Dalil Hannani

Yuting Ma

Takahiro Yamazaki

Julie Dechanet-Merville

Guido Kroemer

Laurence Zitvogel

Harnessing ɣδ T cells in anticancer immunotherapy

ɣδ T lymphocytes are involved in the stress response to injured epithelia and in tissue homeostasis by limiting the dissemination of malignant or infected cells and by regulating the nature of the subsequent adaptive im-mune response. ɣδ T cells have potent MHCunrestricted cytotoxicity, a high potential for cytokine release and broad-spectrum recognition of cancer cells, and as such, are attractive effectors for cancer immunotherapy. Cur-rent expectations are going beyond ex vivo manipulation of the Vɣ9Vδ2 T subset, and target novel ɣδ T cell subsets, properties or receptors, to harness these un-conventional T lymphocytes against cancer. This Opin-ion article discusses novel aspects of ɣδ T cell function during the course of anticancer therapies, as well as new avenues for their clinical implementation.

ɣδ T cell characteristics

. Both human and mouse CD16-expressing ɣδ T cells can also recognize and be activated by antibody-opsonized cells or microorganisms through binding of IgGs, which mediates antibody-dependent cell cytotoxicity (ADCC). On activation, ɣδ T cells expand, acquire cytotoxic functions and secrete an array of proinflammatory (Th1) cytokines. Thus, these cells are attractive for cell therapy strategies against cancer.

In humans, ɣδ T cells represent 3-5% of blood lympho-cytes. Among this population, the Vɣ9Vδ2 T cell subset is predominant (>70%), whereas in other tissues such as gut epithelia, dermis, spleen and liver, ɣδ T cells mostly ex-press the Vδ1 TCR and to a lesser extent the Vδ3 TCR. The Vδ2 -population has received less attention than Vɣ9Vδ2 cells, but recent description of their specific anti-cytomeg-alovirus (CMV) activity and antitumor

crossreactivity has raised a new interest in this subset. Here, we summarize recent advances in our understanding of how the different ɣδ T cell subsets contribute to antitumor responses during different cancer therapies, including treatment with bisphosphonates and immunogenic chemotherapy (Box 1). We highlight the potential therapeutic value of combin-ing immunotherapy to target ɣδ T cells with chemothera-peutic approaches.

ɣδ T cells in cancer immunosurveillance

ɣδ T cells can exert cytolytic functions against a variety of premalignant or malignant primary tumor cells and tumor stem cells in an MHC-dependent or -independent manner [START_REF] Liu | Protective immunosurveillance and therapeutic antitumor activity of gammadelta T cells demonstrated in a mouse model of prostate cancer[END_REF][START_REF] Girardi | Immunosurveillance and immunoregulation by gammadelta T cells[END_REF][START_REF] Ferrarini | Killing of laminin receptor-positive human lung cancers by tumor infiltrating lymphocytes bearing gammadelta(+) T-cell receptors[END_REF]. Activated Vɣ9Vδ2 T cells show in vitro cytolytic activity towards myeloma [START_REF] Ferrarini | Human gammadelta T cells: a nonredundant system in the immune-surveillance against cancer[END_REF][START_REF] Von Lilienfeld-Toal | Activated gammadelta T cells express the natural cytotoxicity receptor natural killer p 44 and show cytotoxic activity against myeloma cells[END_REF][START_REF] Kunzmann | Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma[END_REF], lymphoma cell lines [START_REF] Fisch | Inhibitory MHC class I receptors on gammadelta T cells in tumour immunity and autoimmunity[END_REF][START_REF] Fisch | Control of B cell lymphoma recognition via natural killer inhibitory receptors implies a role for human Vgamma9/Vdelta2 T cells in tumor immunity[END_REF] and solid tumors [START_REF] Viey | Peripheral gammadelta T-lymphocytes as an innovative tool in immunotherapy for metastatic renal cell carcinoma[END_REF][START_REF] Viey | Phosphostim-activated gamma delta T cells kill autologous metastatic renal cell carcinoma[END_REF][START_REF] Mattarollo | Chemotherapy and zoledronate sensitize solid tumour cells to Vgamma9Vdelta2 T cell cytotoxicity[END_REF][START_REF] Liu | Ex vivo expanded human Vgamma9Vdelta2+ gammadelta-T cells mediate innate antitumor activity against human prostate cancer cells in vitro[END_REF]. Ex vivo expanded ɣδ T cells from patients with glioblastoma multiforme (GBM) harbor intact killing functions against GBM cells; however, both ɣδ T cell counts and the mitogen-stimulated prolifer-ative response of circulating ɣδ T cells are decreased when compared to healthy volunteers [START_REF] Bryant | Characterization and immunotherapeutic potential of gammadelta T-cells in patients with glioblastoma[END_REF]. In immunocompro-mised patients, a large number of Vδ2 -ɣδ T cells, which exert crossreactive recognition of both CMV-infected and transformed epithelia cells, are associated with reduced risk of developing cancer. In a longitudinal case-control study involving 18 immunosuppressed recipients who de-veloped cancer between 2 and 6 years after transplantation and 45 recipients who did not, the median percentage of ɣδ T cells among total lymphocytes in patients with malig-nancies was significantly lower (<4%) before the diagnosis of cancer compared with that in control patients. An in-crease in Vδ2 -ɣδ T cell numbers correlates with a lower incidence of cancer only in recipients who experienced pre-or post-graft CMV infection, suggesting a role for the CMV-induced Vδ2 -ɣδ T cells (mainly Vδ1 ɣδT cells) in protection against tumors. Consistent with this, a retrospective fol-low-up of kidney recipients for 8 years revealed that CMV-naive recipients had a fivefold higher risk of developing cancer compared with CMV-seropositive patients [START_REF] Couzi | Cytomegalovirus-induced gammadelta T cells associate with reduced cancer risk after kidney transplantation[END_REF]. CMV replication is also associated with a decrease in relapse risk in acute myeloid leukemia (AML) patients who undergo allogenic stem cell transplantation [START_REF] Elmaagacli | Early human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in acute myeloid leukemia patients[END_REF]. This might rely on the antileukemic activity capacity of Vδ1 ɣδT cells [START_REF] Lamb | Human gammadelta(+) T lymphocytes have in vitro graft vs leukemia activity in the absence of an allogeneic response[END_REF]. Indeed, after bone marrow transplantation in patients suffering from acute lymphoid leukemia (ALL), donor-derived Vδ1 ɣδT cells are activated and proliferate in response to primary recipient ALL blasts. In addition, these cells lyse ALL blasts. Importantly, Vδ1 ɣδT cells do not respond to HLA-mismatch nonleukemic cells, offer-ing promising perspectives for immunotherapy using allo-genic Vδ1 ɣδ T cells.

Whether the presence of ɣδ tumor-infiltrating lympho-cytes (TILs) reflects good or poor prognosis in cancer is still controversial. ɣδ TILs from epithelial tumors in breast, prostate or colon cancer belong to the resident Vδ 2-popu-lation [START_REF] Ferrarini | Killing of laminin receptor-positive human lung cancers by tumor infiltrating lymphocytes bearing gammadelta(+) T-cell receptors[END_REF][START_REF] Groh | Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB[END_REF], whereas in other types of epithelial tumors (such as renal cell carcinoma; RCC) ɣδ T cell infiltrates are found to comprise mainly Vɣ9Vδ2 T cells [START_REF] Viey | Phosphostim-activated gamma delta T cells kill autologous metastatic renal cell carcinoma[END_REF]. ɣδ TILs isolated in various types of cancer, including colorectal, breast, prostate, ovarian and RCC [START_REF] Viey | Phosphostim-activated gamma delta T cells kill autologous metastatic renal cell carcinoma[END_REF][START_REF] Groh | Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB[END_REF][START_REF] Corvaisier | V gamma 9 V delta 2 T cell response to colon carcinoma cells[END_REF], recognize and kill both the autologous tumor and a broad range of related tumors, presumably via the recognition of shared stress-related ligands, but do not kill nontransformed cells [START_REF] Viey | Phosphostim-activated gamma delta T cells kill autologous metastatic renal cell carcinoma[END_REF][START_REF] Corvaisier | V gamma 9 V delta 2 T cell response to colon carcinoma cells[END_REF]. The infiltration of Vɣ1 and Vδ1 ɣδ T cells into necrotizing melanomas (NMMs) significantly correlates with increased survival [START_REF] Bialasiewicz | Alpha/beta-and gamma/delta TCR(+) lymphocyte infiltration in necrotising choroidal melanomas[END_REF], whereas intratumoral ɣδ T cell infiltration in RCC tumors does not [START_REF] Inman | Questionable relevance of gamma delta T lymphocytes in renal cell carcinoma[END_REF]. These studies suggest that ɣδ T cell infiltration might be beneficial; however, the immunoregulatory properties of ɣδ T cells should not be ignored [START_REF] Hayday | Immunoregulation in the tissues by gammadelta T cells[END_REF]. Indeed, in breast and prostate cancer, the Vδ1 + ɣδ T subset of TILs potently suppresses naive and effector T cell responses and blocks the matura-tion and function of DCs. This suppressive function is reversed by addition of TLR8 ligands [START_REF] Peng | Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by a unique toll-like receptor signaling pathway[END_REF]. In addition, ɣδ T cells could prevent tumor elimination in an IL-10-dependent manner [START_REF] Ke | Inhibition of tumor rejection by gammadelta T cells and IL-10[END_REF].

Mouse models suggest that the contribution of ɣδ T cells to anticancer surveillance is nonredundant with ab T cells. Mice lacking ɣδ T cells are vulnerable to cutaneous malignancy when inoculated with carcinoma cells, or when in-duced by chemical carcinogenesis, underlining their crucial role in cancer immunosurveillance [START_REF] Girardi | Immunosurveillance and immunoregulation by gammadelta T cells[END_REF][START_REF] Girardi | The distinct contributions of murine T cell receptor (TCR)gammadelta+ and TCRalphabeta+ T cells to different stages of chemically induced skin cancer[END_REF]. ɣδ T-cell-deficient mice also show that ɣδ T cells strongly contribute to host protection against papillomas, whereas ab T cells have only a moderate effect or even promote the progression of papil-lomas to carcinomas [START_REF] Girardi | Immunosurveillance and immunoregulation by gammadelta T cells[END_REF][START_REF] Girardi | The distinct contributions of murine T cell receptor (TCR)gammadelta+ and TCRalphabeta+ T cells to different stages of chemically induced skin cancer[END_REF]. ɣδ T cells that are probably responsible for restraining malignancy are the NKG2D-expressing, skin-associated NKG2D T cells, suggesting that these cells respond very rapidly to stress-ligand-expressing cells (i.e. transformed cells) [START_REF] Girardi | Immunosurveillance and immunoregulation by gammadelta T cells[END_REF][START_REF] Girardi | The distinct contributions of murine T cell receptor (TCR)gammadelta+ and TCRalphabeta+ T cells to different stages of chemically induced skin cancer[END_REF]. Using the TRAMP trans-genic mouse model of prostate cancer, one group has provid-ed evidence that ɣδ T cells can mediate protective immunosurveillance against spontaneously arising cancer [START_REF] Liu | Protective immunosurveillance and therapeutic antitumor activity of gammadelta T cells demonstrated in a mouse model of prostate cancer[END_REF]. In nude mice inoculated subcutaneously with nasopha-ryngeal carcinoma cell lines, infused human peripheral ɣδ T cells could temporarily accumulate in tumors, causing a transient reduction in tumor growth [START_REF] Zheng | Anti-tumor effects of human peripheral gammadelta T cells in a mouse tumor model[END_REF]. In a preclinical breast tumor model, infused ɣδ T cells traffic to breast tumors in a TCR-dependent manner and prevent tumor growth [START_REF] Beck | Adoptively transferred ex vivo expanded gammadelta-T cells mediate in vivo antitumor activity in preclinical mouse models of breast cancer[END_REF]. Another study using mice transplanted with HER-2 + human breast cancer cell lines has shown that in vivo activation of ɣδ T cells (through bisphosphonates) enhances the efficacy of standard immunotherapy (admin-istration of trastuzumab, a monoclonal antibody directed against HER2/neu receptors) [START_REF] Capietto | Stimulated gammadelta T cells increase the in vivo efficacy of trastuzumab in HER-2+ breast cancer[END_REF]. Also, coadministration of zoledronic acid -a synthetic ɣδ T cell activator -together with intravesical infusion of ɣδ T cells induces antitumor effects against orthotopically inoculated human bladder cancer cells in immunodeficient mice and prolongs survival [START_REF] Yuasa | Intravesical administration of gammadelta T cells successfully prevents the growth of bladder cancer in the murine model[END_REF]. CMV-specific Vδ2 -ɣδ T cells can also target cancer cells in vivo. Prophylactic injections of Vδ2 -ɣδ T cell clones in subcutaneous (s.c.) HT29 tumors established in immunode-ficient mice prevent the development of these colon tumors, whereas Vδ2Vɣ9 ɣδ T cells fail to do this. More importantly, systemic intraperitoneal (i.p.) treatment with Vδ2 - ɣδ T cell clones delays the growth of HT29 s.c. tumors in a chemokine C-C motif receptor 3 (CCR3)-dependent manner. Vδ2 -ɣδ T cell clones express CCR3 and migrate in vitro in response to chemokines (macrophage inflammatory protein-1 d and monocyte chemoattractant protein-4) secreted by HT29 cells [START_REF] Devaud | Antitumor activity of gammadelta T cells reactive against cytomegalovirus-infected cells in a mouse xenograft tumor model[END_REF]. Taken together, these findings suggest that ɣδ T cells can be harnessed for cancer immunotherapy and could synergize with other immunotherapy (such as monoclonal antibodies). IL-17-producing ɣδ T cells (ɣδ T17) contribute to the efficacy of anticancer therapies Bidirectional tumor-host interactions (i.e. tumor-induced immunosuppression vs antitumor immunity) during the course of certain anticancer therapies can influence thera-peutic success [START_REF] Zitvogel | Cancer despite immunosurveillance: immunoselection and immunosubversion[END_REF][START_REF] Zitvogel | Immune parameters affecting the efficacy of chemotherapeutic regimens[END_REF][START_REF] Zitvogel | Immunological aspects of cancer chemotherapy[END_REF]. Indeed, some cytotoxic compounds are capable of triggering an 'immunogenic' cell death, in that chemotherapy-induced cell death elicits a tumor-spe-cific immune response that controls residual tumor cells instead of inducing tolerance [40][START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF][42]. Failure to activate an antitumor immune response compromises the success of conventional anticancer chemotherapies. Anthracy-clines, oxaliplatin and X-rays can trigger immunogenic cell death. Four major checkpoints dictate the immunogenicity of cell death and how the host immune system recognizes dying tumor cells. First, optimal phagocytosis of dying tumor cells requires the translocation of endoplasmic re-ticulum (ER)-resident calreticulin (CRT) [START_REF] Obeid | Ecto-calreticulin in immunogenic chemotherapy[END_REF] and disulfide isomerase ERp57 [START_REF] Panaretakis | The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death[END_REF] to their plasma membrane. The ER stress response, initiated by the phosphorylation of eif2a and culminating in the combined exposure of CRT and ERp57 to the plasma membrane, dictates DC uptake of apoptotic bodies, a crucial step to elicit an immune re-sponse against cancer [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF][START_REF] Panaretakis | Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death[END_REF]. Second, the chromatin-bind-ing high mobility group box 1 protein (HMGB1) is released by dying tumor cells and binds to TLR4 to facilitate antigen processing and presentation to T cells [START_REF] Apetoh | Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy[END_REF]. Third, release of ATP by dying cells is required to engage the purinergic receptor P2RX7, which triggers IL-1b secretion through a cascade involving the Nlrp3 inflammasome, and culmi-nates with IL-1b-dependent ɣδ T cell activation and Tc1 polarization of antigen-specific T cell responses [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF][START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1betadependent adaptive immunity against tumors[END_REF]. The antitumor effects of anthracyclines, oxaliplatin and X-rays are abrogated in mice devoid of T cells or Th1 cytokines (such as IFNg and the IFNg receptor, and IL-1b and IL-1 receptor 1) [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF][START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1betadependent adaptive immunity against tumors[END_REF][START_REF] Mattarollo | Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors[END_REF].

These data suggest that immunogenic cell death in-duced by chemotherapy could influence the composition of the immune infiltrate present in tumors, which in turn could affect the therapeutic response. Supporting this, it has recently been reported that ɣδ T cells that produce IL-17 (ɣδT17), followed by IFNg-producing CD8 + T cells (CTLs), infiltrate tumor beds post-chemotherapy and both ɣδ T cells and CTLs, as well as IL-17 and IFNg, are essential for the therapeutic success of anthracyclines in several preclinical models [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] (Figure 1).

The transcriptional analysis of the cytokine/chemokine profile in regressing tumor beds postanthracycline treat-ment has revealed a four-to fivefold increase in the gene products associated with the typical Th1 signature [Eomes, T-bet, IFN-g, lymphotoxin-b, chemokine C-C motif ligand 5 (Ccl5), chemokine C-X-C motif ligand (Cxcl)10, Cxcl9, and tumor necrosis factor (TNF)-a], as well as gene products encoding IL-7 receptor, IL-21, aryl hydrocarbon receptor (AhR), Cxcl2 and forkhead box p3 (Foxp3). The mandatory role of the IL-17A and IL-17RA pathway has been demon-strated in several transplantable tumor models (EG7 thymoma, MCA205 sarcoma, CT26 colon cancer, and TS/A mammary carcinomas) and in 3methylcholanthrene (MCA)-induced spontaneous sarcoma [START_REF] Mattarollo | Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors[END_REF]. Moreover, doxo-rubicin treatment increases the proportion of both IFN-g-and IL-17-producing TILs (which are CD44 + CD62L -CD69 + granzyme B + CD27 -NKG2D -Vɣ4 + or Vɣ6 + ). Eight days post-chemotherapy in MCA205 sarcomas, the major source of IFN-g is CD8 + T cells, whereas that of IL-17 is mostly TCR d + T cells rather than CD4 + Th17 cells. Doxo-rubicin-based chemotherapy substantially enhances IFN-g production by CD8 + and CD4 + TILs, as well as IL-17 production by ɣδ TILs. ɣδ TILs invade MCA205 tumor beds within 3 days of treatment, rapidly divide and pro-duce IL-17 shortly after chemotherapy. The early induc-tion of IL-17 contrasts with the later induction of IFN-g production by CD8 + T cells on day 8 post-chemotherapy. The percentage of ɣδ T17 cells correlates with the frequen-cy of IFN-g-producing CD8 + T lymphocytes (Tc1) in the tumor microenvironment. Accordingly, in the absence of ɣδ T17 or IL-17, priming of adaptive anticancer Th1/Tc1 responses is hampered, suggesting a helper role of ɣδ T17 cells in the context of immunogenic cell death.

The absence of the TCR d chain, as well as Vɣ4 and Vɣ6 ɣδ T cells, has been found to reduce the efficacy of chemo-therapy. Adoptive transfer of ɣδ T cells from naı¨ve mice enhances chemotherapy efficacy and restores the sensitiv-ity of IL-17 knockout mice, provided that these cells ex-press IL-1 receptor 1 and are competent for IL-17 production. ɣδ T17 cells respond to ex vivo propagated bone-marrow-derived DCs (but not macrophages) loaded with dying tumor cells (but not live tumor cells) for the production of IL-17 and IL-22 in a manner depending on IL-1b [but not IL-23, transforming growth factor (TGF)-b or IL-6] [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF]. It is of note that neutralization of IL-23 fails to abrogate the efficacy of chemotherapy [START_REF] Mattarollo | Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors[END_REF]. A beneficial role of ɣδ T17 cells in cancer is supported by a study showing that ɣδ T17 cells are indispensable for the efficacy of Mycobacterium bovis bacillus Calmette-Guerin (BCG) against bladder cancer by recruiting neutrophils [START_REF] Takeuchi | IL-17 production by gammadelta T cells is important for the antitumor effect of Mycobacterium bovis bacillus Calmette-Guerin treatment against bladder cancer[END_REF]. In contrast to these data, IL-17producing ɣδ TILs could support tumor outgrowth by promoting angiogenesis. Such ɣδ T17 TILs exhibit low levels of perforin and are stimu-lated through TCR, NKG2D and receptors for IL-23, IL-6 and TGF-b to produce IL-17 and cyclooxygenase (COX)-2 in tumor beds [START_REF] Wakita | Tumor-infiltrating IL-17-producing gammadelta T cells support the progression of tumor by promoting angiogenesis[END_REF]. In fact, ɣδ T17 cells from chemotherapy-treated tumor beds depend upon IL-1 receptor 1 to mediate their immunostimulatory effects (instead of TCR, NKG2D and IL-23, as described in [START_REF] Wakita | Tumor-infiltrating IL-17-producing gammadelta T cells support the progression of tumor by promoting angiogenesis[END_REF]).

These findings unravel the capacity of distinct subsets of ɣδT cells, presumably antigen-naı¨ve (as opposed to self-reactive) and IL-1b-responsive [START_REF] Jensen | Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma[END_REF], to link innate signals to adaptive immunity during a sterile inflammation such as chemotherapy-induced cell death. Knowing whether human ɣδT cells invade tumor beds at the onset of chemo-therapy and radiotherapy may help in harnessing such effectors to improve conventional therapies of cancer.

Aminobisphosphonates and bromohydrin pyrophosphate: rationale for the utilization of ɣδ2 T cells as antitumor weapons. Synthetic aminobisphosphonates (pamidronate, zoledro-nate) are well known for treatment of Paget's disease and bone metastases and are potent ɣδ T cell stimulatory compounds that induce cytokine secretion and cell-medi-ated cytotoxicity [START_REF] Kunzmann | Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma[END_REF]. The nitrogen-containing bisphospho-nate zoledronic acid (ZOL) blocks the mevalonate pathway, leading to intracellular accumulation of isopentenyl pyro-phosphate and triphosphoric acid I-adenosin-5 0 -yl ester 3-(3-methylbut-3-enyl) ester (IPP and ApppI) mevalonate metabolites. Vɣ9Vδ2 T cells appear to recognize IPP and ApppI in ZOL-treated cancer cells as tumor phosphoanti-gens in vitro. These findings may also be significant in vivo because there is a correlation between the ability of Vɣ9Vδ2 T cells to kill tumors and the intracellular IPP and ApppI concentrations in ZOL-treated breast cancer cells. In ZOL-treated mice bearing s.c. breast cancer xeno-grafts, Vɣ9Vδ2 T cells traffic to tumor beds containing high concentrations of IPP and ApppI, which lead to tumor regression. These findings suggest that cancers producing high amounts of IPP and ApppI after ZOL administration may benefit from Vɣ9Vδ2 T-cell-mediated immunotherapy [START_REF] Benzaid | High phosphoantigen levels in bisphosphonate-treated human breast tumors promote Vgamma9Vdelta2 T-cell chemotaxis and cytotoxicity in vivo[END_REF].

The antitumor activity of ɣδ T cells in vivo was first tested in a clinical trial in 2003 in which recombinant IL-2 (rIL-2) was combined with pamidronate for the treatment of hematological malignancies (non-Hodgkin lymphoma and multiple myeloma) [START_REF] Wilhelm | Gammadelta T cells for immune therapy of patients with lymphoid malignancies[END_REF][55][START_REF] Dieli | Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer[END_REF]. The combination of pami-dronate and low-dose IL-2 was well tolerated. Preselection of patients based on 'positive in vitro proliferation of ɣδ T cells in response to pamidronate and IL-2' was associated with significant activation/proliferation of ɣδ T cells (in 55% of cases) in vivo and 33% of objective responses (tumor regression). In end-stage breast cancer for which administration of zoledronate and rIL-2 was combined, the number of peripheral Vɣ9Vδ2 T cells correlated signifi-cantly with clinical outcome. The seven patients who failed to sustain Vɣ9Vδ2 T cells showed progressive clini-cal deterioration (PD), whereas three patients who sus-tained robust peripheral Vɣ9Vδ2 cell populations showed declining serum concentrations of the glycoprotein Mucin-1 (MUC-1) tumor-related marker (CA15.3); one patient showed partial remission (PR) and two exhibited stable disease (SD) [55]. In metastatic hormone-refractory pros-tate cancers treated with ZOL and rIL-2, the numbers of effector memory ɣδ T cells and serum levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (produced by activated ɣδ T cells) correlated with declining prostate-specific antigen levels and objective clinical responses that comprised three PR and five SD. By contrast, most patients treated only with zoledronate (seven out of nine) failed to sustain either ɣδ cell numbers or serum TRAIL and showed PD [START_REF] Dieli | Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer[END_REF]. This result is in line with a recent report showing no efficacy of ZOL alone in adjuvant breast cancer [START_REF] Coleman | Breast-cancer adjuvant therapy with zoledronic acid[END_REF].

Adoptive transfer of ɣδ T cells for treatment of some cancers such as metastatic RCC or non-small cell lung carcinoma has been investigated in several clinical trials during the past decade (Table 1). ɣδ T cells can either be infused into patients after ex vivo activation and expansion with phosphoantigens, or without experimental manipula-tion, instead being directly activated in vivo by administra-tion of synthetic aminobisphosphates (such as bromohydrin pyrophosphate, pamidronate or ZOL) and exogenous T cell growth factors [START_REF] Wilhelm | Gammadelta T cells for immune therapy of patients with lymphoid malignancies[END_REF][55][START_REF] Dieli | Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer[END_REF]. These therapies are usually feasible and well tolerated (with the exception of risk for thrombo-embolic events in patients with multiple myeloma [START_REF] Morgan | First-line treatment with zoledronic acid as compared with clodronic acid in multiple myeloma (MRC Myeloma IX): a randomised controlled trial[END_REF]), but therapeutic benefits seem only to occur when used in com-bination with other therapies [START_REF] Viey | Peripheral gammadelta T-lymphocytes as an innovative tool in immunotherapy for metastatic renal cell carcinoma[END_REF][START_REF] Nicol | Clinical evaluation of autologous gamma delta T cell-based immunotherapy for metastatic solid tumours[END_REF], including cytokines (such as IL-2 and IL-21) [START_REF] Wilhelm | Gammadelta T cells for immune therapy of patients with lymphoid malignancies[END_REF][55][START_REF] Dieli | Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer[END_REF][START_REF] Kobayashi | Complete remission of lung metastasis following adoptive immunotherapy using activated autologous gammadelta T-cells in a patient with renal cell carcinoma[END_REF], traditional chemother-apy or monoclonal antibodies, because ɣδ T cells mediate ADCC [START_REF] Li | Effect of ex vivo-expanded gammadelta-T cells combined with galectin-1 antibody on the growth of human cervical cancer xenografts in SCID mice[END_REF]62].

These studies examining the antitumor activity of ɣδ T cells (Table 1) highlight the therapeutic potential of ɣδ T cells in patients in whom Vɣ9Vδ2 T cells can expand and survive. However, more knowledge is needed of ɣδ T cell biology (survival, trafficking patterns, subset variability, ligands and/or TCR identifications) to further such trials [START_REF] Gomes | Targeting gammadelta T lymphocytes for cancer immunotherapy: from novel mechanistic insight to clinical application[END_REF]. Improving ɣδ T cell function for combinatorial regimens with immunotherapy and chemotherapy Vɣ9Vδ2 ɣδ T cell-triggering may be ameliorated by com-bining TCR stimulation with engagement of NK receptors or TLRs [START_REF] O'brien | gammadelta T-cell receptors: functional correlations[END_REF]. In addition, because ɣδ T cells may be inhibited by T regulatory cells [START_REF] Mahan | CD4+ CD25(high) Foxp3+ regulatory T cells downregulate human Vdelta2+ Tlymphocyte function triggered by anti-CD3 or phosphoantigen[END_REF][START_REF] Kunzmann | Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells[END_REF][START_REF] Goncalves-Sousa | Inhibition of murine gammadelta lymphocyte expansion and effector function by regulatory alphabeta T cells is cell-contact-dependent and sensitive to GITR modulation[END_REF], any therapeutic intervention aimed at reducing T regulatory cell (Treg) immunosuppressive functions could be valuable for restor-ing ɣδ T cell activity.

Although Vδ1 ɣδ T cell ligands have not been identified yet, these cells can be triggered using a specific activating anti-Vδ1 TCR antibody coupled with activating signals such as NKG2D ligands (Figure 2). Using this strategy, one group has developed protocols to expand both human peripheral and tumor infiltrating ɣδ T cells, which show cytotoxicity and antitumor activity [START_REF] Zhou | Anti-gammadelta TCR antibody-expanded gammadelta T cells: a better choice for the adoptive immunotherapy of lymphoid malignancies[END_REF][START_REF] Kang | Adoptive immunotherapy of lung cancer with immobilized anti-TCRgammadelta antibody-expanded human gammadelta T-cells in peripheral blood[END_REF][START_REF] Chen | Antitumor activity of expanded human tumor-infiltrating gammadelta T lymphocytes[END_REF]. In addition, Vδ1 ɣδ T cells that are activated by certain CMVinfected cells or CMV-induced autoantigens display crossreactivity against tumor cells. Thus, a protocol for Vδ1 ɣδ T cell amplification using autologous CMV-infected cells may favor in vivo Vδ1 ɣδ T cell antitumor activity [START_REF] Couzi | Cytomegalovirus-induced gammadelta T cells associate with reduced cancer risk after kidney transplantation[END_REF][START_REF] Halary | Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells[END_REF]. In immunocompromised mice, sustained expansion of human peripheral blood ɣδ T cells is observed upon stimulation with anti-ɣδ TCR antibody (which expands both Vδ1 and Vδ2 subsets of ɣδ T cells). Compared to phosphoantigen-stimulated ɣδ T cells, the antibody-expanded cells manifest similar functional phenotypes and cytotoxic activity to-wards tumor cell lines between subsets. Vδ1 T cells are more prone to infiltrate CCL17-or CCL22-expressing lym-phomas than Vδ2 T cells because of high expression levels of CCR4 and CCR8 [START_REF] Zhou | Anti-gammadelta TCR antibody-expanded gammadelta T cells: a better choice for the adoptive immunotherapy of lymphoid malignancies[END_REF]. Vδ1 T cells are also preferentially expanded by extended exposure to concanavalin A in the presence of IL-2. These cells display potent cytotoxicity toward B cell chronic lymphocytic leukemia and represent a new perspective for the treatment of this disease [START_REF] Siegers | Human Vdelta1 gammadelta T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells[END_REF].

Similar to conventional T cells, the polarization of ɣδ T cells crucially influences the nature of the downstream adaptive immune response. In the context of a combinato-rial regimen using both chemotherapy and immunothera-py, IL-1R1 + ɣδ T17 cells appear crucial in that they precede and correlate with trafficking of IFNg + CD8 + T cells to the tumor site [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF]. These cells depend on IL-1b for their triggering and therefore administration of an 'immunogen-ic chemotherapy' (such as oxaliplatin or anthracycline or an X-ray-based regimen) or local delivery of TLR agonists in the tumor microenvironment (which stimulate local DCs, a source of IL-1b) may be instrumental in polariza-tion of these ɣδ TILs. It is of note that targeting of TLR1 and TLR2 (by pathogens or synthetic ligands), as well as dectin-1 on ɣδ T cells bearing a CCR6 + IL-17 + phenotype, promotes (alone or in conjunction with rIL-23) their pro-liferation and increased Th17-like polarization [START_REF] Martin | Interleukin-17-producing gammadelta T cells selectively expand in response to pathogen products and environmental signals[END_REF]. Alter-natively, passive infusion of prepolarized ɣδ T17 cells is conceivable. Co-culture of naive Vɣ9Vδ2 T cells with phos-phoantigens and a cocktail of cytokines (IL-1b, TGF-b, IL-6, and IL-23) leads to selective induction of the transcrip-tion factor Retinoic acid receptor-related Orphan nuclear Receptor (RORgt) and polarization toward IL-17 produc-tion. IL-17 + Vɣ9Vδ2 T cells express granzyme B, TRAIL, FasL, and CD161 and upon activation, rapidly induce CXCL8-mediated migration of neutrophils in vitro. Inter-estingly, an increased percentage of circulating IL-17 + Vɣ9Vδ2 lymphocytes has been found in children with bacterial meningitis [START_REF] Caccamo | Differentiation, phenotype, and function of interleukin-17-producing human Vgamma9Vdelta2 T cells[END_REF].

Combinations of cellular immune-based therapies with chemotherapy and other antitumor agents may be of clini-cal benefit in the treatment of malignancies. High cytotoxicity against solid-tumor-derived cell lines has been reported with combination treatment using Vɣ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate ZOL. Pretreatment with low concentrations of chemother-apeutic agents or ZOL has been shown to sensitize tumor cells to rapid killing by Vɣ9Vδ2 T cells. Vɣ9Vδ2 T cell cytotoxicity and IFNg secretion are mediated by perforin following TCR-dependent and isoprenoid-mediated recog-nition of tumor cells [START_REF] Mattarollo | Chemotherapy and zoledronate sensitize solid tumour cells to Vgamma9Vdelta2 T cell cytotoxicity[END_REF].

Concluding remarks

Abundant IFNg or IL-17 secretion, potent cytotoxicity and MHC-independent targeting of a broad spectrum of tumors make ɣδ T cells promising targets for cancer immunotherapy using high-affinity phosphoantigens and/or anti-Vδ1 (or Vδ2 -TCR) antibodies. Should human data corroborate the findings in mice, the recent demon-stration that ɣδ T cells might participate in the efficacy of chemotherapy will open novel avenues of combinatorial therapies for solid tumors. In the past decade, the safety profile of ɣδ T cells and the doselimiting toxicity of phos-phoantigens and rIL-2 have been demonstrated. Ongoing clinical trials are examining the potential for exploiting the ADCC activity of ɣδ T cells. Manipulation of Vɣδ T cell functions by targeting their activating NKG2D receptor (i.e. by blocking soluble MHC class I chain-related genes (MIC)A/B) or co-stimulatory receptors (CD27, TLR3 or TLR7) or interfering with immunosuppressive check-points [Programmed Death-1 (PD-1) or cytotoxic T lym-phocyte antigen (CTLA)4, Tregs] may be of further interest. Finally, it would be interesting to examine the prospect for predicting 'ɣδ T cell susceptible' hematologi-cal malignancies using specific gene signatures in future work [START_REF] Gomes | Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood gammadelta T cells[END_REF]. IL-17-producing ɣδ T cells can be generated in with IL-1b-secreting DCs exposed to anthracycline-treated tumor cells [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF]. (b) Vɣ9Vδ2 ɣδ T cells can be polarized into IL-17-producing cells using phosphoantigens in the presence of cytokines [START_REF] Caccamo | Differentiation, phenotype, and function of interleukin-17-producing human Vgamma9Vdelta2 T cells[END_REF]. Vδ1 ɣδ T cells can be activated and expanded using either (c) autologous CMV-infected cells [START_REF] Couzi | Cytomegalovirus-induced gammadelta T cells associate with reduced cancer risk after kidney transplantation[END_REF] or (d) anti-Vδ1 TcR antibody. (e) Patients can be treated either by infusion of ex vivo expanded ɣδ T cells (as delineated in a-d) or by direct in vivo ɣδ T cell activation using immunogenic chemotherapy followed by BrHPP + rIL-2 (to trigger Vɣ9Vδ2 ɣδ T cell activation), or anti-Vδ1 TcR antibody (to trigger Vδ1 ɣδ T cells) and/or NKG2D ligands or TLR agonists.

Box 1. Triggering ɣδ T-cell-mediated antitumor activity: focus on aminobiphosphonates and chemotherapy

Aminobiphosphonates are synthetic chemical compounds that potently activate ɣδ T cells and have shown promising efficacy in the treatment of several types of cancer (RCC, metastatic prostate cancer and breast cancer). So far, only a few drugs have been tested, in particular ZOL, pamidronate and bromohydine pyrophosphate (BrHPP; also known as IPH1101). All these synthetic compounds mimic natural ɣδ T cell activating ligands such as IPP or HM-BPP [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate].

Some chemotherapy agents (such as doxorubicin and oxaliplatin) have the capacity to induce an immunogenic tumor cell death, which elicits an antitumor immune response that is crucial for tumor eradication and long-term protection against relapse. ɣδ T cells are recruited to the tumor bed after immunogenic chemotherapy and appear as crucial contributors to chemotherapy efficacy. Doxorubi-cin is an anthracycline antibiotic that works by intercalating DNA. Doxorubicin is commonly used in the treatment of a wide range of cancers, including hematological malignancies, many types of carcinoma, and soft tissue sarcomas.

Oxaliplatin is a platinum-based anticancer agent. The cytotoxicity of platinum compounds results from inhibition of DNA synthesis. Oxaliplatin is mainly used in the treatment of colorectal carcinoma.

Figure 1 .

 1 Figure 1. Contribution of IL-17-producing ɣδ T cells (ɣδ T17) to the efficacy of anticancer chemotherapy. (a) Some chemotherapy regimens induce immunogenic cell death (with release of calreticulin exposure, HMGB1 and ATP by dying tumor cells) leading to (b) tumor cell engulfment by DCs and DC activation through a pathway involving the Nlrp3/Asc/casp-1 inflammasome platform, culminating in (c) IL-1b production by DCs. (d) Within 2-3 days, ɣδ T17 are recruited to the tumor bed and activated in an IL-1b-dependent manner. The IL-17producing ɣδ T cells (e) directly contribute to tumor lysis and may help (f) conventional T cell priming by DCs and/or (g) T cell recruitment into the tumor bed because the accumulation of ɣδ T17 cells precedes and correlates with rapid IFNg-producing CTL infiltration, which is also crucial for tumor eradication.

Figure 2 .

 2 Figure 2. Improving ɣδ T cell functions in combinatorial regimen against cancer. ɣδ T cells can be infused into patients after ex vivo expansion, activation and polarization. (a)IL-17-producing ɣδ T cells can be generated in with IL-1b-secreting DCs exposed to anthracycline-treated tumor cells[START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF]. (b) Vɣ9Vδ2 ɣδ T cells can be polarized into IL-17-producing cells using phosphoantigens in the presence of cytokines[START_REF] Caccamo | Differentiation, phenotype, and function of interleukin-17-producing human Vgamma9Vdelta2 T cells[END_REF]. Vδ1 ɣδ T cells can be activated and expanded using either (c) autologous CMV-infected cells[START_REF] Couzi | Cytomegalovirus-induced gammadelta T cells associate with reduced cancer risk after kidney transplantation[END_REF] or (d) anti-Vδ1 TcR antibody. (e) Patients can be treated either by infusion of ex vivo expanded ɣδ T cells (as delineated in a-d) or by direct in vivo ɣδ T cell activation using immunogenic chemotherapy followed by BrHPP + rIL-2 (to trigger Vɣ9Vδ2 ɣδ T cell activation), or anti-Vδ1 TcR antibody (to trigger Vδ1 ɣδ T cells) and/or NKG2D ligands or TLR agonists.

Table 1 .

 1 Selection of clinical trials assessing safety and efficacy of ɣδ T cell infusions and their stimulatory factors. CR, complete response; PD, progressive disease; PR, partial response; SD, stable disease.

	Immunotherapy	Cancer type	Treatment arm	No. of patients	PD (%)	SD (%)	PR (%)	CR (%)	Ref.
	Autologous ɣδ T	Metastatic RCC	+rIL-2	10	40	60			[76]
	cell infusions		+BrHPP
		Metastatic RCC	+ZOL	1				100	[60]
			+rIL-2
		Solid tumors	+rIL-2	28	ND	ND	ND	ND	[77]
			+BrHPP
		Non-small-cell lung cancer	+ZOL	8	63	37	0		[78]
			+rIL-2
		Non-small-cell lung cancer	+ZOL	15	60	40			[79]
			+rIL-2
		Solid tumors	ɣδ T alone	5	40	40			[80]
			combination	20	30	5	15
		Solid tumors		18	61	17	11	6	[59]
	Bisphosphonate	Refractory low-grade	rIL-2 d6-d8	10	80	10			[54]
	in vivo	non-Hodgkin lymphoma	No preselection
		and multiple myeloma	rIL-2 d1-d6	9	44	22	33
			Preselection
		Metastatic hormone-refractory	Phase I ZOL	9	78	11	11		[56]
		prostate cancer	Phase I ZOL + rIL-2	9	33	44	22
		Advanced stage IV breast cancer	ZOL+rIL-2	10	70	20	10		[55]
			Phase I

Acknowledgments

D.H is supported by Association pour la Recherche sur le Cancer. Y.M is supported by the China Scholarship Council. T.Y is supported by INSERM. JDM is supported by Fondation pour la Recherche Medicale´ (Equipe labellise´e), Ligue Contre le Cancer, Association pour la Recherche contre le Cancer and INCA. GK is supported by Ligue Nationale contre le Cancer (Equipes labellise´e), Agence Nationale pour la Recherche, the AXA Chair for Longevity Research, European Commission (Apo-Sys, ArtForce, ChemoRes, ApopTrain), Fondation Bettencourt-Schueller, Fondation pour la Recherche Me´dicale, Institut National du Cancer and Cance´ropoˆle Ile-de-France. LZ is supported by Ligue contre le Cancer (e´quipe labellise´e), Fondation pour la Recherche Me´dicale, INFLACARE EU grant 2008, INCa and Fondation de France (2009-2011).


PhD ,* † § Dalil Hannani

PhD ,* † § II Antonella Sistigu

PhD Oliver Kepp

PhD Lorenzo Galluzzi

MD Guido Kroemer

¶l

MD Laurence Zitvogel
email: zitvogel@igr.fr.

Prerequisites for the Antitumor Vaccine-Like Effect of Chemotherapy and Radiotherapy

Keywords: Calreticulin, HMGB1, NLRP3, TLR4, ATP, dendritic cells

For a long time, anticancer therapies were believed to work (and hence convey a therapeutic benefit) either by killing cancer cells or by inducing a permanent arrest in their cell cycle (senescence). In both scenarios, the efficacy of anticancer regimens was thought to de-pend on cancer cellYintrinsic features only. More recently, the impor-tance of the tumor microenvironment (including stromal and immune cells) has been recognized, along with the development of therapies that function by modulating tumor cellYextrinsic pathways. In particu-lar, it has been shown that some chemotherapeutic and radiotherapeutic regimens trigger cancer cell death while stimulating an active immune response against the tumor. Such an immunogenic cell death relies on the coordinated emission of specific signals from dying cancer cells and their perception by the host immune system. The resulting tumor-specific immune response is critical for the eradication of tumor cells that may survive therapy. In this review, we discuss the molecular mechan-isms that underlie the vaccine-like effects of some chemotherapeutic and radiotherapeutic regimens, with particular attention to the signaling pathways and genetic elements that constitute the prerequisites for im-munogenic anticancer therapy.

IMMUNOGENIC TUMOR CELL DEATH

Although the armamentarium of anticancer therapies is being constantly ameliorated, the number of people succumbing to cancer has been predicted to drastically rise in the future (www.who.int/cancer). This trend basically reflects the facts that efficient therapies for some prominent neoplasms such as lung cancer (which now is the leading cause of cancer-related deaths worldwide) are still missing and that current anticancer regi-mens are often associated with the insurgence of resistance and therapeutic failure. [START_REF] Zitvogel | The anticancer immune re-sponse: indispensable for therapeutic success?[END_REF] To counteract this dreadful tendency, further insights into the molecular mechanisms underlying the resistance of cancer cells to conventional therapy and into the signaling pathways that govern the host-tumor crosstalk are urgently awaited. This type of information will allow not only for the refinement of the current therapeutic arsenal, but also for a better stratification of cancer patients and the de-velopment of personalized anticancer therapies.

The current clinical approach to cancer most frequently involves surgery (whenever possible) alone or in association with a single-agent or combinatorial treatment based on che-motherapy or radiotherapy. Intriguingly, some cancers can be cured by conventional regimen (such as breast, colon, testicular, prostate, head and neck cancers, Hodgkin and follicular lym-phoma, etc.), whereas others still remain a major medical chal-lenge (such as lung and pancreatic cancers), suggesting that the intrinsic properties of the tumor and/or the specificity of the cytotoxic drugs matter. Minimal residual disease or incom-plete eradication of tumor (stem) cells associated with the arousal of chemoresistant and/or radioresistant metastases [START_REF] Zitvogel | The anticancer immune re-sponse: indispensable for therapeutic success?[END_REF] questioned the bases of our current reasoning. Thus, to improve the clinical outcome of anticancer therapies, it is of the utmost importance to understand how therapy-resistant tumor cells can be efficiently targeted and how therapeutic failure can be predicted.

For a long time, the field of clinical oncology was domi-nated by the notion that efficient anticancer therapies would work exclusively on tumor cells, either by inducing their apo-ptotic demise (cytotoxicity) and immunologically silent clear-ance or by permanently arresting their cell cycle progression (cytostasis). Moreover, several anticancer compounds were known to induce different degrees of immunosuppression, reinforcing the belief that the host immune system plays no role in the fight against transformed cells. Even the official guidelines for-mulated in 1975 by the National Institutes of Health recom-mended that the efficacy of novel anticancer strategies should be evaluated on human cancers xenotransplanted in immuno-deficient mice. [START_REF] Zitvogel | Immunological aspects of cancer chemotherapy[END_REF] More recently, it has been shown that (i) cancer cells engage in a strict crosstalk with their microenvironment (the tumor stroma, including fibroblasts and endothelial and immune cells such as macrophages) and that this interaction can be specifically targeted to induce tumor regression [START_REF] Shao | Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1[END_REF] ; that (ii) highly efficient anticancer regimens can kill tumor cells through nonapoptotic cell death subroutines [START_REF] Okada | Pathways of apoptotic and non-apoptotic death in tumour cells[END_REF] ; and that (iii) apoptosis can also occur in an immunogenic fashion, leading to the elici-tation of an anticancer immune response. [START_REF] Casares | Caspase-dependent immu-nogenicity of doxorubicin-induced tumor cell death[END_REF] Such an immunogenic cell death (ICD) involves the trans-fer of tumor-derived antigens to immune cells that stimulate a tumor-specific immune response. This is critical for the eradi-cation of residual cancer (stem) cells as it operates irrespective of their resistance to therapy. [START_REF] Zitvogel | Immunological aspects of cancer chemotherapy[END_REF] Experiments in suitable animal as genetic interventions whereby BAX, BAK, and/or caspase 8 are removed or depleted, blocks CRT exposure and abolishes the tumor-vaccinating effect of cells undergoing ICD. [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF][START_REF] Panaretakis | Mechanisms of pre-apoptotic calreticulin exposure in cell death[END_REF] Third, approximately 5% to 10% of the endogenous CRT pool is exposed together with ERp57 at the surface of dying cells via SNAP and NSF attachment receptor (SNARE)Y dependent exocytosis. This occurs well before plasma mem-brane permeabilization (which occurs as the final step of apoptosis) and also precedes the translocation of phosphati-dylserine (PS) from the inner to the outer leaflet of the plasma membrane. Phosphatidylserine is the prototypic eat-me sig-nal of apoptotic cells (although it has been implicated also in nonapoptotic cell death), [START_REF] Kroemer | Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009[END_REF][START_REF] Vandenabeele | Molecular mech-anisms of necroptosis: an ordered cellular explosion[END_REF] and the kinetics of its exposure might affect the switch between the silent removal of dying cells by macrophages and the initiation of a cognate immune response by DCs. [START_REF] Kepp | Molecular determinants of immu-nogenic cell death elicited by anticancer chemotherapy[END_REF] The receptor that is responsible for antigen uptake by DCs upon CRT binding remains to be deter-mined. Possible candidates include the major CRT receptor CD91 as well as other CRT-interacting proteins such as scav-enger receptor A, scavenger receptor expressed on endothelial cell I, 22 CD40 ligand, tumor necrosis factorYrelated apoptosis-inducing ligand (tumor necrosis factorYrelated apoptosis in-ducing ligand), or CD95/FAS ligand. [START_REF] Duus | Interaction of calreticulin with CD40 ligand, TRAIL and Fas ligand[END_REF] The CRT-driven uptake of tumor antigens by DCs is per se insufficient to elicit an anti-tumor immune response as internalized antigens must be pro-cessed and re-exposed for the cross-priming of CD4+ and CD8+ T lymphocytes. This implies that other signaling path-ways are involved in ICD.

A systematic study of the response to CDAMPs of dis-tinct Toll-like receptors (TLRs) on naive T cells revealed that TLR4 is both required and sufficient for efficient antigen pre-sentation by DCs. [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] Among other proteins, TLR4 binds the nonYhistone chromatinYbinding nuclear protein high-mobility group box 1 (HMGB1), leading to the activation of the down-stream effector myeloid differentiation primary response 88 (MYD88). [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] This inhibits the fusion between lysosomes and antigen-containing phagosomes, thus facilitating antigen pro-cessing and presentation to T cells. High-mobility group box 1 also stimulates the neosynthesis of proYIL-1A [START_REF] Andersson | High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes[END_REF] but per se does not serve as a DC maturation signal. [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] For a long time, HMGB1 has been thought to exert a proinflammatory function exclu-sively during necrosis, [START_REF] Scaffidi | Release of chromatin protein HMGB1 by necrotic cells triggers inflammation[END_REF] but recent evidence indicates that it also gets released during the late stages of apoptosis. [START_REF] Bell | The extracellular release of HMGB1 during apoptotic cell death[END_REF] The re-lease of HMGB1 from tumor cells succumbing to ICD can be blocked by Z-VADfmk and hence depends on the activation of caspases. [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF][START_REF] Bell | The extracellular release of HMGB1 during apoptotic cell death[END_REF] This process manifests with a dual kinetics whereby HMGB1 first translocates from the nucleus to the cy-toplasm and then, following the breakdown of the plasma membrane, gets released into the extracellular space. [START_REF] Tesniere | Immunogenic death of colon cancer cells treated with oxaliplatin[END_REF] Further insights into the molecular mechanisms that underlie HMBG1 release are missing. However, the addition of recombinant CRT or HMGB1 to dying cancer cells does not suffice to stimulate the presentation of tumor antigens by DCs, [START_REF] Gardai | Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte[END_REF][START_REF] Sancho | Identification of a dendritic cell receptor that couples sensing of necrosis to immunity[END_REF] implying that additional signals are required for the immunogenicity of cell death.

The vaccine-like effect of ICD relies on the elicitation of an IFN-FYpolarized T-cell response, which in turn requires the function of the NLRP3 inflammasome, a multiprotein caspase 1Yactivating complex [START_REF] Kepp | Mitochondrial control of the NLRP3 inflammasome[END_REF] Caspase 1 activation is critical for acti-vating an antitumor immune response as it catalyzes the pro-teolytic maturation of IL-1A. [START_REF] Franchi | The inflammasome: a caspase-1Yactivation platform that regulates immune responses and disease pathogenesis[END_REF] One of the most abundant factors that activate the NLRP3 inflammasome is ATP, [START_REF] Latz | The inflammasomes: mechanisms of activation and function[END_REF] andVat least in DCsVit does so by binding to the purinergic P2RX7 recep-tor on the cell surface. [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1betaYdependent adaptive immunity against tumors[END_REF] ATP also constitutes a CDAMP, as it gets released during the final steps of cell death, possibly via voltage-gated hemichannels of the pannexin 1 or connexin type. [START_REF] Chekeni | Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis[END_REF] Accordingly, the depletion of intracellular or extracel-lular ATP in cells succumbing to ICD abolishes the develop-ment of an IFN-FYpolarized response, and P2RX7-deficient mice fail to mount an immune response against syngenic can-cer cells succumbing to ICD. [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1betaYdependent adaptive immunity against tumors[END_REF] Intriguingly, ATP also serves as a ''find me'' signal for the attraction of immune cells. [START_REF] Elliott | Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[END_REF] Alto-gether, these observations highlight the multifaceted and criti-cal role of ATP for the vaccine-like effects of ICD inducers.

SPATIOTEMPORAL CODE

The spatially and temporally regulated emission of immu-nogenic factors from dying tumor cells accounts for the re-cruitment and activation of immune cells to tumor bed and governs the immune response to cancer cells undergoing ICD (Fig. 1). Thus, the stress conditions that cancer cells confront during chemotherapy and radiotherapy determine whether the subsequent wave of cell death will elicit an antitumor immune response or rather will remain immunologically silent. Normally, cells attempt to cope with stress by arresting normal activities and by activating a series of cytoprotective mechanisms that aim at reestablishing homeostasis. For instance, stressed cells normally arrest protein synthesis, activate DNA repair path-ways, and up-regulate factors for the handling of unfolded pro-teins as well as antioxidant defenses. This is accompanied by alterations of the surface proteome that, in the case of ICD, account for the recognition by immune cells and by the emis-sion of soluble mediators with chemotactic and antichemotactic properties. This is crucial for the ''selection'' and differentiation/ maturation of engulfing cells, which in turn dictates the immu-nogenic or tolerogenic outcome of cell death.

In this sense, the exposure of the DC-specific eat-me sig-nal CRT (well before that of PS) paralleled by the disclosure of other, hitherto uncharacterized ''don't eat me'' signals (such as CD47 35Y37 ) facilitates the recognition and uptake of dying tumor cells by DCs rather than by macrophages. Other authors have described additional molecular components exposed by dying cells that should be recognized for engulfment by specific DC subsets (such as CLEC9A/DNGR1 and HSP70/90) to elicit adaptive immune responses. [START_REF] Sancho | Identification of a dendritic cell receptor that couples sensing of necrosis to immunity[END_REF][START_REF] Spisek | Bortezomib enhances dendritic cell (DC)-mediated induction of immunity to human myeloma via exposure of cell surface heat shock protein 90 on dying tumor cells: therapeutic implications[END_REF][START_REF] Sancho | Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin[END_REF] However, additional stimuli released by dying tumor cells in the proximity of DCs are indispensable for the induction of a tumor-specific immune response. Thus, HMGB1 and ATP facilitate antigen processing/presentation and the release of IL-1A, which is necessary for IFN-FYpolarized T-cell responses. Based on these observations, the spatially restricted and temporally ordered appearance of CRT, HMGB1, and ATP might constitute a ''key'' that would precisely fit into a series of pattern recognition receptors expressed by DCs (the ''lock'') for the conversion of nonimmunogenic into ICD and for the elicitation of an anti-cancer immune response. [START_REF] Zitvogel | Decoding cell death signals in inflam-mation and immunity[END_REF]

THE PERCEPTION OF IMMUNOGENIC CELL DEATH BY THE IMMUNE SYSTEM

Oncogenesis is a multistep mechanism that also involves an escape from immunosurveillance; that is, often cancer cells that sustain tumor growth are poorly immunogenic (immu-noselection) and/or they actively inhibit immune functions (immunosubversion). [START_REF] Zitvogel | Cancer despite immunosurveillance: immunoselection and immunosubversion[END_REF] In this context, there are (at least) 2 different pathways whereby the immune system can be re-cruited against tumors: via direct immunomodulatory thera-pies that relieve immunosuppression or indirectly upon the induction of ICD.

By shaping T-cell responses, DCs are the first-line deci-sion makers of the innate immune system, and their role in immunogenic chemotherapy has been deeply investigated. Ex-periments in transgenic mice that express the diphtheria toxin receptor under the control of a DC-specific promoter (allowing for in vivo DC depletion) [START_REF] Jung | In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens[END_REF] revealed the essential role of DCs in the perception and decoding of ''come and get me'' signals emitted by dying tumor cells during ICD. [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] Similarly, the in vivo depletion of CD8+ T cells with specific antibodies has been instrumental to highlight the critical role of this lympho-cyte subset for the vaccine-like effect of chemotherapy and radiotherapy in a large panel of murine tumor models, including CT26 colon cancer, EL4 thymomas, TS/A mammary carci-nomas, MCA205 fibrosarcomas, Glasgow osteosarcoma osteosarcomas, [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 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] and spontaneous methylcholanthrene-induced sarcomas. [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF][START_REF] Mattarollo | Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors[END_REF] In line with these observations, CD8+ T cells have been shown to mediate potent anticancer immune effects in clinical settings, for instance, in colorectal tumors, where im-mune infiltration might serve as a prognostic factor. [START_REF] Pages | Immune infiltration in human tumors: a prognostic factor that should not be ignored[END_REF] Moreover, it has recently been shown that a precise orchestration of the T-cell response is required for immune effectors to eradicate tumors. [START_REF] Ma | Contribution of IL-17Yproducing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF] In this context, the IL-1AYdependent activation of IL-17Ysecreting F/C T cells had to precede the infiltration of tumors by Tc1 lymphocytes for the efficacy of immunogenic chemotherapy in vivo. [START_REF] Ma | Contribution of IL-17Yproducing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF] Thus, a finely regulated crosstalk be-tween components of the innate (DCs) and cognate (F/C and CD8+ T cells) immune system is required for cell death to be perceived as immunogenic, for the elicitation of an anticancer immune response, and for complete tumor eradication leading to therapeutic success (Fig. 2). However, how resident macrophages and/or adverse inflammatory monocytes, which contribute to the proangiogenic and protumoral microenvironment and dominate the scenario before chemotherapy, become ''transformed'' and/or ''overruled'' by a subset of antigen presenting cells capable of eliciting a protective anticancer responses in the context of ICD remains to be established.

IMMUNOGENIC ANTICANCER CHEMOTHERAPY

The current definition of immunogenic chemotherapy is based on the ability of a limited array of antineoplastic drugs to elicit ICD rather than the stereotypical, immunologically silent or even tolerogenic apoptotic pathway. A plethora of precli-nical 44Y51 and clinical [START_REF] Hirschowitz | Autologous dendritic cell vaccines for non-small-cell lung cancer[END_REF] studies revealed that DCs can take up apoptotic tumor cells and cross-present the internalized anti-gens on major histocompatibility complex class I molecules to CD8+ T cells, thus eliciting a productive immune response.

These studies suggest that the immunogenic outcome of cell death is influenced (among other factors) by the nature of the tumor cell as well as by the type of cell death inducer. In this context, a wide arsenal of stimuli including ER stressors (thapsigargin, tunicamycin, brefeldin), lysosometargeting agents (bafilomycin A1), mitochondrion-targeting compounds (arse-nite, betulinic acid, ceramide), proteasome inhibitors (MG132, lactacystin, ALLN), and DNA damaging molecules (Hoechst 33 342, camptothecin, etoposide, mitomycin) is per se not immu-nogenic. [START_REF] Casares | Caspase-dependent immu-nogenicity of doxorubicin-induced tumor cell death[END_REF] Conversely, some cytotoxic chemicals that are currently used for anticancer therapy such as anthracyclines, oxaliplatin (but not cisplatin), and cyclophosphamide induce a type of cell death that is immunogenic, yet is accompanied by all known biochemical and morphologic hallmarks of apoptosis. [START_REF] Obeid | Ecto-calreticulin in immu-nogenic chemotherapy[END_REF][START_REF] Schiavoni | Cyclophosphamide synergizes with type I interferons through systemic dendritic cell reactivation and induction of immunogenic tumor apoptosis[END_REF] Tumor cells that have been killed in vitro with such chemothera-peutic agents elicit a vaccine-like effect when they are injected subcutaneously into immunocompetent mice. This leads to the long-term protection of mice against subsequent rechallenges with live tumor cells of the same type. Cancer cells respond to DNA damaging agents (which constitute an important class of clinically used chemotherapeutics) with a complex signaling pathway that either allows for DNA repair (if the damage is lim-ited) or engages apoptotic mechanisms (if the damage is exces-sive). [START_REF] Pallis | DNA repair pathways and their implication in cancer treatment[END_REF] Prominent players of the DNA damage response include the ataxia telangiectasia mutated (ATM) and the ATM-related kinases, checkpoint kinases 1 and 2 (CHK1 and CHK2), [START_REF] Vitale | Mitotic catastrophe: a mechanism for avoiding genomic instability[END_REF] and the tumor suppressor protein TP53. [START_REF] Galluzzi | Targeting p53 to mitochondria for cancer therapy[END_REF] Beyond their role in the DNA damage response, ATM and CHK1 are known to induce the expression of natural killer (NK) cell group 2D (NKG2D) ligands, thus sensitizing tumor cells to NKmediated lysis. 58Y61 In addition, TP53 might mediate NKG2D ligand-independent immunogenic effects by inducing cell senescence, a status that has been surmised to be recognized by NK cells and macro-phages, leading to tumor eradication. [START_REF] Xue | Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas[END_REF] Recently, multiple chemotherapeutic agents have been shown to up-regulate the expression of mannose-6-phosphate receptors on the surface of tumor cells, thereby promoting a perforin-independent in-crease in the permeability to granzyme B released by CD8+ lymphocytes. [START_REF] Ramakrishnan | Chemotherapy enhances tumor cell susceptibility to CTL-mediated killing during cancer immu-notherapy in mice[END_REF] Altogether, these observations suggest that mul-tiple anticancer agents that are currently used in the clinical set-ting induce or at least facilitate ICD. Some chemotherapeutic agents improve anticancer immu-nity by exerting direct immunomodulatory effects. For instance, cyclophosphamideVwhich is widely used against lymphomas and leukemiaVselectively reduces the frequency of tumor-induced regulatory T cells, [START_REF] Ghiringhelli | Metronomic cyclophospha-mide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients[END_REF] induces the differentiation of T H 17 cells, [START_REF] Viaud | Cyclophosphamide induces dif-ferentiation of T H 17 cells in cancer patients[END_REF] enhances the long-term survival and prolifera-tion of lymphocytes, [START_REF] Proietti | Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice[END_REF][START_REF] Schiavoni | Cyclophosphamide induces type I interferon and augments the number of CD44(hi) T lymphocytes in mice: implications for strategies of chemoimmunotherapy of cancer[END_REF] and resets DC homeostasis. 54,68Y71 Along similar lines, imatinibVa tyrosine kinase inhibitor used in bcr/abl and Kit-induced malignanciesVactivates NK-dependent antitumor effects in mouse models [START_REF] Borg | Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects[END_REF] and stimu-lates the NKmediated secretion of IFN-F in patients with gastrointestinal stromal tumor, thus improving longterm sur-vival. [START_REF] Menard | Natural killer cell IFN-gamma levels predict long-term survival with imatinib mesylate therapy in gastroin-testinal stromal tumor-bearing patients[END_REF] Thus, several chemotherapeutic agents provide thera-peutic benefits not only via tumor cellYintrinsic, on-target effects but also by modulating immune responses in an off-target fashion.

RADIOTHERAPY AS A POTENT ANTICANCER VACCINE INDUCER

Focused ionizing radiations induce cancer cell death upon the induction of DNA damage and the overgeneration of re-active oxygen species. [START_REF] Shao | Targeted cytoplasmic irradia-tion induces bystander responses[END_REF] For many years, the direct cytotoxic effect of radiotherapy has been considered as the sole determi-nant of its therapeutic success. However, multiple lines of evi-dence have accumulated suggesting that the therapeutic effects of radiotherapy cannot be accounted for by tumor cell death alone and hence might dependVat least in partVnot only on endothelial cells but also on the host immune system. [START_REF] Formenti | Systemic effects of local radiotherapy[END_REF][START_REF] Formenti | Effects of chemoradiation on tumor-host in-teractions: the immunologic side[END_REF] Thus, radiotherapy appears to be more efficient in immuno-competent mice than in their immunodeficient counterparts. [START_REF] Meng | Successful combination of local CpG-ODN and radiotherapy in malignant glioma[END_REF] Moreover, the irradiation of primary tumors is known to in-hibit the growth of nonirradiated metastases that are localized at distant sites (a phenomenon known as ''abscopal effect''). [START_REF] Formenti | Systemic effects of local radiotherapy[END_REF] In line with this notion, the irradiation of primary 4T1 tumors (mouse breast cancer that spontaneously metastasizes) induced a CD8+ T cellYmediated immune response that controlled the growth of lung micrometastases when combined with an in-hibitor of cytotoxic T lymphocyteYassociated antigen 4 (CTLA-4 receptors (to overcome tumor-induced T-cell tolerance). [START_REF] Demaria | Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer[END_REF] Recently, these findings have been corroborated in other mu-rine models of cancer, namely, in TSA-derived breast can-cer and MCA-38Yderived colon carcinoma. In these settings, fractioned radiotherapy (but not single dose) combined to a CTLA-4Yblocking antibody led to potent abscopal effects that were paralleled by the production of consistent levels of IFN-F. [START_REF] Dewan | Fractionated but not single-dose radiotherapy induces an immunemediated abscopal effect when combined with antiYCTLA-4 antibody[END_REF] Importantly, the frequency of CD8+ T cells elicited by radiotherapy appears to correlate with the intensity of the abscopal effect. Interestingly, radiotherapy influences the chemokine pattern of the tumor microenvironment (promot-ing CXCL16 secretion by irradiated tumor cells), facilitating the entry of effector CD8+CXCR6+ T cells into irradiated tu-mor beds. [START_REF] Matsumura | Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells[END_REF] Altogether, these studies demonstrate that radiotherapy induces a T cellYdependent antitumor effect, by inducing and/or recruiting tumor-specific T cells into the tumor bed. [START_REF] Schaue | T-cell responses to survivin in cancer patients undergoing radiation therapy[END_REF][START_REF] Lugade | Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor[END_REF] Recently, we have demonstrated that tumor cells succumbing upon irradiation elicit a cognate tumor-specific response when injected subcutaneously into syngenic mice, thereby exerting a vaccine-like effect and protecting mice against subsequent chal-lenges with living tumor cells of the same type. [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF][START_REF] Obeid | Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC lightYinduced apoptosis[END_REF] Irradiation-induced ICD, which might account for, at least part of, the abscopal effect, [START_REF] Ma | Chemotherapy and radiotherapy: cryptic anticancer vaccines[END_REF][START_REF] Ma | How to improve the immunogenicity of chemotherapy and radiotherapy[END_REF] also turned out to rely on the preapoptotic exposure of CRT and the TLR4/Myd88 pathway (see above). Alternatively, it has been suggested that dormant antitumor im-munity might get reactivated by radiotherapy-induced inflammation and cytokine release, which together would trigger the recruitment of T cells into the tumor bed. [START_REF] Matsumura | Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells[END_REF][START_REF] Matsumura | Up-regulation of the pro-inflammatory che-mokine CXCL16 is a common response of tumor cells to ionizing ra-diation[END_REF] Irrespective of the fact that the molecular mechanisms underlying the abscopal ef-fect remain poorly understood, radiotherapy appears as a potent trigger of ICD.

GENETIC BACKGROUND AND CLINICAL OUTCOME

During ICD, TLR4 and P2RX7 on DCs are critical for sensing and decoding the immunogenic message conveyed by the release of HMGB1 and ATP, respectively. [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1betaYdependent adaptive immunity against tumors[END_REF][START_REF] Zitvogel | Decoding cell death signals in inflam-mation and immunity[END_REF][START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] Both TLR4 and P2RX7 present several nonsynonymous single nucleotide polymorphisms, [START_REF] Sluyter | Glu496 to Ala polymorphism in the P2X7 receptor impairs ATP-induced IL-1 beta release from human monocytes[END_REF][START_REF] Arbour | TLR4 mutations are associated with endotoxin hyporesponsiveness in humans[END_REF] and loss-of-function TLR4 and P2RX7 mutants (Asp299Gly and Gly496Ala, respectively) display low ligand-binding affinity. In line with the fact that TLR4 plays a critical role during ICD (see above), the TLR4 Asp299Gly allele as been shown to negatively affect the progression-free survival of breast cancer patients who received anthracycline-based adjuvant chemotherapy [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] as well as of patients bearing colorectal cancers that were treated with oxaliplatin-based regi-mens. [START_REF] Tesniere | Immunogenic cancer cell death: a key-lock paradigm[END_REF] Furthermore, among breast cancer patients who carried wild-type TLR4, the P2RX7 Gly496Ala allele was associated with shortened progression-free survival. [START_REF] Ghiringhelli | Activation of the NLRP3 inflammasome in dendritic cells induces IL-1betaYdependent adaptive immunity against tumors[END_REF] These results strongly suggest that the defect in the molecular mechanisms by which ICD is perceived by the immune system limits the efficacy of anticancer chemotherapy. This also provides further support to the concept that anticancer immune responses and hence all the genetic and environmental factors that affect such responsesVare crucial for therapeutic success.

In view of these considerations, it is tempting to specu-late that detailed information on the patient and tumor genetic background would allow for the design of tailored anticancer regimens with optimal efficacy and limited adverse effects. In particular, such information might (at least partially) predict the proficiency of a tumor to undergo ICD and elicit a cog-nate immune response and, if required, suggest the develop-ment of interventions aimed at restoring the immunogenicity of cell death. For instance, defects in the ER stress module that is required for CRT exposure during CRT might be cor-rected by the direct absorption of recombinant CRT to the tumor. [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF] Along similar lines, TLR4 loss-of-function mutations (which result in the deficient perception of HMGB1-conveyed signals) might be compensated by combining chemotherapy with alternative TLR agonists or with lysosomal inhibitors such as chloroquine, [START_REF] Apetoh | Toll-like receptor 4Ydependent contribution of the immune system to anticancer chemotherapy and ra-diotherapy[END_REF] whereas defects in P2RX7 signaling might be reverted by the administration of exogenous IL-1A 10 or apyrase inhibitors.

Alternative approaches for the elicitation of an antican-cer immune response focus on the reversal of tumor-induced tolerance by means of immunomodulatory agents such as mo-noclonal antibodies targeting suppressive pathways (such as CTLA-4, PD-1, Lag3, Tim-3) or engaging activating recep-tors (such as CD40, CD27, 4-1BB), cytokines, and cell based-approaches (T and DC) in combination with conventional therapies. 89Y91 In preclinical models, these strategies have been shown to enhance the vaccine-like effect of both chemothera-peutic and radiotherapeutic regimens, thereby constituting pro-mising approaches. [START_REF] Agarwala | Current experience with CTLA4-blocking mono-clonal antibodies for the treatment of solid tumors[END_REF][START_REF] Dewan | Fractionated but not single-dose radiotherapy induces an immunemediated abscopal effect when combined with antiYCTLA-4 antibody[END_REF] Gulley and colleagues [START_REF] Gulley | Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer[END_REF] reported that, in prostate cancer patients, the combination of radiotherapy with an admixture of a recombinant vaccine against prostate-specific antigen (PSA) and B7.1 costimulatory molecules in-duced a significant increase in PSA-specific and MUC1-specific T cells. The expansion of MUC1-specific T cells indicates that a tumor antigen cross-priming occurs in vivo following radio-therapy. Brody and colleagues reported a phase I/II trial where low-dose radiotherapy combined with in situ TLR9 agonists induced lymphoma remission, not only at the site of the treated lesion but also at distant sites, associated with detectable an-ticancer T-cell responses. [START_REF] Brody | In situ vaccination with a TLR9 agonist induces systemic lymphoma regression: a phase I/II study[END_REF] Taken together, these studies suggest that efficient anti-cancer regimens should combine immunogenic chemotherapy or radiotherapy with immunomodulatory agents that overcome tumor-induced immunosuppression. Moreover, whenever re-quired, the defects in the molecular machinery for the execution and perception of ICD should be compensated to obtain the complete eradication of tumors and long-term tumor-free sur-vival (Fig. 3).

FIGURE 1 .

 1 FIGURE 1. Immunogenic signals emitted by dying cells form a spatiotemporal code unlocking DCs to mount a potent immune response toward tumor cells. (i) Early exposure of ecto-CRT by dying tumor cells, which facilitates engulfment by DCs. (ii) HMGB1 released from dying cells binds to TLR4 on DCs, thus favoring antigen cross-presentation and up-regulating proYIL-A (proYIL-1A). (iii)ATP liberated from dying cells binds to the purinergic receptor P2RX7 on DCs, activates the NLRP3 inflammasome, and leads to the secretion of active IL-1A, which polarizes CD8+ T cells toward IFN-F production. (iv) An additive DC maturation factor remains to be characterized.

FIGURE 2 .

 2 FIGURE 2. Precise orchestration of antitumor T-cell responses elicited following ICD: After (immunogenic) chemotherapy, tumor material is phagocytosed by DCs. These later are also activated by ICD signals emitted by dying tumor cells. Within 2 days, IL-17Yproducing FC T cells are recruited to the tumor bed in an IL-1AYdependent manner. Their arrival precedes and correlates with the IFN-FYproducing CD8 + T cells infiltration, which is critical for tumor eradication.

FIGURE 3 .

 3 FIGURE 3. Schematic view of tailored anticancer regimens: To achieve therapeutic success, (i) the anticancer regimen should be able to induce ICD, that is, to induce an ER stress before cell death and ATP release. Nonimmunogenic cytotoxic drug can be combined with ER stressors to restore immunogenicity. (ii) The tumor of the patient should have conserved the intrinsic capacities to emit all the immunogenic signals. If not, the defective signals could be identified and then compensate by recombinant CRT or rIL-1A or ATP superagonists. (iii) The loss-of-function mutation of key receptors involved in the perception of ICD signals might also be compensated by triggering alternative TLR pathway or supplementation with the appropriate cytokine. (iv) The combination of immunotherapy and immunogenic chemotherapy enhances the vaccine-like effect of chemotherapy and radiotherapy by overcoming the tumor-induced immunosuppression. Thus, tailored anticancer regimens should be designed by taking into account the genetic background of both the tumor and the host, with an aim to correctly unlock the immune system to obtain the complete eradication of the tumor and long-term tumor-free survival.

PERSPECTIVES

As we have discussed above, some (but not all) chemo-therapeutic and radiotherapeutic regimens induce the immu-nogenic death of tumor cells that, in specific circumstances, lead to the elicitation of a potent anticancer immune response. This vaccine-like effect is critical for both therapeutic success and long-term tumor-free survival. The ability of anticancer drugs to induce an ER-stress response that precedes cell death, the intrinsic capacity of tumor cells to emit immunogenic CDAMPs in a defined spatiotemporal order, and the ability of the host to perceive these signals and to overcome tumor-induced immunosuppression appear as fundamental prerequisites for the vaccine-like effect of radiotherapy or chemotherapy. We believe that future anticancer regimens should be tailored to each pa-tient and tumor's genetic background to take into account all these elements, as this will result in combination therapies with optimal cytotoxic and immunogenic profiles and limited adverse effects.

Conflicts of Interests and Sources of