Tumor Cell Death and ATP Release Prime Dendritic Cells and Efficient Anticancer Immunity

Published OnlineFirst January 19, 2010; DOI: 10.1158/0008-5472.CAN-09-3566 Published Online First on January 19, 2010 as 10.1158/0008-5472.CAN-09-3566

Review Cancer Research Tumor Cell Death and ATP Release Prime Dendritic Cells and Efficient Anticancer Immunity

Laetitia Aymeric1,2,3, Lionel Apetoh1,2,3, François Ghiringhelli1,2,3,4,5, Antoine Tesniere2,3,6, Isabelle Martins2,3,6, Guido Kroemer2,3,6, Mark J. Smyth7, and Laurence Zitvogel1,2,3

Abstract By destroying tumor cells, conventional anticancer therapies may stimulate the host immune system to elim- inate residual disease. Anthracyclines, oxaliplatin, and ionizing irradiation activate a type of tumor cell death that elicits efficient anticancer immune responses depending on interferon γ (IFNγ)andtheIFNγ receptor. Thus, dying tumor cells emit danger signals that are perceived by dendritic cells (DC), which link innate and cognate immune responses. Recently, we observed that ATP was released by tumor cells succumbing to chemotherapy. ATP activates purinergic P2RX7 receptors on DC, thus activating the NLRP3/ASC/caspase-1 inflammasome and driving the secretion of interleukin-1β (IL-1β). IL-1β then is required for the adequate polarization of IFNγ-producing CD8+ T cells. These results imply a novel danger signal, ATP, and a novel receptor, P2RX7, in the chemotherapy-elicited anticancer immune response. Cancer Res; 70(3); 855–8. ©2010 AACR.

Setting the Stage Results

Some regimens of chemotherapy mediate direct cytotoxic Components of the immune system required for thera- effects on the tumor and, in addition, elicit indirect antitumor peutic effects. Immunosurveillance of tumors involves lym- effects resulting from the immunogenicity of cell death (1, 2). phocytes and requires an intact interferon γ (IFNγ)/IFNγR Anthracyclines, oxaliplatin, and ionizing irradiation can trig- pathway (8–10). To investigate the contribution of these ger immunogenic cell death, whereas many other anticancer immune effectors in the efficacy of chemotherapy, we com- agents fail to do so. We delineated the molecular mechanisms pared chemotherapy-induced tumor growth retardation in underlying the recognition of dying tumor cells by dendritic immunocompetent mice and in mice bearing various immu- − − cells (DC) and found two major checkpoints that dictate nodefects affecting B and/or T cells [Rag / , nu/nu, or wild- the immunogenicity of cell death. First, optimal phagocytosis type (WT) mice treated with a depleting anti-CD8 mAb] or − − − − of chemotherapy or radiotherapy-treated tumor cells by the IFNγ/IFNγR system (Ifng / , IfngR / , or WT treated with DC requires the translocation of endoplasmic reticulum neutralizing anti-IFNγ antibodies). The depletion of CD8+ (ER)-resident calreticulin and disulfide isomerase ERp57 to T cells or the removal of the IFNγ/IFNγR pathway reduced the plasma membrane of dying tumor cells (3–5). Second, the therapeutic effects of oxaliplatin against CT26 colon can- the chromatin-binding high mobility group box 1 protein cer, EL4 thymoma, and abolished the antitumor effects of (HMGB1) must be released by dying tumor cells and must anthracyclines (doxorubicin or mitoxanthrone) against bind to its receptor toll-like receptor 4 (TLR4) on DC to facil- CT26 cells or MCA-induced sarcomas. In contrast, other mo- itate antigen processing of the phagocytic cargo (6). However, lecules such as IL-12Rβ2, perforin, and TRAIL were dispens- additional signals emanating from dying tumor cells are able for the efficacy of these cancer chemotherapies in the same required for the full-blown maturation and differentiation tumor models. Chemotherapy promoted a tumor-specific T-cell of DC and T cells, respectively. Therefore, we addressed the con- response that was detectable in the tumor-draining lymph tribution of damage-associated molecular patterns (DAMP) to nodes (LN), 7 to 10 days after the systemic administration the efficacy of chemotherapy in mouse tumor models (7). of oxaliplatin. After ex vivo restimulation with tumor antigens, oxaliplatin-induced CD4+ T cells mainly produced interleukin-2 (IL-2), whereas chemotherapy-induced CD8+ Authors' Affiliations: 1INSERM, U805; 2Institut Gustave Roussy; γ 3Université Paris-Sud, Villejuif, France; 4AVENIR team INSERM CRI- T cells produced high levels of IFN . These chemotherapy- 866; 5Centre Georges Francois Leclerc, Dijon, France; 6INSERM, U848, induced T-cell responses apparently resulted from local can- Villejuif, France; and 7Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia cer cell death because they were detectable in draining LN (but not distant LN) and because dying (but not living) tumor Note: G. Kroemer, M.J. Smyth, and L. Zitvogel share senior co-authorship. cells inoculated in the footpad could mimic these immune Corresponding Author: Laurence Zitvogel, U805 and CIC BT507 IN- SERM, Institut Gustave Roussy, 39 rue Camille Desmoulins, F-94805 effects (7). Villejuif, France. Phone: 33-1-42-11-50-41; Fax: 33-1-42-11-60-94; In conclusion, tumor regression promoted by oxaliplatin or E-mail: [email protected]. anthracyclines elicits a tumor-specific IFNγ-polarized CD8+ doi: 10.1158/0008-5472.CAN-09-3566 T-cell immune response, which is critical for the efficacy of ©2010 American Association for Cancer Research. chemotherapy.

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The inflammasome NLRP3 is mandatory for the efficacy lease of ATP in vitro from dying tumor cells, by 8 to 20 hours of chemotherapy. Inoculation of anthracycline or oxalipla- postexposure (7, 23). This ATP release became undetectable tin-treated tumor cells in the footpad induced a potent when cells were incubated with the ATP-degrading enzyme IFNγ-polarized CD8+ T-cell response relying on TLR4 signal- apyrase or with inhibitors of ATP synthesis such as antimycin ing in DC (6). We investigated the possible contribution of A plus deoxyglucose (A/D) or the oxidative phosphorylation other receptors for DAMPs that could account for the prim- uncoupler 2,4-dinitrophenol (DNP). When oxaliplatin-treated ing of IFNγ-producing T cells during anticancer chemother- EG7 or anthracycline-treated CT26 cells were admixed with apy. In addition to TLR, NOD-like receptors (NLR) recognize ATP scavengers (A/D, apyrase, DNP) or nonselective P2× re- DAMPs and link inflammation to innate immunity (11, 12). ceptor antagonists (iso-pyridoxalphosphate-6-azophenyl- In macrophages, the so-called “inflammasome” serves as a 2′,5′-disulphonate or oxidized ATP), they lost their capacity central sensor for DAMPs (13). The NLRP3 (CIAS1/CRYO- to elicit protective antitumor immune responses upon subcu- PYRIN)-inflammasome is a multimeric protein complex that taneous inoculation into normal mice. ATP depletion or P2× activates the protease caspase-1, which mediates the proces- receptor blockade also abolished the capacity of oxaliplatin- sing and secretion of pro-inflammatory cytokines (IL-1β, treated EG7 cells [which express the model antigen ovalbu- IL-18, IL-33). The NLRP3 inflammasome can be activated by min (OVA)] to prime OVA-specific cells for IFNγ production endogenous danger signals (DAMPs) or pathogen-associated (7). Altogether, we generalized the finding that dying tumor molecular patterns (PAMP), which all act in concert to induce cells release ATP, which is indispensable for their immunoge- K+ efflux (14–17). Gain-of-function mutations in the human nicity. In contrast, the immunizing capacity of candidate NLRP3 gene cause autoinflammatory disease that can be antigen proteins admixed in TLR adjuvants is not blocked suppressed by the systemic administration of IL-1R antago- by P2× receptor antagonists and hence is likewise indepen- nists (18, 19). To investigate the putative role of the NLRP3 dent of endogenous ATP. inflammasome in anticancer chemotherapy, we compared Because the high affinity receptor for ATP is the purinergic − − tumor growth kinetics of EL4 sarcomas treated with oxalipla- receptor P2RX7, we assessed the ability of p2rx7 / mice to tin in WT mice versus mice deficient in each component of mount a chemotherapy-induced IFNγ polarized CD8+ T-cell the inflammasome [NLRP3, its adaptor molecule apoptosis- response and to control the growth of established tumors associated speck-like protein containing a caspase recruit- after chemotherapy. Importantly, oxaliplatin failed to control − − ment domain (ASC) or caspase-1] or its final product IL-1β. tumor progression in p2rx7 / mice, and, similarly, oxalipla- Each individual player of this complex was required for the tin-treated OVA expressing EG7 could not elicit OVA-specific − − success of chemotherapy. The key contribution of IL-1β in T-cell responses in LN harvested from p2rx7 / mice. Next, the antitumor effects mediated by anthracyclines and/or ox- we showed that host DC were the cells harboring functional aliplatin was also shown in CT26 colon cancer and more P2RX7 receptors during chemotherapy. In transgenic mice importantly in spontaneous tumors induced by MCA (7). expressing the diphtheria toxin receptor on their DC (under To analyze the role of the NLRP3 inflammasome in the the control of the CD11c promoter), diphtheria toxin depletes antitumor immune response elicited by chemotherapy, we LN DC, and this maneuver readily abolishes the CD8+ T-cell inoculated live versus dying tumor cells exposed to chemo- response elicited by dying EG7 cells. The adoptive transfer of − − therapy ex vivo and determined the capacity of draining LN T bone marrow-derived WT (but not in p2rx7 / )DCloaded cells to produce IFNγ in response to tumor antigens. In these with dying EG7 restored the OVA-specific CD8+ T-cell − − circumstances, T cells from mice deficient in individual com- response in p2rx7 / mice. Accordingly, WT DC loaded with ponents of the inflammasome/IL-1β axis (NLRP3, Casp-1, oxaliplatin-treated EG7 cells in vitro produced IL-1β in a IL-1R1) failed to mount optimal IFNγ polarized CD8+ T-cell NLRP3-, ASC-, and casp-1-dependent fashion, in conditions responses. Moreover, caspase-1-deficiency resulted in defi- in which IL-12p40 secretion was independent of the NLRP3 cient IFNγ production by tumor antigen-specific CD8+ T inflammasome. The PAMP lipopolysaccharide (a TLR4 cells, whereas normal mice mounted such T-cell responses ligand) and the DAMP ATP reportedly act in concert to ignite in the draining LN of established tumors treated by systemic the NLRP3 inflammasome in macrophages (17). Accordingly, oxaliplatin injections (7). Hence, we showed that the NLRP3 we found that the TLR4 ligand HMGB1 and ATP, which are inflammasome is critical for the immunogenicity of cell both released by tumor cells exposed to cytotoxic agents, death triggered by anthracyclines, oxaliplatin, or x-rays. act in concert to promote IL-1β secretion by DC ex vivo. Key contribution of ATP and purinergic receptors. The treatment of DC with oxidized ATP or anti-HMGB1- Multiple distinct bacterial products or endogenous damage neutralizing antibodies prior to loading with dying tumor signals (such as toxins, ATP, uric acid crystals, alum, silica) cells completely abolished IL-1β secretion (7). stimulate the NLRP3 inflammasome resulting in the proteolyt- The link between inflammation and cognate immunity. ic auto-activation of caspase-1 (15, 20, 21). One of the most As discussed above, ATP released by dying tumor cells en- pleiotropic activators of the NLRP3 inflammasome is extracel- gages P2RX7 receptors on DC, thereby triggering the NLRP3 lular ATP, which is released from stressed cells and acts on inflammasome that culminates in IL-1β production and DC- purinergic receptors, mostly of the P2 × class (22). To our sur- mediated IFNγ polarized CD8+ T-cell response. How then prise, multiple distinct cell death inducers (cadmium, etopo- could IL-1β contribute to the immunogenicity of cell death? side, mitomycin C, oxaliplatin, cis-platin, staurosporine, We hypothesized that IL-1β may control and/or switch thapsigargin, mitoxanthrone, doxorubicin) induced the re- the quality of the priming of naive CD8+ T cells during the

856 Cancer Res; 70(3) February 1, 2010 Cancer Research

Immune Response against Dying Tumor Cells

treatment of a tumor by chemotherapy (7). We produced In conclusion, IL-1β contributes to the full differentiation three lines of evidence supporting this contention. of IFNγ polarized CD8+ T cells during the priming of anti- First, DC loaded with dying EG7 cells induced the polari- tumor immune responses that are elicited as a byproduct zation of naive OT-1 cells (which express a transgenic T-cell of anticancer chemotherapy. receptor that recognizes an OVA-derived peptide, SIINFEKL) into IFNγ producing lymphocytes in a caspase-1- and IL-1β- Discussion dependent manner in vitro and in vivo. IL-1β receptor antag- onist or neutralizing anti-IL-1β antibodies suppressed IFNγ Our results identify tumor-derived ATP as a new DAMP, polarization in this in vitro priming system. Similarly, OT-1 which is required for cancer cell death to be immunogenic. cells were activated (CD69 expression) upon their adoptive Our data are compatible with a scenario (Fig. 1) in which − − transfer into WT mice, but not into casp-1 / mice, following ATP activates P2RX7 receptors on DC, thereby stimulating immunization with dying EG7 cells. IL-1RA also prevented the aggregation and/or activation of the NLRP3/ASC/Casp-1 the expansion of SIINFEKL/Kb tetramer-specific T cells inflammasome, the proteolytic maturation of caspase-1, pro- in LN draining the immunization with dying EG7. Second, IL-1β cleavage, and consequent IL-1β release. IL-1β then is activation of naive CD3+ T lymphocytes by a cocktail of required for the priming of IFNγ-producing tumor antigen- anti-CD3/anti-CD28 mAb in the presence of IL-1β (but not specific CD8+ T cells (7). In accord with previous studies IL-6 or TNFα) resulted in a IFNγ polarizing effect that was (24), IFNγ, rather than perforin or TRAIL-dependent cyto- similar to the one described for IL-12 (7). Third, the failure of toxic activities, mediates the anticancer activity of T lympho- − − − − p2rx7 / and casp-1 / mice to mount OVA-specific IFNγ- cytes that have been primed in a P2RX7/NLRP3/ASC/Casp-1/ polarized CD8+ T-cell response after immunization with IL-1β-dependent fashion. oxaliplatin-treated EG7 could be overcome by recombinant New links between inflammasomes and cognate immunity IL-1β coinjected with the vaccine (7). are being progressively unraveled. Eisenbarth and colleagues

Figure 1. The integrity of the NLRP3 inflammasome is required for IFNγ polarized CD8+ T-cell response with dying tumor cells. During a hit with chemotherapy, dying tumor cells are captured and processed by host DC triggering a tumor-DC crosstalk dictated by ATP/P2RX7 and HMGB1/TLR4 molecular interactions. ATP and HMGB1 act in concert to ignite the NLRP3 inflammasome in DC, culminating in IL-1β processing and secretion in the extracellular milieu. IL-1β then contributes to the TCR-driven IFNγ polarized CD8+ T-cell response, a mandatory step for the efficacy of chemotherapy.

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(25) showed that the NLRP3 inflammasome pathway stimu- compromises the efficacy of anthracycline-based chemother- lated by aluminum-based adjuvant (alum) directs a humoral apy in breast cancer patients, implying that the pathway adaptive immune response. Mice deficient in Nlrp3, ASC,or we elucidated has clinical relevance and will presumably be caspase-1 failed to mount an antibody response to antigen clinically implemented for a better management of “person- administered with alum, yet generated normal responses alized chemotherapy.” against antigens emulsified with complete Freund's adjuvant. The Nlrp3 inflammasome was required for the production of Disclosure of Potential Conflicts of Interest IL-1β and IL-18 by macrophages in response to alum (25). It is noteworthy that in response to dying tumor cells, IL-18 was not produced by P2RX7/NLRP3-activated DC and No potential conflicts of interest were disclosed. IL-18R was not required for the immunogenicity of cell death in vivo (7). During lung infection with influenza A virus, ASC Grant Support and caspase-1 (but not NLRP3) were required for CD4+ and + CD8 protective T-cell immunity, mucosal IgA production, G. Kroemer and L. Zitvogel are supported by grants from the Ligue and systemic IgG antibody responses (26), suggesting that Nationale contre le Cancer (équipes labellisées), Fondation pour la Recherche Médicale, European Union, Cancéropôle Ile-de-France, Institut National du each immune response relies on a specific mode of inflam- Cancer, Association for International Cancer Research, and Agence Nationale masome activation and perhaps a specific pattern of inflam- pour la Recherche. M.J. Smyth is supported by the National Health and Medical masome-dependent cytokines. Research Council of Australia and the Victorian Cancer Agency. A. Tesniere is Importantly, we showed that a loss-of-function allele of supported by INSERM (poste d'accueil). P2RX7 that reduces the affinity of P2RX7 receptor for ATP Received 10/16/09; accepted 10/18/09; published OnlineFirst 1/19/10.

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858 Cancer Res; 70(3) February 1, 2010 Cancer Research

Tumor Cell Death and ATP Release Prime Dendritic Cells and Efficient Anticancer Immunity

Laetitia Aymeric, Lionel Apetoh, François Ghiringhelli, et al.

Cancer Res Published OnlineFirst January 19, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-3566

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