exclusively during necrosis (132), but recent evidence indicates that it also gets released by cells undergoing the late stages of apoptosis [START_REF] Bell | The extracellular release of HMGB1 during apoptotic cell death[END_REF]148). Indeed, many investigators have found that nuclear DNA is released in a time-dependent manner during apoptosis [START_REF] Choi | Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro[END_REF]. Moreover, binding of HMGB1 to DNA is increased during apoptosis, in line with the fact that late-stage apoptotic cells can release both DNA and HMGB1. Thus, genotoxic agents that activate polyadenosylribosyl polymerase (PARP), including chemotherapeutic agents, can stimulate the release of HMGB1 from its association with chromatin, presumably as a result of the direct PARP-mediated polyadenosylribosylation of HMGB1 [START_REF] Ditsworth | Activation of poly(ADP)-ribose polymerase (PARP-1) induces release of the pro-inflammatory mediator HMGB1 from the nucleus[END_REF]. Outside the cell, HMGB1 could also favor the establishment of T-cell-dependent, protective immune responses [START_REF] Kono | How dying cells alert the immune system to danger[END_REF][START_REF] Krysko | From regulation of dying cell engulfment to development of anti-cancer therapy[END_REF]106), acting as ''alarmin'' or damaged-associated molecular pattern (DAMP) during cell death. Once passively released from necrotic cells in the extracellular space, HMGB1 acts as a chemoattractant for inflammatory leukocytes and stem cells [START_REF] Chavakis | Highmobility group box 1 activates integrin-dependent homing of endothelial progenitor cells[END_REF]108), and is able to trigger the functional maturation of DCs and to support the clonal expansion of IFNc-producing Th1 cells [START_REF] Dumitriu | Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products[END_REF][START_REF] Moser | Receptor for advanced glycation end products expression on T cells contributes to antigen-specific cellular expansion in vivo[END_REF]. It also influences the migration of DCs in secondary lymphoid organs [START_REF] Dumitriu | The secretion of HMGB1 is required for the migration of maturing dendritic cells[END_REF][START_REF] Manfredi | Maturing dendritic cells depend on RAGE for in vivo homing to lymph nodes[END_REF]. Besides the roles of HMGB1 within the nucleus and in the extracellular environment, a third life as a cytosolic chaperon has been recently described, modulating the ability of cancer cells to die via apoptosis or to activate the autophagic machinery. For instance, it has been recently demonstrated that HMGB1/p53 complexes are able to regulate the cytoplasmic localization of the reciprocal protein, and subsequent levels of autophagy and apoptosis [START_REF] Livesey | p53/HMGB1 complexes regulate autophagy and apoptosis[END_REF].

HMGB1 appears to have an alternate paradoxical role during tumorigenesis. Indeed, HMGB1 recognition can favor tumor progression through neoangiogenesis by attracting pro-angiogenic macrophages and endothelial cells in tumor beds (155) (Fig. 2A). Extracellular HMGB1 can trigger cell proliferation, migration, and sprouting of endothelial cells [START_REF] Mitola | Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine[END_REF]136). In addition, other studies have suggested a direct role of HMGB1 in tumor cell motility and invasion [START_REF] Fages | Regulation of cell migration by amphoterin[END_REF]114,115,121). Altogether, HMGB1 was considered a factor of tumor progression that was associated with a dismal prognosis in cancer (100, 117). Accordingly, HMGB1-RAGE interactions mediated invasion, migration, and the growth of implanted tumors in glioma models (165). In most of the studies, HMGB1 and/or RAGE expression was studied at protein or mRNA levels. Overexpression (compared with healthy tissues) as well as cytoplasmic localization of HMGB1 have been reported in many cancer types, including gastrointestinal stromal tumors [START_REF] Choi | Overexpression of high mobility group box 1 in gastrointestinal stromal tumors with KIT mutation[END_REF], colon (157), gastric [START_REF] Kuniyasu | Expression of receptors for advanced glycation end-products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer[END_REF], pancreatic (144), melanoma, prostate, and cervical carcinomas [START_REF] Hao | Expression and clinical significance of HMGB1 and RAGE in cervical squamous cell carcinoma[END_REF][START_REF] Ishiguro | Receptor for advanced glycation end products (RAGE) and its ligand, amphoterin are overexpressed and associated with prostate cancer development[END_REF][START_REF] Jacob | In vitro modulation of human melanoma cell invasion and proliferation by all-trans-retinoic acid[END_REF]116,117,157). Moreover, in prostate cancer, HMGB1 and RAGE expressions were increased in metastatic sites compared with primary tumors of nonmetastatic patients [START_REF] Kuniyasu | Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer[END_REF]. Enhanced expression of HMGB1 and RAGE is associated with aggressive tumor phenotype and poor prognosis at any stage in colon carcinoma (130). One possible scenario is that neoplastic cells expressing higher amounts of HMGB1 also release the pro-metastatic and pro-angiogenic cytokine in the tumor microenvironment [START_REF] Aychek | E-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances HMGB1 release[END_REF][START_REF] Ellerman | Masquerader: high mobility group box-1 and cancer[END_REF]. A recent and elegant study demonstrated that HMGB1 can bind to TIM3 in tumor DCs, thereby suppressing the immunostimulatory pathway in DC mediated through nucleic acids released by dying tumor cells (20) (Fig. 2B).

Surprisingly, the relevance of HMGB1 expression has been poorly investigated in BC. In one study, an increased expression of HMGB1 proteins in the tumor beds compared with healthy breast tissues was reported [START_REF] Flohr | Variation of HMGB1 expression in breast cancer[END_REF]. Of note, nuclear HMGB1 markedly enhances DNA binding and transcriptional activity of the estrogen receptors in the estrogen responsive elements [START_REF] Das | High mobility group B proteins facilitate strong estrogen receptor binding to classical and half-site estrogen response elements and relax binding selectivity[END_REF][START_REF] Flohr | Variation of HMGB1 expression in breast cancer[END_REF][START_REF] Melvin | The role of the C-terminal extension (CTE) of the estrogen receptor alpha and beta DNA binding domain in DNA binding and interaction with HMGB[END_REF], suggesting its potential relevance in the biology of estrogen-sensitive BC. Northern blots analyses of the 1.4 kb and the 2.4 kb transcripts of HMGB1 in 13 BC samples revealed strong variations that could potentially account for the response to endocrine therapy in estrogen receptor-positive BC [START_REF] Flohr | Variation of HMGB1 expression in breast cancer[END_REF]. Since a few data FIG. were available on HMGB1 expression in BC and normal breast tissues using immunohistochemistry (IHC), we carried out the IHC analysis of the pattern of HMGB1 expression in a series of BC patients from Institut Gustave Roussy, Villejuif, France, and Center George Franc ¸ois Leclerc, Dijon, France. We observed that this pattern of expression is variable among tumors and peculiar in cancer versus healthy cells. In our hands, in the vast majority of cases, normal mammary glands and stromal cells harbor a strong nuclear staining (Fig. 3A-C). On the contrary, approximately one-third of breast tumors exhibit a similar nuclear staining (Fig. 4A). The vast majority of BC has lost this nuclear expression and only harbors a weak cytoplasmic or perinuclear staining (Fig. 4B), while in situ carcinoma presents with a mixed heterogeneous pattern of nuclear expression (Fig. 3D). We are currently addressing the prognosis value of HMGB1 nuclear expression in various molecular subtypes of BC and its relevance for the response to conventional therapies.

However, HMGB1/RAGE expression was not found to be elevated in all histological types, especially in lung carcinoma in which RAGE and HMGB1 levels are down-regulated compared with benign tumors and normal control tissues [START_REF] Bartling | Down-regulation of the receptor for advanced glycation end-products (RAGE) supports non-small cell lung carcinoma[END_REF][START_REF] Donato | Intracellular and extracellular roles of S100 proteins[END_REF][START_REF] Huttunen | Receptor for advanced glycation end productsbinding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis[END_REF]. Besides its putative role in the promotion of tumor progression, recent preclinical and clinical data revealed that HMGB1 seems to trigger protective anti-cancer T-cell responses. Pioneering studies have shown that this extracellular HMGB1 release could activate DCs (94, 125) and promote the induction of protective immune responses against poorly immunogenic apoptotic lymphoma cells (125). Therefore, when an appropriate antigenic transfer to DCs has occurred, the acute release of HMGB1 may contribute to elicit immune responses in cancer-bearing patients (4, 124, 126). Moreover, chemotherapeutic agents such as alkylating agents and taxanes appear to be more effective than others to trigger the translocation from the nucleus and the subsequent exodus of HMGB1 in the extracellular milieu [START_REF] Xda | High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy[END_REF][START_REF] Formenti | Effects of chemoradiation on tumor-host interactions: the immunologic side[END_REF], suggesting that HMGB1 could behave as a DAMP or a CDAMP during ICD.

The multiple and controversial biological functions of HMGB1 are context dependent and could be explained by the redox modifications induced by the cell or the extracellular milieu. Redox-associated modulations of HMGB1 bioactivity oscillate between a chemokine-like molecule (fully reduced HMGB1), a pro-inflammatory cytokine (in case of disulfidebond possessing HMGB1), and inactivated DAMP (fully oxidized HMGB1). Activation of caspases during apoptosis can amplify mitochondrial ROS production, which may neutralize the DAMP activity of HMGB1 (Fig. 2). Of note, recent studies suggest that the redox modulation might finely control the DAMP activity of HMGB1, regulating leukocyte recruitment or their induction to produce inflammatory cytokines by adopting mutually exclusive redox states (154).

The second face of HMGB1 in cancer: role in processing and cross-presentation during ICD

The fact that some anticancer treatments appear to be more efficient in immunocompetent rather than in immunodeficient mice prompted the search for a link between innate and adaptive immune responses during ICD and led to the systematic screening for a role of toll-like receptors (TLRs) in the efficacy of chemotherapy or radiotherapy. Indeed, tumor cell death triggered by cytotoxic agents may lead to the release of danger signals that favor the development of anticancer immunity [START_REF] Matzinger | Tolerance, danger, and the extended family[END_REF]151). Although TLRs were originally described to detect microbial products (145), it has also been shown that endogenous danger signals from dying cells can bind TLRs and, thus, modulate adaptive immune responses [START_REF] Marshak-Rothstein | Toll-like receptors in systemic autoimmune disease[END_REF]. Indeed, when TLRs on the surface of DCs recognize pathogenassociated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), signaling pathways are ignited that eventually inhibit the fusion between phagosomes and lysosomes, thereby facilitating antigen processing and the initiation of cognate immunity (141).

To address whether TLRs could be involved in ICD, the immunogenicity of oxaliplatin-exposed tumor cells was tested in TLR-deficient mice. Only those tlr4 deficient mice failed to mount a tumor-specific protective immunity. Next, we tested the efficacy of chemotherapy (anthracyclines or oxaliplatin) and radiotherapy in wild-type versus TLR4-deficient animals in 4 different tumor models (transplantable CT26, EG7, TS/A cell lines, and the graft of an osteosarcoma GOS). Although these cytotoxic compounds significantly reduced tumor growth in wild-type (WT) hosts, they failed to do so in tlr4 -/ -mice, underlining the absolute requirement for TLR4 and its downstream effector MyD88 in the chemotherapyinduced control of tumor progression [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF][START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. T cells were also mandatory for the observed antitumor effects, as athymic nu/ nu mice failed to mount an efficient antitumor response after local treatment with radiotherapy or systemic chemotherapy [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Interestingly, TLR4 appears to be required for efficient DC-mediated antigen presentation [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF][START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. In fact, TLR4 has been shown to inhibit the lysosome-dependent degradation of phagosomes in macrophages (141). Accordingly, the kinetics of fusion between phagosomes and lysosomes was slower in WT DCs than in TLR4-deficient DCs loaded with dying tumor cells, which suggests that the TLR4 defect causes accelerated lysosomal degradation of the phagocytic cargo (Fig. 5). Thus, TLR4-deficient bone marrow-derived DCs were unable to present dying-cell-associated tumor antigens (engulfed by phagocytosis), although they retained the capacity to present antigens from soluble proteins (taken up by pinocytosis) [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF][START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Accordingly, the priming of CD8 + T lymphocytes by DCs pulsed with cancer cells un-dergoing ICD was markedly compromised in tlr4 -/ -DC. Altogether, these data suggest that the TLR4/MyD88 pathway contributes to the success of ICD-mediated anticancer therapy. Identification of the danger molecules that contribute to the immunogenicity of dying tumor cells was performed using the systematic comparison of putative TLR4 ligands released by oxaliplatin or anthracycline-treated tumor cells. These experiments revealed that HMGB1 molecules but not other known TLR4 ligands (such as heat shock proteins, fibrinogen, fibronectin, and beta-defensin 2) selectively accumulated at late time points in the supernatant of tumor cells undergoing ICD (as shown in Western Blotting and ELISA) [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF]. The release of HMGB1 from cancer cells that succumb to anthracycline or ionizing radiation was measurable within 18h after ICD induction and resulted, similar to CRT exposure, from a caspase-dependent process [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF]. HMGB1 first translocates from the nucleus to the cytoplasm and then, after the disruption of the plasma membrane, gets released into the FIG. 5. HMGB-1 and adenosine tri-phosphate (ATP) release act as an immunogenic signal through TLR4 and P2RX7, respectively, culminating in enhanced antigen presentation and IL-1b production. After radiotherapy or immunogenic chemotherapy treatment, (immunogenic) tumor cell death leads to an acute HMGB-1 release. The ligation of HMGB-1 to its receptor, TLR4, inhibits the fusion of phagosomes (containing tumor antigens) and lysosomes, thereby allowing an enhanced antigen processing and presentation. ATP is released in an autophagy-dependent manner. Dendritic cells are recruited and activated via the purinergic receptor P 2 RX 7 . The ligation of ATP with its receptor leads to the activation and the aggregation of NLRP3 inflammasome that will, in turn, cleave the pro-caspase 1 into active caspase1. The recognition of HMGB-1 by TLR4 leads to the synthesis of pro-IL-1b, which will be split by caspase 1 into active IL-1b. Thus, HMGB-1 and ATP act in concert for activating the NLRP3 inflammasome in DCs, culminating in the secretion of IL-1b in the extracellular milieu. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars.)

CRT, HMGB1, AND ATP IN IMMUNOGENIC CELL DEATH extracellular space (147). The binding of HMGB1 to TLR4 was previously shown using fluorescence resonance energy transfer analyses and immunoprecipitation studies (112).

The release of HMGB1 and its interaction with TLR4 on the surface of DCs appears to be mandatory to prevent the fusion of phagosomes with lysosomes and the rapid degradation of tumor antigens [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Hence, further experiments clearly showed that the ligation of TLR4 by HMGB1 is crucial for the priming of T cells by dying tumor cells. When either HMGB1 or TLR4 was absent from the system, dying tumor cells lost their immunogenicity in vivo. While usually engulfed by DCs, tumor cells were genetically modified to express an siRNA HMGB1 that exhibited reduced immunogenic potential on exposure to doxorubicine. Moreover, a neutralizing antibody directed against HMGB1 blunted antigen presentation of OVA antigens from dying EG7 cells to T cells in vivo [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF].

The clinical relevance of the HMGB1/TLR4 pathway as a key determinant for the immunogenicity of dying tumor cells has been analyzed in retrospective clinical studies in N + BC patients treated with adjuvant anthracyclines. Indeed, a TLR4 single nucleotide polymorphism (SNP) rs4986790 has been associated with decreased responses to lipopolysaccharide (LPS: a well-known ligand of TLR4) [START_REF] Arbour | TLR4 mutations are associated with endotoxin hyporesponsiveness in humans[END_REF]. The single nucleotide substitution ( + 896A/G) in the tlr4 gene leads to the replacement of an aspartic acid by a glycine (Asp299Gly) in the extracellular domain of TLR4 and is present in 12%-15% of Caucasians. Hence, alveolar epithelial cells from individuals bearing the Asp299Gly variant form of TLR4 lost their capacity to produce IL-1a after LPS stimulation [START_REF] Arbour | TLR4 mutations are associated with endotoxin hyporesponsiveness in humans[END_REF]. Coimmunoprecipitation experiments showed that the expression of the variant form of TLR4 significantly decreases the binding of recombinant HMGB1 to TLR4 [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Moreover, although monocyte-derived DCs from individuals bearing the normal allele of TLR4 could cross-present antigens from dying melanoma cells to T cells in an HMGB1-dependent manner, DCs from individuals bearing the TLR4 Asp299Gly mutation failed to do so [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. In a retrospective analysis of the outcome of 280 localized-BC patients treated with local radiotherapy followed by adjuvant anthracycline-based chemotherapy, patients bearing the Asp299Gly variant TLR4 SNP exhibited a markedly increased incidence of metastatic relapse, when compared with patients bearing the normal TLR4 allele [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. In a multivariate analysis, carrying the TLR4 variant remained independently associated with poorer metastasisfree survival [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. The relevance of the TLR4 Asp299Gly SNP was corroborated in metastatic colon cancers treated with oxaliplatin-based chemotherapy (147).

Altogether, these results identified the interaction of HMGB1 released by dying tumor cells with the TLR4 receptor present in DCs as a major determinant for the cross-presentation of tumor antigens and the promotion of tumor-specific Tc1/Th1-cell responses, culminating in the success of some anticancer treatments (such as anthracyclines and oxaliplatin). It is interesting to note that other investigators have identified alternate DAMP which could mediate antigen processing of cell-associated antigens. Hence, Reis E Sousa's team demonstrated the critical role of DNGR1/CLEC9A expressing CD8a DC in the crosspresentation of necrotic tumor cells to T cells. Their team identified F-actin as the pivotal CLEC9A ligand promoting the processing of the phagocytic cargo by DC, highlighting how an evolutionary conserved cytoskeletal component can repre-sent a relevant DAMP/CDAMP emanating from necrotic cells [START_REF] Ahrens | F-actin is an evolutionarily conserved damage-associated molecular pattern recognized by DNGR-1, a receptor for dead cells[END_REF]128,129).

However, neither the addition of recombinant CRT nor HMGB1 as a stand-alone DAMP to live cancer cells is sufficient to induce an efficient presentation of tumor antigens by DCs, suggesting that additional factors should be exchanged between dying cells and DCs to induce a full-blown anticancer immune response.

ATP and Inflammasomes in Carcinogenesis and Cancer Immunogenicity

The possible contribution of receptors for DAMPs other than TLRs, which could account for the priming of IFNcproducing T cells during anticancer chemotherapy, led to an investigation of the putative role of NOD-like receptors (NLRs) that could recognize putative CDAMP and link inflammation to innate immunity [START_REF] Fritz | Nod-like proteins in immunity, inflammation and disease[END_REF][START_REF] Medzhitov | Toll-like receptors and innate immunity[END_REF]. Indeed, some of the NLRs also sense nonmicrobial danger signals and form large cytoplasmic complexes called the inflammasome platforms (reviewed in [START_REF] Martinon | The inflammasomes: guardians of the body[END_REF]138)). Inflammasomes are multiprotein complexes that operate as platforms activating caspase-1 and mediating the proteolytic maturation and secretion of proinflammatory cytokines (such as IL-1 and IL-18) (61, 138). Inflammasomes can be categorized according to their main constituent: NLRP1, NLRP3, NLRC4, NLRP6, and AIM2. In response to danger signals, the NLR, such as NLRP3, interact with the adaptor molecule apoptosis-associated speck like protein (ASC) to form the inflammasome, the principal caspase-1 activation complex.

Similar to HMGB1, these caspase-1 activation complexes have contrasting roles in the setting of cancer [reviewed in (169)]. First, inflammasomes were associated with procarcinogenic functions, especially in human cancers linked to chronic inflammatory processes such as gastric cancers (caused by Helicobacter pylori infection), liver carcinoma (resulting from viral infection), and colorectal cancer (favored by inflammatory bowel diseases) [START_REF] Grivennikov | Immunity, inflammation, and cancer[END_REF]133). In these contexts, preclinical and clinical data indicate that IL-1b can directly drive oncogenesis [START_REF] Krelin | Interleukin-1beta-driven inflammation promotes the development and invasiveness of chemical carcinogeninduced tumors[END_REF]123,150) or suppress immunosurveillance mechanisms through the recruitment and the activation of myeloid-derived suppressor cells (MDSCs) [START_REF] Bunt | Reduced inflammation in the tumor microenvironment delays the accumulation of myeloidderived suppressor cells and limits tumor progression[END_REF]150,152), thus facilitating tumor development. In melanoma or multiple myeloma, tumor cells are able to produce and secrete IL-1b that confers a proliferative advantage to cancer cells through autocrine or paracrine mechanisms [START_REF] Lust | Induction of a chronic disease state in patients with smoldering or indolent multiple myeloma by targeting interleukin 1{beta}-induced interleukin 6 production and the myeloma proliferative component[END_REF]105,119). Similarly, IL-1b can also convert fibroblasts into tumor-associated fibroblasts, generating a tumor microenvironment that favors cancer progression (134). IL-1 may also favor tumor invasiveness, metastatic spreading, and tumordriven angiogenesis [START_REF] Elaraj | The role of interleukin 1 in growth and metastasis of human cancer xenografts[END_REF][START_REF] Nakao | Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1 beta-induced neovascularization and tumor growth[END_REF]135,156,160). Moreover, IL-18 secreted by tumor cells can suppress the antimetastatic functions of NK cells in mice (146).

Conversely, in the setting of cancer cell demise, there is growing evidence which suggests that the inflammasome or its products (particularly IL-1b) can also sustain immune response against dying tumor cells, especially in chemotherapy-elicited anticancer immune responses. Indeed, the inflammasome NLRP3 appeared mandatory for the efficacy of some chemotherapies [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. In oxaliplatin-treated mouse tumor models, each individual player of this complex (NLRP3, its adaptor molecule ASC, or caspase-1 or IL-1b/IL-1R1), or its final 8 LADOIRE ET AL.

product IL-1b, was required for the immunogenicity of cell death as well as for the success of chemotherapy [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF]. Similar results were obtained in other tumor models treated with anthracyclines [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. Hence, caspase-1-deficiency resulted in reduced IFN-c production by tumor antigen-specific CD8 + T cells in tumor draining lymph nodes [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. IL-1b appears to be a key contributor for the full differentiation of IFN-c polarized CD8 + T cells during the priming phase of antitumor immune responses in these models. Indeed, in the absence of the IL-1R1 or in the presence of IL-1 receptor antagonist, dying tumor cells failed to prime cancer-specific IFN-c-producing CD8 + T cells [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. Multiple distinct bacterial products or endogenous damage signals (such as toxins, ATP, uric acid crystals, alum, and silica) are able to stimulate the NLRP3 inflammasome, resulting in the proteolytic auto-activation of caspase-1 [START_REF] Kanneganti | Critical role for Cryopyrin/ Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA[END_REF][START_REF] Mariathasan | Cryopyrin activates the inflammasome in response to toxins and ATP[END_REF]103). One of the most abundant activators of the NLRP3 inflammasome is extracellular ATP, which is released from stressed cells and acts on purinergic receptors [START_REF] Virgilio | Liaisons dangereuses: P2X(7) and the inflammasome[END_REF]. ATP has an important pro-inflammatory role as a danger signal, especially when released in the extracellular environment in response to tissue damage and cellular stress. Through the activation of P 2 • and P 2 Y receptors, extracellular ATP enhances tissue repair, promotes the recruitment of immune phagocytes and DCs, and acts as a co-activator of NLRP3 inflammasome. ATP may be released from the mitochondria of necrotic cells [START_REF] Iyer | Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome[END_REF] or can be secreted in an active, caspaseand autophagy-dependent way by apoptotic cells [START_REF] Martins | Chemotherapy induces ATP release from tumor cells[END_REF][START_REF] Michaud | Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice[END_REF]. Once in the extracellular space, ATP may attract and activate innate effector cells of the immune system into the tumor bed [START_REF] Idzko | Nucleotides in-duce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors[END_REF], at least in part by binding P 2 Y 2 purinergic receptors [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF], thus also constituting a ''find me'' signal [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF]. Accordingly, expression of the ectonucleoside triphosphate diphosphohydrolase (ENTPD1, an ATP-degrading enzyme) reduced the chemoattractiveness of apoptotic cells [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF].

Recent findings indicate that ATP is released by dying tumor cells succumbing to many chemotherapeutic agents (such as cadmium, etoposide, mitomycin C, oxaliplatin, cisplatin, staurosporine, thapsigargin, mitoxanthrone, or doxorubicin) [START_REF] Martins | Chemotherapy induces ATP release from tumor cells[END_REF][START_REF] Michaud | Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice[END_REF]. This ATP release became undetectable when cells were incubated with the ATP-degrading enzyme apyrase or with inhibitors of ATP synthesis such as antimycin A plus deoxyglucose (A/D) or the oxidative phosphorylation uncoupler 2,4-dinitrophenol. When oxaliplatin-or anthracyclinetreated cancer cell lines were admixed with these ATP scavengers or nonselective P 2 • receptor antagonists (iso-pyridoxalphosphate-6-azophenyl-2¢,5¢-disulphonate or oxidized ATP), they lost their capacity to elicit protective antitumor immune responses on subcutaneous inoculation into immunocompetent mice [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. Thus, ATP appears to be mandatory for the efficacy of some chemotherapeutic agents, especially immunogenic treatments (radiotherapy, anthracyclines, and oxaliplatin). Tumor cells manipulated to express the ATP ectonucleotidase CD39 on their surface or to be deficient in essential proteins of the autophagic machinery can usually undergo apoptosis in vitro and in vivo, yet failed to trigger an ICD pattern and to respond to chemotherapy [START_REF] Michaud | Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice[END_REF]. Importantly, the immunogenicity of autophagy-deficient or CD39-overexpressing tumor cells could be restored by the administration of inhibitors of extracellular ATPases (which increase pericellular ATP concentrations) or by the administration of recombinant IL-1b (170).

The high-affinity receptor for ATP present in DCs is the purinergic P 2 RX 7 receptor [START_REF] Ferrari | The P2X7 receptor: a key player in IL-1 processing and release[END_REF]. ATP stimulates P 2 RX 7 , thus activating the NLRP3/ASC/caspase-1 inflammasome and driving the secretion of IL-1b (49) (Fig. 5). The IFN-c-polarized CD8 + T-cell response of P 2 RX 7 -/mice to oxaliplatin is reduced, and these animals fail to control the progression of established tumors under oxaliplatin treatment [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. Functional P 2 RX 7 receptor on host DCs was mandatory for the chemotherapy-elicited anticancer immune response, as the adoptive transfer of bone marrow-derived WT (but not in P 2 RX 7 -/ -) DCs loaded with dying tumor cells restored the tumor antigen-specific CD8 + T-cell response that had been originally impaired in P 2 RX 7 -/mice (49). Altogether, these results are compatible with a scenario in which a danger signal, ATP, activates P 2 RX 7 receptors on DCs, thereby stimulating the aggregation and/or activation of the NLRP3/ASC/Casp-1 inflammasome; then the proteolytic maturation of caspase-1, pro-IL-1b cleavage, and consequent IL-1b release (Fig. 5). Then, IL-1b is required for the priming of IFNc-producing tumor antigen-specific CD8 + T cells (Th1/Tc1), and, thus, for the chemotherapy-elicited anticancer immune response [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. These data suggest that ICD induced by chemotherapy could influence the composition of the immune infiltrate present in tumors, which, in turn, could affect the therapeutic response. Supporting this assumption, we reported that autophagy-deficient tumors failed to attract DC and to accumulate IL-17-producing cd T cells as well as IFN-c-producing CD8 + T cells in tumor beds postimmunogenic chemotherapy, unless apyrase inhibitors be supplied to the tumor microenvironment (Fig. 6) [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF][START_REF] Mattarollo | Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors[END_REF]. When binding to IL-1R1, IL-1b (most likely produced by antigen-presenting cells exposed to dying bodies) attracts cd T cells to the tumor site, leading to local production of IL-17 and the secondary recruitment of IFN-c-producing CD8 + ab T cells, eventually causing the lysis of tumor cells. Although the molecular links between cd T17 and cytotoxic T lymphocytes (CTLs) were not elucidated, the percentage of cd T17 cells appeared closely correlated with the frequency of IFN-cproducing CD8 + T lymphocytes in the tumor microenvironment. Accordingly, in the absence of cd T17 or IL-17, the priming of adaptive anticancer Th1/Tc1 responses was hampered, suggesting a helper role of cd T17 cells in the context of ICD [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF][START_REF] Mattarollo | Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors[END_REF].

Accordingly, the TLR4 ligand HMGB1 and ATP, both of which are released by tumor cells exposed to cytotoxic agents, act in concert to promote IL-1b secretion by DCs. The treatment of DCs with oxidized ATP or anti-HMGB1-neutralizing antibodies before loading with dying tumor cells completely abolished IL-1b secretion [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. As for the HMGB1/TLR4 interaction, the integrity of this ATP/P 2 RX 7 pathway appeared to be clinically relevant, at least in N + BCs, as loss-of-function alleles of P 2 RX 7 , which confer a reduced affinity of P 2 RX 7 receptor for ATP, compromised the efficacy of anthracyclinebased chemotherapy [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF]. Supporting this notion, relative elevated expression of ENTPD1 mRNA (which encodes the ectonucleotidase CD39) was associated with a poor outcome in colorectal cancer [START_REF] Kunzli | Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer[END_REF]. Similarly, CD73, an ecto-5¢ nucleotidase that acts downstream of CD39 to convert AMP into adenosine, was overexpressed in some human tumors (Table 1). CD73-deficient mice were protected against metastatic spreading, and harbor increased antitumor immunity (142). Together, these observations suggest that ATP-dependent, P 2 RX 7 -mediated activation of the inflammasome might also be clinically relevant for the efficacy of anticancer therapies in patients [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors[END_REF].

Perspectives

ICD has now entered the field of cancer immunization and vaccines [START_REF] Krysko | Immunogenic cell death and DAMPs in cancer therapy[END_REF]. The comprehensive understanding of the mechanisms governing the immunogenicity of cancer cell death could have a profound impact on the current management of cancer patients. It pertains not only to the use of distinct chemotherapy compounds or various regimens of radiotherapy but also to the ear of ''targeted'' drugs. As an example, trastuzumab efficacy stems from a functional TLR4 pathway and is boosted by TLR4 agonists (113,159,164).

The first obvious and direct clinical implementation of these observations is the subversion of an ''immune checkpoint blockade.'' Tumors have evolved many immunosuppressive mechanisms to turn down the innate and the effector arms of the immune system. After the pioneering success of Ipilimumab in metastatic melanoma, investigators have conducted trials that are aimed at curtailing the other immune checkpoints. Both CTLA4 and the B7-H1 (PD-L1)/PD-1/B7.1 axis are best understood among the inhibitory B7-H family members featuring in the relevant ''immune checkpoint blockers.'' The therapeutic potential of novel immune checkpoint blockers (14, 149) has been recently highlighted in advanced cancer patients, including patients harboring lung or brain metastases. ICD may lead to anticancer immune responses that will be unleashed by such immune checkpoint blockers. The current issue is to analyze which regimen of combinatorial therapies (dose and scheduling) will be most suitable to achieve a synergistic anticancer efficacy. Second, since human cancers carry hundreds of nonsynonymous mutations (several dozens among which lead to the generation of tumor-specific MHC class I-restricted epitopes), every patient's tumor harbors a highly specific mutational and antigenic signature. This ''mutanome'' can be identified by deep sequencing and can lead to individualized immunotherapies by means of ICD-inducing compounds and/or tailored vaccines [START_REF] Kroemer | Can the exome and the immunome converge on the design of efficient cancer vaccines?[END_REF]. Clinical trials are ongoing and are aimed at combining anthracyclines or oxaliplatin and mutanome-derived mRNA epitopes in BCs (Biontech.de).

As already demonstrated, the propensity of tumor cells to undergo ICD in vitro can be exploited toward the therapeutic success of DC-based anticancer vaccines. In indolent B-cell lymphoma patients, antitumor responses were not observed when autologous tumor vaccines failed to harbor the ICDrelated hallmarks (166). As mentioned earlier, in cohorts of breast or colon cancer patients treated with adjuvant anthracycline and local radiotherapy or oxaliplatin, respectively, genetic defects in the TLR4 or P 2 RX 7 axis compromised the efficacy of these treatments (6, 49, 147). However, it is anticipated that exploiting the ICD concept will require a ''complete profiling'' of the drug-tumor-host putative interactions (Fig. 7). First, compounds should be screened for their capacity to trigger an ICD pattern. The most prominent bottleneck is the failure of most anticancer drugs to trigger an ER stress response, culminating in CRT exposure [START_REF] Menger | Cardiac glycosides exert anticancer effects by inducing immunogenic cell death[END_REF]. Hence, although cisplatin alone does not induce the preapoptotic exposure of CRT, it can do so in combination with the ER stressor thapsigargin, de facto rendering cisplatininduced cell death immunogenic [START_REF] Martins | Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress[END_REF]. Many novel ER stressors are currently developed and may enter the clinical armamentarium (137). Otherwise, recombinant CRT directly supplied into the tumor bed can compensate for the lack of ER stress response of a given pro-apoptotic compound (101). Second, tumors should exhibit the appropriate machinery to undergo proper ER stress, autophagy, and apoptosis responses when subjected to a tumoricidal compound. Hence, tumors defective in ERp57, HMGB1, or Atg5 will not die in an immunogenic manner and need appropriate compensatory therapies (6, 95, 110) (Fig. 7). We could speculate that failure of HMGB1 release could be compensated by administration of other potent TLR4/Myd88 agonists. Pharmacological inhibitors of extracellular ATPases, which can increase ATP ALL, acute lymphoblastic leukemia; T-ALL, T-lineage ALL; C-ALL, common ALL; CR, complete response; M1, distant metastases; M0, no distant metastases; R, relapse within a 10 year period; NR, without relapse within a 10 year period; CLL, chronic lymphoblastic leukemia; CRC, colorectal cancer; OS, overall survival; EOC, epithelial ovarian carcinoma; GBM, glioblastoma; PTC, papillary thyroid carcinoma. concentration during ICD, could favor an efficient IFN-cpolarized T-cell response against tumor cells [START_REF] Michaud | Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice[END_REF]. Novel antibodies targeting CD39 (10) could be of particular interest should the safety profile be ascertained. Third, hosts presenting with genetic defects compromising sensing of danger signals may fail to optimally respond to cell death in an immunogenic fashion. Compensation of TLR4 deficiency may rely on the administration of other TLR ligands to boost antitumor immune responses. Thus, administration of specific ligands of TLR3 or TLR9 after irradiation could restore potent antitumor effects in TLR4-deficient mice [START_REF] Apetoh | The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy[END_REF].

It is our hope that future anticancer strategies could be tailored to each patient and a tumor's genetic background, taking into account not only the cytotoxic properties of anticancer agents, but also their capacity to stimulate a host's innate and acquired immune responses. Hence, taking into account these parameters will lead either to a stratification of patients according to various categories pertaining to the tumor profile for ICD hallmarks only or to a personalization according to a complex algorithm retaining in the model the host genetic defects, the resistance to apoptosis, the lack of ICD, and the mutational status of the cancer. FIG. 7. Compensatory strategies to achieve immunogenic cell death. To achieve immunogenic cell death, drugs should be able to induce ER stress, autophagy, and cell death. Tumors should be able to expose the CRT and to secrete ATP and HMGB-1. Finally, the host should be able to sense all these immunogenic signals, through appropriate receptors, leading to the recruitment of immune effector cells. Thus, the lack of one of the parameters listed earlier would compromise the response to chemotherapy. In such a scenario, compensatory strategies can be proposed to specifically complement the missing factor, when possible, with the aim of restoring the immunogenicity of tumor cell death. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars.)

FIG. 2 .

 2 FIG. 2. Pro-tumor role of high mobility group box 1 protein (HMGB-1). (A) At the center of a growing tumor, hypoxia leads to tumor cell death and chronic release of HMGB-1. By recruiting macrophages and endothelial cell precursors, HMGB-1 activates neo-angiogenesis, thus promoting tumor growth and metastatic spreading. The oxidation of HMGB-1 by the hypoxia-induced oxidative milieu inactivates it, impairing this alarmin function. (B) HMGB-1 can bind Tim-3 that is expressed in tumor-associated dendritic cells (DCs). The ligation of HMGB-1 with Tim-3 inhibits acid nucleic-mediated DC activation (through TLR3, TLR7, and TLR9), and subsequent cytokine production (Type I IFN, IL-12), impairing the generation of anti-tumor responses. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars.)

FIG. 4 .

 4 FIG. 4. Immunohistochemical HMGB1 staining of paraffin-embedded breast cancers. Representative results are shown for (A) breast cancer cells harboring strong nuclear staining, and (B) breast cancer cells that have lost nuclear staining (inset: normal mammary tissue harboring nuclear staining on the same slide). Magnification • 20. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars.)

FIG. 6 .

 6 FIG. 6. Induction of an antitumor immune response by ICD. (i) Chemotherapy induces early exposure of ecto-CRT, which acts as an ''eat-me'' signal and facilitates the recognition and uptake of tumor cells by DC. HSP70 and HSP90 enhance antigen cross-presentation and the release of pro-inflammatory cytokines. The release of F-actin also enhances antigen crosspresentation. (ii) The release of HMGB1 and ATP by tumor cells promotes the antigen-processing/presentation and the release of IL-1b by DC. (iii) This spatiotemporal code licences the polarization of CD8 + T cells toward IFNc production, leading to complete tumor eradication. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertonline.com/ars.)

Table 1 .

 1 Studies Correlating CD73 or CD39 Expression and Human Tumor Prognosis

	Histology	CD39 & CD73 expression	Prognosis values	Number of patients (n)	Ref
	ALL	Significant lower expression	CD73 expression is not correlated	n = 411	(162)
		of CD73 in T-ALL compared	with prognosis
		with pre-B-ALL & C-ALL
			Higher percentage of CR	n = 79	(153)
			in CD73 + ALL than
			in CD73-ALL ( p < 0.05)
	Breast cancer Higher CD73 enzymatic	Poor outcome associated	n = 21 M1; 9 MO; 15 R + 15 NR	(75)
		activity in tumors versus	with high level of CD73	paired
		healthy tissues	expression in primary tumor
				n = 9 stage II; 6 stage IIIa;	(16)
				9 noncancerous tissues
				from stage II
	CLL	32% of CLL expressed CD73	CD39 expression is associated	n = 34 & 31 healthy controls (118)
			with late stages of disease,	n = 299	(140)
			whereas CD73 + clones are
			associated with early stages of
			disease
	CRC	Higher CD73 enzymatic	CD73 + CRC correlated with a	training cohort: n = 223	(163)
		activity in primary tumors	poor OS	validation cohort: n = 135
		than in normal mucosa		n = 38	(38)
			Higher CD39 + CRC are	n = 63	(73)
			associated with a lower OS
	EOC	29/36 EOC are positive for CD39	Overexpression of CD73 EOC	n = 167	(104)
		expression, and 31/34 are	is associated with better	n = 36	(54)
		positive for CD73 expression	prognosis (73% of 5 years
			OS versus 50% for CD73 +
			and CD73, respectively
	Gastric cancer Higher CD73 enzymatic activity		n = 7 grade I-II; 8 grade III-IV;	(34)
		in cancerous tissues compared		9 noncancerous tissues
		with the adjacent healthy		from stages I to II
		tissues
	GBM	95% of GBM are CD73 +		n = 165	(78)
	PTC	CD73 expression by PTC		n = 3	(63)

LADOIRE ET AL.

Acknowledgments

This study was supported by the Ligue Nationale Contre le Cancer (Equipes Labellise ´e; L.Z. and G.K.), Agence Nationale pour la Recherche (L.Z. and G.K.), Fondation pour la Recherche Me ´dicale (L.Z. and G.K.), Institut National du Cancer (L.Z. and G.K.), Cance ´ropo ˆle Ile-de-France (L.Z. and G.K.), the European Commission (Apo-Sys (L.Z., G.K.), ArtForce (L.Z. and G.K.), ChemoRes (L.Z. and G.K.) and Death-Train (L.Z. and G.K.)), LabEx OncoImmunology (L.Z. and G.K.) and Institut National de la Sante ´et de la Recherche Me ´dicale (O.K.), and Association pour la recherche sur le cancer (D.H.).


Centre R Etienne Merlin
email: e_merlin@chu-clermontferrand.fr

Dalil Hannani

Richard Veyrat-Masson

Jacques Chassagne

Fra Ç Oise Gabert

Marc Berger

Fran Ç Ois

Dem É Ocq

Jo Ë L Plumas

Justyna Kanold

Cryopreservation of mononuclear cells before extracorporeal photochemotherapy does not impair their anti-proliferative capabilities

Keywords: alloreactivity, carboxyfl uorescein succinimidyl ester, human, pre-clinical study

Background aims. The clinical benefi ts of extracorporeal photochemotherapy (ECP) are well recognized, but its clinical use is limited by logistical diffi culties, especially because of the need to perform repeated aphereses. The cryopreservation of mononuclear cells could allow maintenance of the ECP schedule while reducing the number of aphereses. The aim of this work was to assess whether previous cryopreservation impairs the immunomodulatory function of ECP-treated peripheral blood mononuclear cells (PBMC). Methods. Fresh or previously cryopreserved PBMC were exposed to ECP and added on day 0 into a mixed leukocyte reaction. Proliferation of alloreactive lymphocytes was measured by carboxyfl uorescein succinimidyl ester (CFSE) dye dilution. Apoptosis was quantifi ed by annexin -7AAD staining. Results . ECP-induced apoptosis was slightly increased in cryopreserved cells but the kinetics of apoptosis were similar to fresh cells. Lymphocytes stimulated in the presence of ECP-treated PBMC displayed a signifi cant decrease in proliferation. The suppression was enforced when ECP-treated cells had been activated previously by allogeneic stimulation. Cryopreservation before ECP exposure did not impact apoptosis triggering or anti-proliferative properties of ECP-treated cells. Conclusions. Cryopreservation before ECP does not impair the immunomodulatory effects of treated cells. These data warrant investigation of the clinical use of cryopreserved PBMC for ECP.

Introduction

Extracorporeal photochemotherapy (ECP) is an immunomodulatory cellular therapy relying on the injection of autologous cell products exposed to ultraviolet (UV) in the presence of a photosensitizer. The cell product is obtained by the isolation of white blood cells by cytapheresis and their subsequent ex vivo exposure to 8-methoxypsoralen (8-MOP) and UV-A radiation (1). Treated cells are then reinfused into the patient. Several injections of the cell products are needed to obtain clinical improvement.

How ECP is able to induce immune tolerance without immunosuppression has still not been elucidated fully. Its mechanism of action involves lymphocyte (2,3) and monocyte (4) apoptosis, and several mechanisms have been hypothesized in humans, such as the involvement of tolerogenic antigen-presenting cells, production of immunosuppressive cytokines and generation of regulatory T cells (5,6). However, until now none has been clearly related to the effi cacy of ECP in humans.

To date, the main three indications are cutaneous T-cell lymphoma (CTCL) (7), graft-versus-host disease (GvHD) (6) and solid organ rejection (5). However, its original mechanism of action combined with the absence of generalized immunosuppression or side-effects makes ECP a very attractive approach for safely treating a wide variety of T-cell mediated pathologies (8). Depending on the clinical situation, the treatment schedule varies from three sessions a week to two sessions a month. Hence, the main obstacles to overcome for the widespread use of ECP are the logistical and psychologic issues secondary to the performance of several apheresis procedures per week for several weeks.

The French technique for ECP, as described by Georges Andreu in 1991 (9), comprises two distinct steps: (a) collection of peripheral blood mononuclear cells (PBMC) by apheresis and (b) treatment of the collection bag in the cell therapy laboratory. This technique makes it possible to cryopreserve the PBMC before UV irradiation. This approach could usefully diminish the number of apheresis runs but it is not known whether cryopreservation affects the properties of ECP-treated cells. This in vitro study investigated whether cryopreservation of PBMC prior to ECP impairs their in vitro immunomodulatory properties after UV exposition. Using a model of allogeneic mixed leukocyte reaction (MLR), we show that cryopreserved cells exhibit similar antiproliferative capabilities to fresh cells.

Methods

Model

The aim of our model was to reproduce an allogeneic confl ict resembling the situation where a patient with GvHD is treated with ECP (Figure 1). In this situation, the ECP-treated bag contains alloreactive and resting mononuclear donor cells. The study enrolled six pairs of unrelated healthy volunteers; in each pair, one volunteer was considered as the donor (D) and the other as the recipient (R). To obtain alloreactive cells, donor cells were primarily exposed to recipient cells. This step consisted of a fi rst-run MLR. After a 7-day culture, the alloreactive cells were harvested. Naive ECP-treated cells (D ECP ) or alloreactive ECPtreated cells (D SENS ϩ ECP ) were added to a second MLR together with naive fresh cells from D and R. Our judgment criterion was the intensity of the alloreactive proliferation in this second MLR.

PBMC sampling and isolation

Blood donations in this study conformed with local ethical committee requirements, and informed consent was obtained. Venous blood (30 mL) was collected in ethylene diamine tetra acetic acid (EDTA). PBMC were isolated by density-gradient centrifugation over Ficoll -Histopaque (Saint-Quentin Fallavier Sigma-Aldrich, St-Quentin Fallavier, France). The isolated cells were then washed twice in phosphatebuffered saline (PBS; Gibco, Cergy-Pontoise, France). Stimulating PBMC were irradiated at 30 Gy.

Generation of D to R alloreactive cells (fi rst MLR)

A fi rst-run MLR was performed to obtain alloreactive cells from D cells stimulated by R cells; 10 ϫ 10 6 reactive PBMC from D were cultured with 10 ϫ 10 6 30-Gy irradiated stimulator PBMC R * for 7 days in a Falcon tube in 10 mL Dutch-modifi ed RPMI-1640 (Gibco) supplemented with 10% fetal calf serum (FBS; Gibco, Cergy-Pontoise, France), L -glutamine, streptomycin and penicillin (Sigma-Aldrich). After 7 days, the cells were centrifuged, washed twice in PBS and treated with ECP.

Generation of immunomodulatory donor cells (psoralen and UV-A (PUV-A) exposure/ECP)

ECP was performed using the Vilber -Lourmat procedure, as described previously (10). Briefl y, 5 ϫ 10 6 PBMC were diluted in 20 mL normal saline solution to obtain a fi nal hematocrit Ͻ 1%, and 8-methoxypsoralen was added at 200 ng/mL. After a 3-min incubation, cells were transferred into a UV-A transparent bag (Macopharma, Tourcoing, France) and exposed to UV-A light (340 nm) at 2 J/cm 2 for 10 min (UVA-matic; Macopharma, Tourcoing, France).

Carboxyfl uorescein succinimidyl ester labeling

Carboxyfl uorescein succinimidyl ester (CFSE) is a green fl uorochrome that is retained in the cells for several weeks without inducing problems of viability (at suitable concentrations). During each cellular division, CSFE is distributed in a strictly dichotomic fashion between both daughter cells. Cytometric analysis of its decrease thus makes it possible to follow cell proliferation (11). This method has proved suitable for lymphocyte studies in vitro (12). In our experiments, 2.5 ϫ 10 6 PBMC from D were suspended in 2 mL PBS (0.1%)/bovine serum albumin (BSA) buffer. Cells were labeled with 1 μ mol/L CFSE (Molecular Probes, Eugene, OR, USA) for 5 min at 37 ° C. Labeling was stopped by adding 30 mL cold RPMI containing 10% FCS, after which the cells were maintained in ice for 5 min. Cells were centrifuged (800 g for 10 min at 20 ° C) and the supernatant removed. Cells were washed twice in PBS then suspended in 5 mL RPMI with 10% FCS.

Cryopreservation and thawing of PBMC

We used the same protocol as for hematopotietic progenitor cell cryopreservation in clinical routines (13); 8 ϫ 10 6 density-gradient isolated PBMC were resuspended in 5 mL PBS ϩ 5% FCS and transferred into 10-mL cryobags (Baxter, Maurepas, France). Five milliliters of ice-cold cryoprotective solution containing 7% dimethyl sulfoxide (DMSO), 10% serum albumin and 83% hydroxyethylstarch were added slowly. Immediately after the cryoprotective solution was added, specimens were transferred to a -80 ° C freezer overnight. This simplifi ed method of uncontrolled-rate freezing lowers specimen temperatures by approximately 1 -2 ° C/min (13). Speci-mens were kept in the freezer for at least 24 h before being thawed and treated with PUV-A.

Frozen specimens were thawed in a 37 ° C water bath with continuous agitation until completely melted, and then 40 mL warm RPMI-1640 ϩ 10% FCS was added directly into the cryobag. Cells were transferred into a Falcon 50 and centrifuged (800 g for 10 min at 25 ° C) to remove the DMSO, then washed twice in 50 mL PBS. The cells were resuspended in RPMI-1640 and exposed For personal use only.

Statistical analysis

The experiments were repeated six times. The data presented show the mean of these six experiments. The error bars on the bar charts gives standard deviations (SD). Comparisons between fresh and frozen cells were run by paired t -test using SEM software ( 14).

Results

ECP induces cell apoptosis

Induction of lymphocyte apoptosis is a hallmark of ex vivo exposure to ECP. As soon as 2 h after ECP, the proportion of cells staining positive for annexin and negative for 7AAD was higher in ECP-treated cells compared with non-treated cells (24.8 Ϯ 4% versus 18.4 Ϯ 5%, P Ͻ 0.05). Annexin/7AAD staining was performed on each day of a 6-day culture. The percentage of annexin ϩ cells remained higher in ECP-treated cells from day 1 (69.1 Ϯ 4% versus 25.7 Ϯ 2%, respectively) until day 6 (95.9 Ϯ 4% versus 52.3 Ϯ 5%, P Ͻ 0.001). The rate of late apoptotic cells (staining positive for both annexin and 7AAD) among ECP-treated cells was also increased compared with non-treated cells (2 h, 3.9 Ϯ 0.4% versus 2.7 Ϯ 0.9%, P ϭ NS; 6 days, 55.5 Ϯ 5% versus 29.5 Ϯ 6%, P Ͻ 0.01) (Figure 2).

Prior cryopreservation does not impair ECP-induced lymphocyte apoptosis

Rapid and massive lymphocyte apoptosis is thought to be of major importance for the action of ECP. It was therefore necessary to assess whether ECP triggers apoptosis in cryopreserved cells as in fresh cells. We used fresh cells collected from six donors. Cells to PUV-A. The cells (frozen/thawed PBMC) were then assessed for viability by trypan blue dye exclusion, and counted.

Immunomodulation with PUV-A-treated donor cells (second MLR)

After a 7-day culture, cells were harvested from the fi rst MLR and treated by ECP. A second MLR was performed with 5 × 10 4 fresh stimulator cells (irradiated at 30 Gy) from the same recipient as the initial MLR (R * ) and 1 ϫ 10 5 CFSE-labeled cells from D (D CFSE ). 1 ϫ 10 5 naive or previously sensitized ECP-treated ' immunomodulatory ' cells from D were added on day 0 of the culture.

The cell ratio was 1:2:2. Cells were cultured for 7 days in 96-well round-bottom plates in 250 μ L Dutch-modifi ed RPMI-1640 supplemented with 10% FCS, L -glutamine, streptomycin and penicillin, and then cells were analyzed by fl ow cytometry (Beckman-Coulter EPICS, Roissy, France). Each point was performed in triplicate.

Apoptosis

Fluroescein isothiocyanate (FITC)-conjugated annexin-V (annexin) coupled with phycoerythrin (PE)-labeled 7-aminoactinomycin D (7AAD; Beckman-Coulter) was used to detect apoptosis. Cells were washed twice in PBS then stained with annexin/7AAD (in 100 μ L labeling solution) and incubated for 10 min in the dark according to the manufacturer ' s protocol. Then 400 μ L buffer were added, and stained cells ( Ͼ 10 00 events) were analyzed by fl ow cytometry (Beckman-Coulter EPICS). This staining protocol makes it possible to differentiate cells in early apoptosis (annexin ϩ /7AAD -) and late apoptosis (annexin ϩ /7AAD ϩ ) and necrotic cells (annexin -/7AAD ϩ ) from viable cells (annexin -/ 7AAD -).

Mitogen-induced proliferation assay of ECP-treated cells

After thawing, cells were placed on RPMI with phytohemagglutinin (PHA) at 10 μ g/mL. After a 3-day incubation period, 1 μ Ci 3 H-thymidine (Amersham, Pantin, France) was added to each well. Eight hours later, DNA was transferred onto fi lter paper using a multiwell harvester, and 3 Hthymidine incorporation was measured in counts per minute (c.p.m.) using a gas -ionization counter. Proliferation was calculated as the mean value of triplicates, and inhibition was calculated as: For personal use only.

Cryopreservation before ECP does not impair the inhibition of PHA-induced lymphocyte proliferation

PHA is a non-specifi c mitogen activator of T cells. It binds the T-cell receptor and is able to trigger a massive activation and proliferation of the whole lymphocytic population. The impairment of PHA-induced lymphocyte proliferation is a currently used quality were exposed to ECP after having been cryopreserved or not, then cultured for 6 days and apoptosis measured at different time points. In cryopreserved ECP-treated cells, the rate of early apoptosis was slightly increased compared with ECP-treated fresh cells immediately after ECP (27.1 Ϯ 4% versus 20.0 Ϯ 2%, P Ͻ 0.05). This small increase in apoptosis persisted for 3 days, and was no longer signifi cant thereafter (Figure 3). At any time of the study, the rate of necrotic cells (annexin -/7AAD ϩ ) was less than 1%.

ECP-treated cells slightly inhibit alloreactive proliferation

CFSE-loading makes it possible to quantify precisely the level of alloreactive proliferation by measuring the proportion of dividing CFSE low and quiescent CFSE bright cells in the gate of viable lymphocytes (11). Quiescent cells (CFSE bright ) were higher in the autologous controls (D CFSE ϫ D * ) than in the allogeneic MLR (D CFSE ϫ R * ), refl ecting a higher proliferation rate in the second: 81.2 Ϯ 2.6% versus 50.3 Ϯ 8.5%, respectively, P Ͻ 0.01 (Figure 4). When ECP-treated naive D cells were added (on day 0) to the culture well, proliferation was slightly but signifi cantly decreased (CFSE bright , 57.9 Ϯ 9.3% versus 50.3 Ϯ 8.5%, respectively, P Ͻ 0.05).

Prior sensitization optimizes the ECP-induced anti-proliferative effect

ECP is believed to result in the selective neutralization of pathogenic T-cell clones. Moreover, activated lymphocytes are more susceptible to cell death after ECP exposure (D. Hannani, manuscript in preparation) and their immunomodulatory capabilities exceed those of non-activated cells (15). When a patient with GvHD is treated with ECP, the ECP-treated bag contains alloreactive and resting mononuclear donor cells. Thus, to get closer to the clinical setting of active GvHD, we considered mimicking in vitro the step of pre-ECP alloreactive activation. To obtain alloreactive activated cells, donor cells were primarily exposed to receiver cells in a fi rst MLR (Figure 1). We therefore assessed whether prior allogeneic stimulation could enforce the anti-proliferative effect of ECP-treated cells. In our model, the inhibition of alloreactive proliferation was stronger when the ECP-treated mononuclear cells had been activated previously by cells from the same donor than with naive cells (CFSE bright non-reactive cells, 77.5 Ϯ 16.3% versus 57.9 Ϯ 9.3%, respectively, P Ͻ 0.01) (Figure 4). For personal use only.

Previously cryopreserved cells retain their anti-proliferative properties when treated by ECP after thawing

The ultimate aim of our study was to determine whether cryopreserved PBMC retain their immunomodulatory properties when treated by ECP after being thawed. Knowing the activated state of lymphocyte responsible of active GvHD, we used the same manipulation with a fi rst MLR to sensitize PBMC from D to R, followed by a second MLR to measure the inhibition of alloreactive proliferation provided by ECP-treated cells. After the second MLR, the rate of non-dividing CFSE bright cells was 47.8 Ϯ 12% in allogeneic controls. The inhibition of alloreactive proliferation induced by ECP-treated cells resulted in a signifi cant increase of non-dividing cell rate. This inhibition was similar whether ECP-treated cells had been previously cryopreserved or not: non-dividing CFSEbright cells 68.6 Ϯ 26% versus 64.5 Ϯ 10% with cells sensitized before ECP ( P ϭ 0.8) and 60.6 Ϯ 9% versus 57.9 Ϯ 9% with ECP-treated cells without previous sensitization ( P ϭ 0.7) (Figure 5). The inhibition of proliferation was given by the following formula: percentage inhibition ϭ 100 ϫ (CFSE bright ECP -CFSE bright control )/CFSE bright control ; where ECP is the culture with ECP-treated cells and control is the control MLR without ECP-treated cells. Inhibition was similar between fresh and cryopreserved cells (24.6 Ϯ 16% versus 32.2 Ϯ 30%, respectively, P ϭ 0.6).

Discussion

ECP is a fascinating immunotherapy that uniquely provides a selective immune regulation without generalized immune suppression (2). The benefi cial effects of the method have been observed in a variety of T-cell mediated infl ammatory diseases, such as GvHD, transplant rejection and certain T-cell mediated autoimmune diseases (17). However, its clinical use is limited by logistical diffi culties, especially because of the need to perform repeated aphereses. This problem is of particular importance for small children for whom reducing exposure to conventional immunosuppressive therapies is especially warranted (10).

One approach to reducing the number of apheresis sessions while maintaining the treatment schedule is to use cryopreserved cells, as illustrated in Figure 6. For example, the cell product harvested in one apheresis can be divided into three aliquots: two can be cryopreserved while the third can be treated immediately and reinfused. This approach offers four advantages: (a) it makes ECP easier for the patients; (b) it takes advantage of the temporary use of a central venous line to harvest a large quantity of cells; (c) it allows patients from other care centers to benefi t easily from ECP control for the procedure of ECP ( 16). We therefore assessed whether prior cryopreservation would impact this effect of ECP. Using PHA at a concentration of 10 μ g/mL, proliferation was achieved in all cryopreserved samples (99357 Ϯ 8524 c.p.m. versus 133 Ϯ 55 without PHA). After the ECP process, inhibition was complete (132 Ϯ 61 c.p.m. versus 32 Ϯ 25 without PHA, 99% inhibition) (data not shown). For personal use only.

The main drawback of cryopreservation is that it implies an overall decrease in total cell dose. However, if the aphereses are spaced out, the price to pay to harvest more cells (stronger platelet depletion) becomes acceptable. In our center, the mean duration of one apheresis for ECP is about 100 min. If the frequency of aphereses decreases from three a week to one a week, it becomes possible to lengthen one session to up to 150 min ( ϩ 50%). Moreover, increasing the total volume collected can enhance collection effi ciency by about 20%. Overall, the total cell dose can be maintained at about 60% for each reinfusion while the treatment schedule is maintained with three times fewer aphereses sessions. Although suggested previously (18), the effect of the cell dose has never yet been clearly established. By allowing a standardization of the cell dose for each reinfusion, cryopreservation can provide insightful data regarding this question. Another theoretical issue can stem from the toxicity of repeated DMSO infusions. In our procedure, cells were frozen in a low DMSO cryoprotective solution (only 3.5%) and the cells washed before reinfusion. As a result, we assume that the toxicity of DMSO would be negligible, but this warrants rigorous assessment in clinical safety trials.

The prospects for clinical use make it critical to determine the in vitro impact of cryopreservation on the properties of ECP-treated cells as a pre-clinical measure. The fi rst reported phenomenon is the massive induction of lymphocyte apoptosis after ECP therapy; (d) it allows standardization of the quantity of cells infused at each session. Here we have shown that cryopreserved cells retain their immunomodulatory capabilities when treated by ECP. exposure (19). Apoptotic cells are engulfed by treated or untreated phagocytes and dendritic cells, which are thought to trigger a regulatory immune response.

In certain conditions, cryopreservation is known to trigger lymphocyte apoptosis (20) and to preserve the stimulatory function of dendritic cells (21,22). To assess whether cryopreserved cells retain their immunomodulatory properties after ECP, we built an in vitro model that is applicable to healthy humans and is easy to exploit. It is known that the adjunction of ECP-treated cells in an allogeneic MLR results in an inhibited alloreactive proliferation (23). As the mechanism of action of ECP remains largely unknown, we chose to measure functional inhibition of alloreactive proliferation. Our model demonstrates a signifi cant inhibition of alloreactive proliferation by ECP-treated cells, and this effect is retained even when cells have been cryopreserved before ECP. We therefore believe that the advantages and disadvantages of using cryopreserved PBMC for ECP in clinical practice deserve to be investigated.

Figure 1 .

 1 Figure 1. Scheme of the model of immunomodulation by ECP-treated cells in an alloreactivity setting. The aim of this model was to mimic the situation were a patient with GvHD is treated with ECP. Six couples of healthy volunteers were enrolled in the study; in each pair, one was considered the donor (D) and the other as the recipient ® . To obtain alloreactive cells, we employed a fi rst step of sensitizing D to R in a MLR with responsive cells from D and 30-Gy irradiated stimulator cells from R (D ϫ R * ). After a 7-day culture, the alloreactive 'sensitized' cells (D SENS ) were harvested and treated with ECP (D SENSϩECP ). Either fresh cells (D ECP ) or D SENSϩECP were added in a second MLR (D ϫ R * ). Reactive cells were labeled with CFSE (D CFSE ) to allow measurement of cell proliferation. In each experiment, we measured the inhibition of alloreactive proliferation as a result of the presence of ECP-treated cells. Cytotherapy Downloaded from informahealthcare.com by INSERM on 08/04/10For personal use only.

Figure 2 .

 2 Figure 2. ECP induces apoptosis. PBMC were collected from healthy donors and cultured in 96-well plates in RPMI supplemented with FCS, glutamine and ATB. After 6 days of culture, apoptosis was measured by annexin/7AAD labeling, allowing differentiation of cells in early apoptosis (annexin ϩ /7AAD -) and late apoptosis (annexin ϩ /7AAD ϩ ) from viable cells (annexin - /7AAD -). * * P Ͻ 0.01. Cytotherapy Downloaded from informahealthcare.com by INSERM on 08/04/10For personal use only.

Figure 3 .

 3 Figure3. Cryopreservation slightly increases the percentage of apoptotic cells after exposure to ECP for 3 days. PBMC were collected from six healthy donors and frozen at -80°C in an icecold cryoprotective solution containing 7% DMSO, 10% serum albumin and 83% hydroxyethylstarch. After 24 h at -80°C, cells were thawed, washed twice in PBS and exposed to ECP. Apoptosis was then measured at various time points. (A) One representative experiment in presented. Fresh or cryopreserved ECP-treated cells were labeled with annexin and 7AAD at day 2. The staining protocol made it possible to differentiate cells in early apoptosis (annexin ϩ /7AAD -), cells in late apoptosis (annexin ϩ /7AAD ϩ ) and necrotic cells (annexin -/7AAD ϩ ) from viable cells (annexin - /7AAD -). (B) The kinetics of apoptosis of fresh and cryopreserved cells treated or not by ECP are presented for 2 h and 1, 2, 3 and 6 days. Upper panel, percentage of early apoptotic cells (annexin ϩ /7AAD -); lower panel, percentage of total apoptotic cells (annexin ϩ ); * P Ͻ 0.05 (cryopreserved cells versus fresh cells). Cytotherapy Downloaded from informahealthcare.com by INSERM on 08/04/10For personal use only.

Figure 4 .

 4 Figure 4. ECP-treated cells inhibit allogeneic proliferation in an MLR. PBMC were collected from six couples of healthy volunteers. Responsive D cells were labeled with 1 μmol/L CFSE (D CFSE ) and cultured for 7 days with stimulator cells R * (irradiated at 30 Gy) and ECP-treated cells (added on day 0 of culture). ECPtreated cells were from D and were naive or had been previously sensitized in a previous 7-day MLR with R * . The ratio between R * , D CFSE and ECP-treated cells was 1:2:2. (A) One representative experiment; (B) results of the six experiments. Compared with allogeneic control: * P Ͻ 0.05; * * P Ͻ 0.01. Cytotherapy Downloaded from informahealthcare.com by INSERM on 08/04/10For personal use only.

Figure 5 .Figure 6 .

 56 Figure5. Previously cryopreserved mononuclear cells retain their anti-proliferative properties when treated with ECP. Six couples of healthy volunteers were enrolled. PBMC from healthy volunteer D were labeled with CFSE, whereas PBMC from healthy R were irradiated at 30 Gy and used as stimulators. ECP-treated cells were added on day 0 of the 7-day culture. The ratio between R * , D CFSE and ECP-treated cells was 1:2:2. ECP-treated cells were:(1) fresh naive cells from D; (2) cryopreserved naive cells from D;(3) fresh cells from D previously sensitized by a 7-day MLR, D ϫ R * ; (4) cryopreserved cells from D previously sensitized by a 7-day MLR, D ϫ R * . * 1, 2, 3 and 4 versus allogeneic control, P Ͻ 0.05; 1 versus 2 and 3 versus 4, P ϭ NS.

Cytotherapy Downloaded from informahealthcare.com by INSERM on 08/04/10For personal use only.

Acknowledgements

This work was supported by the association ' Capucine ' (grant attributed under the control of the association ' Centpoursanglavie ' ). We thank Magalie Divin é , Florence Jacomet and Camille Confl and for technical assistance.

Disclosure of interest:

The authors have no confl ict to declare.


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Identifying uncertainty contributions to the seismic fragility assessment of a nuclear reactor steam line

Pierre Gehl, Marine Macilhac-Fradin, Jeremy Rohmer, Yves Guigueno, Nadia Orléans, France p.gehl@brgm.fr, j.rohmer@brgm.fr 2

IRSN

Fontenay-aux-Roses, France marine.marcilhacfradin@irsn.fr, yves.guigueno@irsn.fr, nadia.rahni@irsn.fr, julien.clement@irsn.fr

ABSTRACT

In nuclear applications, fragility curves are an essential element of the seismic probabilistic safety assessment that is performed at the level of the power plant. These statistical tools establish the link between the probabilistic seismic hazard loading at the site and the required performance of the plant's safety functions. In this context, the fragility model formalized by [START_REF] Kennedy | Seismic fragilities for nuclear power plant studies[END_REF] proposes to account for the aleatory randomness and the epistemic uncertainty generated by various sources of variability, such as the representation of the seismic input or the assumptions in the structural model and in the loading patterns.

Therefore, this study investigates the relative contributions of such variables to the dispersion of the resulting fragility functions, while ensuring the separation between aleatory and epistemic uncertainty sources, as advocated by the standards in effect in the nuclear industry. To this end, a coupled model of a supporting structure and an anchored steam line is considered and computed with the finite-element CAST3M software [START_REF] Rahni | An original approach to derived seismic fragility curves -Application to a PWR main steam line[END_REF]. The failure criterion is related to the exceedance of a given effort value at a point of the steam line corresponding to a vertical stop. A coupled of hundreds of threecomponent ground-motion records are applied at the base of the model, in order to perform non-linear timehistory analyses. The record selection follows the conditional spectrum method [START_REF] Lin | Conditional spectrum-based ground motion selection -Part I[END_REF], in order to scale the records while ensuring the hazard consistency, through the identification of reference earthquake scenarios for each predefined hazard level.

As a result, dozens of ground-motion parameters are tested as potential intensity measures (IMs), with respect to state-of-the-art indicators measuring efficiency or sufficiency. Some couples of ground-motion parameters are assembled as vector-valued IMs, in order to generate so-called fragility surfaces: these multivariate functions may then be reinterpreted as single-IM fragility curves, where the conditional distribution of the secondary IM with respect to the main one generates additional confidence intervals for the fragility curve. It is thus shown that the aleatory randomness due to record-to-record variability may be partially transferred to an epistemic uncertainty contribution. This variability is then put in perspective with other sources of uncertainty due to modelling assumptions, such as the variability in the mechanical parameters of the model or the angle of loading of the seismic input.

  Rahni, Julien Clément To cite this version: Pierre Gehl, Marine Macilhac-Fradin, Jeremy Rohmer, Yves Guigueno, Nadia Rahni, et al.. Identifying uncertainty contributions to the seismic fragility assessment of a nuclear reactor steam line. COMPDYN 2019 -7th International Conference on Computational Methods in Structural Dynamics and Earthquake Engineering, Jun 2019, Heraklion -Crète, Greece. pp.47 -68. hal-02047529Identifying uncertainty contributions to the seismic fragility assessment of

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