Consensus guidelines for the detection of immunogenic cell death

none82siApoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.Kepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G; Cirone, Mara; Colombo, Maria I; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G; Faggioni, Alberto; Formenti, Silvia C; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong-Min; Huang, Xing; Illidge, Tim M; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V; Loi, Sherene; Lowenstein, Pedro R; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean-Ehrland; Riganti, Chiara; Rovere-Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T; Yang, Haining; Zong, Wei-Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, LorenzoKepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G; Cirone, Mara; Colombo, Maria I; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G; Faggioni, Alberto; Formenti, Silvia C; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong Min; Huang, Xing; Illidge, Tim M; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V; Loi, Sherene; Lowenstein, Pedro R; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean Ehrland; Riganti, Chiara; Rovere Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T; Yang, Haining; Zong, Wei Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenz

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches

Mécanismes de l’immunogénicité des cellules cancéreuses hyperploïdes

An at least transient increase of ploidy, usually by whole genome duplication, is a frequent event in oncogenesis. Hyperploidization triggers an endoplasmic reticulum (ER) stress characterized by the hyperphosphorylation of the eukaryotic initiation factor 2α (eIF2α), with leads to the exposure of calreticulin to the cell surface. Calreticulin is acting like an "adjuvant" effect as a "eat me" signal, for dendritic cells, ultimately eliciting immune control by CD8 + T lymphocytes. We investigated the possibility that hyperploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. However, vaccination with candidate peptides was unable to elicit tumor growth-inhibitory responses against hyperploid cells in vivo. We conclude that hyperploidy increases the immunogenicity of cancer cells mostly through their adjuvanticity (through calreticulin exposure) rather than their antigenicity. We also show that when developed in Rag2 - / - γc - / - immunodeficient mice (but not in WT mice) fibrosarcomas induced by methylcholanthrene (MCA) are particularly hyperploid, correlating with higher DNA content, increased nuclear surface, as well as the hyperphosphorylation of eIF2α. These cells are able to form tumors in Rag2 - / - γc - / - recipients (which lack T, B and NK cells) as well as in Rag2 - / - recipients (which only lack T and B lymphocytes) without any difference between the two strains, suggesting that the absence of B and T cells is enough to allow these cells to grow. To measure these parameters, we developed a morphometric analysis tool that is applicable to immunohistochemistry. This software automatically identifies and quantifies the surface of nuclei and the intensity of eIF2α phosphorylation within a perinuclear region of interest. Comparative analyzes validated the accuracy of this method, which can be used to investigate ploidy and ER stress in cancers in situ.Une augmentation au moins transitoire de la ploïdie est un événement fréquent de l'oncogenèse. L'hyperploïdisation déclenche un stress du réticulum endoplasmique (RE) caractérisé par l'hyperphosphorylation du facteur d'initiation eucaryote 2α (eIF2α) qui induit l'exposition consécutive de la calréticuline à la surface cellulaire. La calréticuline exerce un effet « adjuvant » en tant que un signal "mange-moi", pour les cellules dendritiques, entrainant un contrôle immunitaire de la tumeur par les lymphocytes T CD8+. Nous avons étudié la possibilité que l'hyperploïdisation puisse également affecter l'antigénicité des cellules cancéreuses en modifiant l'immunopeptidome. Cependant, la vaccination avec des peptides candidats n’a pas réussi à induire des réponses inhibitrices de la croissance tumorale de cellules hyperploïdes in vivo. Nous concluons donc que l'hyperploïdie augmente principalement l'immunogénicité des cellules cancéreuses en affectant leur adjuvanticité (via l’expression de calréticuline) plutôt que leur antigénicité. Nous montrons de plus que des fibrosarcomes induits par méthylcholanthrène (MCA), développés dans des souris Rag2-/-γc-/- immunodéficientes (mais pas dans des souris sauvages), sont particulièrement hyperploïdes, en corrélation avec une teneur en ADN plus élevée, une augmentation de la surface nucléaire, ainsi que l'hyperphosphorylation de eIF2α,. De telles cellules forment des tumeurs dans les souris Rag2-/-γc-/- (dépourvues de cellules T, B et NK) ainsi que dans les souris Rag2-/- (dépourvues de lymphocytes T et B) sans différence entre les deux souches de souris, suggérant que l'absence de cellules B et T est suffisante pour que ces cellules prolifèrent. Pour mesurer ces paramètres, nous avons développé un outil d'analyse morphométrique applicable à l'immunohistochimie. Cet algorithme identifie et quantifie automatiquement la surface des noyaux et l'intensité de la phosphorylation de eIF2α dans une région d'intérêt périnucléaire. Des analyses comparatives ont validé la précision de cette méthode, qui peut être utilisée pour étudier la ploïdie et le stress du RE dans les cancers in situ

Mechanisms of immunogenicity in polyploid cancer cells

Une augmentation au moins transitoire de la ploïdie est un événement fréquent de l'oncogenèse. L'hyperploïdisation déclenche un stress du réticulum endoplasmique (RE) caractérisé par l'hyperphosphorylation du facteur d'initiation eucaryote 2α (eIF2α) qui induit l'exposition consécutive de la calréticuline à la surface cellulaire. La calréticuline exerce un effet « adjuvant » en tant que un signal "mange-moi", pour les cellules dendritiques, entrainant un contrôle immunitaire de la tumeur par les lymphocytes T CD8+. Nous avons étudié la possibilité que l'hyperploïdisation puisse également affecter l'antigénicité des cellules cancéreuses en modifiant l'immunopeptidome. Cependant, la vaccination avec des peptides candidats n’a pas réussi à induire des réponses inhibitrices de la croissance tumorale de cellules hyperploïdes in vivo. Nous concluons donc que l'hyperploïdie augmente principalement l'immunogénicité des cellules cancéreuses en affectant leur adjuvanticité (via l’expression de calréticuline) plutôt que leur antigénicité. Nous montrons de plus que des fibrosarcomes induits par méthylcholanthrène (MCA), développés dans des souris Rag2-/-γc-/- immunodéficientes (mais pas dans des souris sauvages), sont particulièrement hyperploïdes, en corrélation avec une teneur en ADN plus élevée, une augmentation de la surface nucléaire, ainsi que l'hyperphosphorylation de eIF2α,. De telles cellules forment des tumeurs dans les souris Rag2-/-γc-/- (dépourvues de cellules T, B et NK) ainsi que dans les souris Rag2-/- (dépourvues de lymphocytes T et B) sans différence entre les deux souches de souris, suggérant que l'absence de cellules B et T est suffisante pour que ces cellules prolifèrent. Pour mesurer ces paramètres, nous avons développé un outil d'analyse morphométrique applicable à l'immunohistochimie. Cet algorithme identifie et quantifie automatiquement la surface des noyaux et l'intensité de la phosphorylation de eIF2α dans une région d'intérêt périnucléaire. Des analyses comparatives ont validé la précision de cette méthode, qui peut être utilisée pour étudier la ploïdie et le stress du RE dans les cancers in situ.An at least transient increase of ploidy, usually by whole genome duplication, is a frequent event in oncogenesis. Hyperploidization triggers an endoplasmic reticulum (ER) stress characterized by the hyperphosphorylation of the eukaryotic initiation factor 2α (eIF2α), with leads to the exposure of calreticulin to the cell surface. Calreticulin is acting like an "adjuvant" effect as a "eat me" signal, for dendritic cells, ultimately eliciting immune control by CD8 + T lymphocytes. We investigated the possibility that hyperploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. However, vaccination with candidate peptides was unable to elicit tumor growth-inhibitory responses against hyperploid cells in vivo. We conclude that hyperploidy increases the immunogenicity of cancer cells mostly through their adjuvanticity (through calreticulin exposure) rather than their antigenicity. We also show that when developed in Rag2 - / - γc - / - immunodeficient mice (but not in WT mice) fibrosarcomas induced by methylcholanthrene (MCA) are particularly hyperploid, correlating with higher DNA content, increased nuclear surface, as well as the hyperphosphorylation of eIF2α. These cells are able to form tumors in Rag2 - / - γc - / - recipients (which lack T, B and NK cells) as well as in Rag2 - / - recipients (which only lack T and B lymphocytes) without any difference between the two strains, suggesting that the absence of B and T cells is enough to allow these cells to grow. To measure these parameters, we developed a morphometric analysis tool that is applicable to immunohistochemistry. This software automatically identifies and quantifies the surface of nuclei and the intensity of eIF2α phosphorylation within a perinuclear region of interest. Comparative analyzes validated the accuracy of this method, which can be used to investigate ploidy and ER stress in cancers in situ

Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer

Current preclinical models to investigate human HR + breast cancer progression and response to immunotherapy in vivo are limited. Here, the authors demonstrate that mammary tumours driven by a synthetic progestin combined with an oral carcinogen recapitulate several immunobiological features of human HR + breast cancers

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine

Autoimmunity affecting the biliary tract fuels the immunosurveillance of cholangiocarcinoma

International audienceCholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract

Caloric Restriction Mimetics Enhance Anticancer Immunosurveillance

International audienceCaloric restriction mimetics (CRMs) mimic the biochemical effects of nutrient deprivation by reducing lysine acetylation of cellular proteins, thus triggering autophagy. Treatment with the CRM hydroxycitrate, an inhibitor of ATP citrate lyase, induced the depletion of regulatory T cells (which dampen anticancer immunity) from autophagy-competent, but not autophagy-deficient, mutant KRAS-induced lung cancers in mice, thereby improving anticancer immunosurveillance and reducing tumor mass. Short-term fasting or treatment with several chemically unrelated autophagy-inducing CRMs, including hydroxycitrate and spermidine, improved the inhibition of tumor growth by chemotherapy in vivo. This effect was only observed for autophagy-competent tumors, depended on the presence of T lymphocytes, and was accompanied by the depletion of regulatory T cells from the tumor bed