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• W1994826332 abstract "•STING but not MyD88 or TRIF is essential for therapeutic radiation•cGAS-STING axis mediates dendritic cell sensing of irradiated-tumor cells•STING is required for effective adaptive immune responses to radiation•Exogenous cGAMP treatment promotes antitumor efficacy of radiation Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-β induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-β treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy. Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-β induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-β treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy. Radiotherapy used alone or in combination with surgery or chemotherapy is employed to treat the primary and metastatic tumors in approximately 50%–60% of all cancer patients (Begg et al., 2011Begg A.C. Stewart F.A. Vens C. Strategies to improve radiotherapy with targeted drugs.Nat. Rev. Cancer. 2011; 11: 239-253Crossref PubMed Scopus (772) Google Scholar, Liauw et al., 2013Liauw S.L. Connell P.P. Weichselbaum R.R. New paradigms and future challenges in radiation oncology: an update of biological targets and technology.Sci. Transl. Med. 2013; 5: 173sr172Crossref Scopus (154) Google Scholar). The biological responses of tumors to radiation have been demonstrated to involve DNA damage, modulation of signal transduction, and alteration of the inflammatory tumor microenvironments (Begg et al., 2011Begg A.C. Stewart F.A. Vens C. Strategies to improve radiotherapy with targeted drugs.Nat. Rev. Cancer. 2011; 11: 239-253Crossref PubMed Scopus (772) Google Scholar, Liauw et al., 2013Liauw S.L. Connell P.P. Weichselbaum R.R. New paradigms and future challenges in radiation oncology: an update of biological targets and technology.Sci. Transl. Med. 2013; 5: 173sr172Crossref Scopus (154) Google Scholar). Indeed, radiotherapy has been recently shown to induce antitumor adaptive immunity, leading to tumor control (Apetoh et al., 2007Apetoh L. Ghiringhelli F. Tesniere A. Obeid M. Ortiz C. Criollo A. Mignot G. Maiuri M.C. Ullrich E. Saulnier P. et al.Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.Nat. Med. 2007; 13: 1050-1059Crossref PubMed Scopus (2292) Google Scholar, Lee et al., 2009Lee Y. Auh S.L. Wang Y. Burnette B. Wang Y. Meng Y. Beckett M. Sharma R. Chin R. Tu T. et al.Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment.Blood. 2009; 114: 589-595Crossref PubMed Scopus (982) Google Scholar). Based on this concept, the blockade of immune checkpoints improves the efficacy of radiotherapy on local and distant tumors in experimental systems and more recently in clinical observations (Deng et al., 2014Deng L. Liang H. Burnette B. Beckett M. Darga T. Weichselbaum R.R. Fu Y.X. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice.J. Clin. Invest. 2014; 124: 687-695Crossref PubMed Scopus (1325) Google Scholar, Postow et al., 2012Postow M.A. Callahan M.K. Barker C.A. Yamada Y. Yuan J. Kitano S. Mu Z. Rasalan T. Adamow M. Ritter E. et al.Immunologic correlates of the abscopal effect in a patient with melanoma.N. Engl. J. Med. 2012; 366: 925-931Crossref PubMed Scopus (1535) Google Scholar). Furthermore, radiotherapy sculpts innate immune response in a type I interferon (IFN)-dependent manner to facilitate adaptive immune response (Burnette et al., 2011Burnette B.C. Liang H. Lee Y. Chlewicki L. Khodarev N.N. Weichselbaum R.R. Fu Y.X. Auh S.L. The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity.Cancer Res. 2011; 71: 2488-2496Crossref PubMed Scopus (568) Google Scholar). However, the molecular mechanism for host type I IFN induction following local radiation has not yet been defined. The innate immune system is the major contributor to host-defense in response to pathogen invasion or tissue damage (Takeuchi and Akira, 2010Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (5701) Google Scholar). The initial sensing of infection and injury is mediated by pattern-recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (Chen and Nuñez, 2010Chen G.Y. Nuñez G. Sterile inflammation: sensing and reacting to damage.Nat. Rev. Immunol. 2010; 10: 826-837Crossref PubMed Scopus (2045) Google Scholar, Desmet and Ishii, 2012Desmet C.J. Ishii K.J. Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination.Nat. Rev. Immunol. 2012; 12: 479-491Crossref PubMed Scopus (292) Google Scholar, Kono and Rock, 2008Kono H. Rock K.L. How dying cells alert the immune system to danger.Nat. Rev. Immunol. 2008; 8: 279-289Crossref PubMed Scopus (1292) Google Scholar). The first-identified and well-characterized class of PRRs are the Toll-like receptors (TLRs), which are responsible for detecting PAMPs and DAMPs outside the cell and in endosomes and lysosomes (O’Neill et al., 2013O’Neill L.A. Golenbock D. Bowie A.G. The history of Toll-like receptors - redefining innate immunity.Nat. Rev. Immunol. 2013; 13: 453-460Crossref PubMed Scopus (1131) Google Scholar). Under the stress of chemotherapy and targeted therapies, the secretion of HMGB-1, which binds to TLR4, has been reported to contribute to antitumor effects (Apetoh et al., 2007Apetoh L. Ghiringhelli F. Tesniere A. Obeid M. Ortiz C. Criollo A. Mignot G. Maiuri M.C. Ullrich E. Saulnier P. et al.Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.Nat. Med. 2007; 13: 1050-1059Crossref PubMed Scopus (2292) Google Scholar, Park et al., 2010Park S. Jiang Z. Mortenson E.D. Deng L. Radkevich-Brown O. Yang X. Sattar H. Wang Y. Brown N.K. Greene M. et al.The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity.Cancer Cell. 2010; 18: 160-170Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). However, whether the same mechanism dominates radiotherapy remains to be determined. Four endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) that respond to microbial and host-mislocalized nucleic acids in cytoplasm have more recently been revealed (Desmet and Ishii, 2012Desmet C.J. Ishii K.J. Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination.Nat. Rev. Immunol. 2012; 12: 479-491Crossref PubMed Scopus (292) Google Scholar). Through interaction of the adaptor proteins, myeloid differentiation primary-response protein 88 (MyD88), and TIR-domain-containing adaptor protein inducing IFN-β (TRIF), the activation of these four endosomal TLRs leads to significant induction of type I IFN production (Desmet and Ishii, 2012Desmet C.J. Ishii K.J. Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination.Nat. Rev. Immunol. 2012; 12: 479-491Crossref PubMed Scopus (292) Google Scholar, O’Neill et al., 2013O’Neill L.A. Golenbock D. Bowie A.G. The history of Toll-like receptors - redefining innate immunity.Nat. Rev. Immunol. 2013; 13: 453-460Crossref PubMed Scopus (1131) Google Scholar). Given that radiation induces production of type I IFNs, it is conceivable that radiation causes tumor cell nucleic acids and/or stress proteins to trigger the activation of TLRs and MyD88 and TRIF signaling. A recently defined endoplasmic-reticulum-associated protein STING (stimulator of IFN genes) has been demonstrated to be a mediator for type I IFN induction by intracellular exogenous DNA in a TLR-independent manner (Burdette and Vance, 2013Burdette D.L. Vance R.E. STING and the innate immune response to nucleic acids in the cytosol.Nat. Immunol. 2013; 14: 19-26Crossref PubMed Scopus (341) Google Scholar). Cytosolic detection of DNA activates STING in the cytoplasm, which binds to TANK-binding kinase 1 (TBK1) and IκB kinase (IKK), which in turn activate the transcription factors IFN regulatory factor 3 (IRF3), signal transducer and activator of transcription (STAT6), and nuclear factor-κB (NF-κB), respectively (Paludan and Bowie, 2013Paludan S.R. Bowie A.G. Immune sensing of DNA.Immunity. 2013; 38: 870-880Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). Subsequently, nuclear translocation of these transcription factors leads to the induction of type I IFNs and other cytokines that participate in host defense (Chen et al., 2011Chen H. Sun H. You F. Sun W. Zhou X. Chen L. Yang J. Wang Y. Tang H. Guan Y. et al.Activation of STAT6 by STING is critical for antiviral innate immunity.Cell. 2011; 147: 436-446Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Paludan and Bowie, 2013Paludan S.R. Bowie A.G. Immune sensing of DNA.Immunity. 2013; 38: 870-880Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). In the past 6 years, STING has been demonstrated to be essential for the host protection against DNA pathogens through various mechanisms (Chen et al., 2011Chen H. Sun H. You F. Sun W. Zhou X. Chen L. Yang J. Wang Y. Tang H. Guan Y. et al.Activation of STAT6 by STING is critical for antiviral innate immunity.Cell. 2011; 147: 436-446Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1945) Google Scholar, Ishikawa et al., 2009Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1664) Google Scholar). STING is also a mediator for autoimmune diseases, which are initiated by the aberrant cytoplasmic DNA (Ahn et al., 2012Ahn J. Gutman D. Saijo S. Barber G.N. STING manifests self DNA-dependent inflammatory disease.Proc. Natl. Acad. Sci. USA. 2012; 109: 19386-19391Crossref PubMed Scopus (351) Google Scholar, Gall et al., 2012Gall A. Treuting P. Elkon K.B. Loo Y.M. Gale Jr., M. Barber G.N. Stetson D.B. Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease.Immunity. 2012; 36: 120-131Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, Gehrke et al., 2013Gehrke N. Mertens C. Zillinger T. Wenzel J. Bald T. Zahn S. Tüting T. Hartmann G. Barchet W. Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing.Immunity. 2013; 39: 482-495Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Following the recognition of cytosolic DNA, cGAMP synthase (cGAS) catalyzes the generation of 2′ to 5′ cyclic GMP-AMP (cGAMP), which binds to and activates STING signaling (Li et al., 2013Li X.D. Wu J. Gao D. Wang H. Sun L. Chen Z.J. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects.Science. 2013; 341: 1390-1394Crossref PubMed Scopus (702) Google Scholar, Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339: 786-791Crossref PubMed Scopus (2508) Google Scholar, Wu and Chen, 2014Wu J. Chen Z.J. Innate immune sensing and signaling of cytosolic nucleic acids.Annu. Rev. Immunol. 2014; 32: 461-488Crossref PubMed Scopus (762) Google Scholar, Wu et al., 2013Wu J. Sun L. Chen X. Du F. Shi H. Chen C. Chen Z.J. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA.Science. 2013; 339: 826-830Crossref PubMed Scopus (1344) Google Scholar). More recently, cGAS has been considered as a universal cytosol DNA sensor for STING activation, such as in the setting of viral infection and lupus erythematosus (Gao et al., 2013aGao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Gehrke et al., 2013Gehrke N. Mertens C. Zillinger T. Wenzel J. Bald T. Zahn S. Tüting T. Hartmann G. Barchet W. Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing.Immunity. 2013; 39: 482-495Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, Lahaye et al., 2013Lahaye X. Satoh T. Gentili M. Cerboni S. Conrad C. Hurbain I. El Marjou A. Lacabaratz C. Lelièvre J.D. Manel N. The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells.Immunity. 2013; 39: 1132-1142Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, Liang et al., 2013Liang H. Deng L. Chmura S. Burnette B. Liadis N. Darga T. Beckett M.A. Lingen M.W. Witt M. Weichselbaum R.R. Fu Y.X. Radiation-induced equilibrium is a balance between tumor cell proliferation and T cell-mediated killing.J. Immunol. 2013; 190: 5874-5881Crossref PubMed Scopus (123) Google Scholar). On the basis of these considerations, it has become important to determine whether innate immune sensing following tumor radiation is mediated through TLR pathways or the alternative STING pathway. Here, we demonstrate that innate immune sensing following radiotherapy is dominated by the cGAS-STING-dependent cytosolic DNA sensing pathway, which drives the adaptive immune response to radiation. Our study provides insight through better understanding of the mechanism of radiation-mediated tumor regression and forms the basis for new strategies to improve radiotherapy efficacy in cancer patients. We previously demonstrated that antitumor effects of radiation were dependent on type I IFN signaling by utilizing IFNAR1-deficient mice (Burnette et al., 2011Burnette B.C. Liang H. Lee Y. Chlewicki L. Khodarev N.N. Weichselbaum R.R. Fu Y.X. Auh S.L. The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity.Cancer Res. 2011; 71: 2488-2496Crossref PubMed Scopus (568) Google Scholar). To rule out the possibility that the failure of tumors to respond to radiation was due to the intrinsic or developmental deficiency of IFNAR1-deficient mice, we administered blocking antibody against IFNAR1 in wild-type (WT) mice following radiation. The results were similar to the effects observed in the knockout (KO) mice in that the antitumor effect of radiation was greatly attenuated by the neutralization of type I IFN signaling with antibodies (Figure 1A). It has been demonstrated that MyD88 is essential for antitumor immunity of chemotherapy and targeted therapy with anti-HER2 (Apetoh et al., 2007Apetoh L. Ghiringhelli F. Tesniere A. Obeid M. Ortiz C. Criollo A. Mignot G. Maiuri M.C. Ullrich E. Saulnier P. et al.Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.Nat. Med. 2007; 13: 1050-1059Crossref PubMed Scopus (2292) Google Scholar, Park et al., 2010Park S. Jiang Z. Mortenson E.D. Deng L. Radkevich-Brown O. Yang X. Sattar H. Wang Y. Brown N.K. Greene M. et al.The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity.Cancer Cell. 2010; 18: 160-170Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, Stagg et al., 2011Stagg J. Loi S. Divisekera U. Ngiow S.F. Duret H. Yagita H. Teng M.W. Smyth M.J. Anti-ErbB-2 mAb therapy requires type I and II interferons and synergizes with anti-PD-1 or anti-CD137 mAb therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 7142-7147Crossref PubMed Scopus (353) Google Scholar). To investigate whether MyD88 is required to mediate response to radiation therapy, we implanted tumor cells on flanks of WT and MyD88-deficient mice. The inhibition of tumor growth postradiation was comparable between WT and MyD88-deficient mice (Figure 1B), demonstrating that host MyD88 was dispensable for the antitumor effect of radiation. To examine whether TRIF might be involved in the antitumor effects of radiation, we implanted tumor cells into WT and TRIF-deficient mice. Absence of host TRIF also failed to prevent the antitumor activity of radiation (Figure 1C), consistent with previous data (Burnette et al., 2011Burnette B.C. Liang H. Lee Y. Chlewicki L. Khodarev N.N. Weichselbaum R.R. Fu Y.X. Auh S.L. The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity.Cancer Res. 2011; 71: 2488-2496Crossref PubMed Scopus (568) Google Scholar). Similar to chemotherapy and targeted antibody therapies, radiotherapy induces cell stress and results in the secretion of DAMPs. Next, we examined whether HMGB-1 secretion might be involved in the antitumor effect of radiation. We blocked HMGB-1 by administering specific antibodies along with radiation. However, tumor control by radiation was unaffected by anti-HMGB-1 treatment (Figure 1D), suggesting that HMGB-1 secretion is also not required for the antitumor effect of radiation. The cathelicidin-related antimicrobial peptide (CRAMP in mice and LL37 in human) has been identified as a mediator of type I IFN induction by binding self-DNA to trigger the TLR9-MyD88 pathway (Diana et al., 2013Diana J. Simoni Y. Furio L. Beaudoin L. Agerberth B. Barrat F. Lehuen A. Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes.Nat. Med. 2013; 19: 65-73Crossref PubMed Scopus (318) Google Scholar, Lande et al., 2007Lande R. Gregorio J. Facchinetti V. Chatterjee B. Wang Y.H. Homey B. Cao W. Wang Y.H. Su B. Nestle F.O. et al.Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.Nature. 2007; 449: 564-569Crossref PubMed Scopus (1360) Google Scholar). To test the possibility that CRAMP might be responsible for the radiation response, we inoculated tumor cells into WT and CRAMP-deficient mice (CRAMP is encoded by Camp). Absence of host CRAMP also failed to prevent the antitumor effect of radiation (Figure 1E). Taken together, these data indicate that well-characterized TLR-dependent molecular mechanisms involved in chemotherapy and targeted antibody therapies are not responsible for the antitumor efficacy of radiation. Also, these results raise the possibility that a unique molecular mechanism that is TLR-independent for type I IFN induction mediates the antitumor effect of radiation. Recently, the STING-mediated cytosolic DNA sensing cascade has been demonstrated to be a major mechanism of TLR-independent type I IFN induction. To determine the role of STING in the radiation response, we implanted tumor cells on flanks of WT and STING-deficient mice (STING is encoded by Tmem 173) and monitored tumor growth. We found that, while tumor burden was significantly reduced by radiation in WT mice, absence of host STING significantly impaired the antitumor effect of radiation (Figure 1F), demonstrating that STING signaling is essential for the maximal antitumor effect of radiation. The antitumor effects of radiation were also impaired in STING-deficient mice when two doses of radiation treatment were utilized (see Figure S1 available online). Taken together, these results suggest that the STING-dependent cytosolic DNA sensing pathway is critical for the therapeutic effect of radiation in vivo. To test whether STING was responsible for type I IFN induction following radiation, we measured the amount of IFN-β protein in tumors. The induction of IFN-β in tumors was significantly abrogated in the absence of STING in the host after radiation (Figure 2A). As further confirmation, we found that the amount of CXCL10, a type I IFN-stimulated gene (Ablasser et al., 2013Ablasser A. Schmid-Burgk J.L. Hemmerling I. Horvath G.L. Schmidt T. Latz E. Hornung V. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP.Nature. 2013; 503: 530-534Crossref PubMed Scopus (378) Google Scholar, Holm et al., 2012Holm C.K. Jensen S.B. Jakobsen M.R. Cheshenko N. Horan K.A. Moeller H.B. Gonzalez-Dosal R. Rasmussen S.B. Christensen M.H. Yarovinsky T.O. et al.Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING.Nat. Immunol. 2012; 13: 737-743Crossref PubMed Scopus (174) Google Scholar), was also markedly diminished in tumors after radiation in STING-deficient hosts (Figure 2B). These results indicate that host STING is required for type I IFN induction by radiation. Next, to determine in which cell population STING mediates type I IFN induction, we performed quantitative real-time PCR assay of IFN-β in different sorted cell populations isolated from tumors after radiation. The phenotype of CD11c+ cells in tumors was characterized (Figure S2A). We observed that CD11c+ DCs were the major producer of IFN-β after radiation, compared to the CD45− population and the rest of myeloid cells (Figure 2C). In contrast, radiation-mediated induction of IFN-β mRNA by DCs was abolished in STING-deficient hosts (Figure 2C). Together, these data suggest that host STING controls radiation-mediated type I IFN induction in tumors and that the presence of STING in tumor-infiltrating DCs plays a major role in type I IFN induction after radiation. To determine whether STING signaling is activated by irradiated-tumor cells and whether it is essential for DC-mediated cross-priming of CD8+ T cells, a cross-priming assay was conducted with BMDCs from WT and STING-deficient mice. The phenotype of CD11c+ cells from GM-CSF stimulated bone marrow cells was characterized (Figure S2B). The functional capability of DCs to cross-present antigen was augmented by the stimulation of irradiated-tumor cells compared to nonirradiated-tumor cells, whereas the deficiency of STING in DCs resulted in failed responses of DCs to cross-prime T cells (Figure 3A). In contrast, CD19+ B cells isolated from the spleen of naive mice were unable to cross prime T cells (Figure S3A). To determine whether DCs differentiated in vivo were also functionally affected by STING, we isolated CD11c+ cells from the spleen of WT and STING-deficient mice to perform cross-priming assays. Similar to in vitro generated DCs, splenic DCs were impaired in the absence of STING (Figure S3B). To confirm whether IRF3 is essential to the function of DCs by the stimulation of irradiated-tumor cells, we performed cross-priming assay with WT BMDCs and IRF3-deficient BMDCs. Similar to STING-deficient BMDCs, IRF3-deficient BMDCs failed to cross-prime CD8+ T cells in response to stimulation with irradiated-tumor cells (Figure 3B). These results indicate that the STING-IRF3 axis in DCs is activated by irradiated-tumor cells and is the predominant innate signaling pathway needed for cross-priming by DCs. To determine whether exogenous IFN-β treatment rescues the functions of STING-deficient BMDCs, we added IFN-β to cocultured BMDCs and irradiated-tumor cells. The ability of STING-deficient BMDCs to cross-prime specific T cells was restored in the presence of exogenous IFN-β treatment (Figure 3C). Recently, it has been demonstrated that DMXAA, a small molecule inducing cytokine production and disrupting tumor vascularization, binds to murine STING and activates STING signaling to induce type I IFN production (Gao et al., 2013bGao P. Ascano M. Zillinger T. Wang W. Dai P. Serganov A.A. Gaffney B.L. Shuman S. Jones R.A. Deng L. et al.Structure-function analysis of STING activation by c[G(2′,5′)pA(3′,5′)p] and targeting by antiviral DMXAA.Cell. 2013; 154: 748-762Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). DMXAA failed to rescue the function of STING-deficient BMDCs; confirming activation of STING is required to increase cross-priming through IFN pathway (Figure 3C). Next, to rule out the possibility that the impaired capacity of STING-deficient DCs and IRF3-deficient DCs for priming is due to intrinsic defects of these cells, a direct priming assay was performed with peptide stimulation. No difference was observed between WT BMDCs and STING-deficient BMDC function in priming 2C cells with the stimulation of SIY peptide (Figure S3C). This result suggests that STING-deficient DCs do not have an intrinsic defect in direct priming of T cells. IRF3-deficient DCs also retained the ability to directly prime 2C T cells with SIY peptide stimulation (Figure S3C). To determine whether STING signaling might be activated by irradiated-tumor cells, we assessed the production of IFN-β by WT and STING-deficient BMDCs stimulated by irradiated-tumor cells. While the amount of IFN-β induction in response to irradiated-tumor cells was less than that induced by STING pathway agonists such as DMXAA (data not shown), we were nonetheless able to characterize the molecular requirements for this induction. The amount of IFN-β induced by irradiated-tumor cells (MC38) in vitro was reduced in STING-deficient BMDCs compared to WT BMDCs (Figure 3D). A similar difference was also observed when WT and STING-deficient BMDCs were stimulated by another tumor cell line (B16) in vitro (Figure S3D). These results suggest that activation of STING by irradiated-tumor cells controls type I IFN induction in DCs and that this process is crucial for the ability of DCs to cross-prime CD8+ T cells. These results also raise the possibility that STING molecules in DCs are stimulated by a component provided by irradiated-tumor cells, presumably DNA. To interrogate whether cGAS (encoded by Mb21d1) is required for DC sensing of irradiated-tumor cells to stimulate adaptive immunity, we compared the function of BMDCs from WT and cGAS-deficient mice. In contrast to WT BMDCs, cGAS-deficient BMDCs failed to cross-prime 2C cells in response to stimulation by irradiated-tumor cells (Figure 4A). To validate that the phenotype of cGAS-deficient BMDCs is not due to intrinsic or developmental defects, we silenced cGAS in WT BMDCs using siRNA. The silencing of cGAS in BMDCs diminished cross-priming by DCs compared to the silencing of nontarget controls, when stimulated with irradiated-tumor cells (Figure 4B). The results confirmed that cGAS is essential for sensing of irradiated-tumor cells by DCs. To map whether the cGAS-STING-type I IFN axis is needed for cross-priming by BMDCs, we performed bypass experiments in which either DCs were cocultured with irradiated-tumor cells in the presence of exogenous IFN-β or isolated DCs from the coculture were additionally stimulated with DMAXX. The cross-priming by cGAS-deficient BMDCs were restored with IFN-β and DMXAA treatment, respectively (Figure 4C). To further assess whether cGAS is required for sensing of irradiated-tumor cells by BMDC, we determined the production of IFN-β by WT BMDCs and by cGAS-deficient BMDCs after stimulation with irradiated-tumor cells. Indeed, the amount of IFN-β induced by irradiated-tumor cells was decreased in cGAS-deficient BMDCs compared to WT BMDCs (Figure 4D). Mb21d1 mRNA was detected in CD11c+ cells from tumors and increased after radiation in vivo (Figure 4E). We also performed the cross-priming assay using irradiated-human tumor cells expressing SIY and again found the cross-priming by DCs was impaired in the absence of STING or cGAS (Figure S4A). Thus cGAS responds to irradiated-murine and -human tumor cells and initiates type I IFN production to enhance DC cross-priming activity. The results suggest that DNA from irradiated-tumor cells might gain access to the cytosolic DNA sensing pathway to trigger STING-dependent type I IFN induction. DNA from irradiated-tumor cells could be delivered into the cytosol of DCs as free DNA or as membrane-associated DNA transferred by membrane fusion. The priming ability of DCs in response to irradiated-tumor cells was not impaired by the presence of DNase I (Figure S4B), suggesting that DCs do not engulf free DNA fragments. To test whether DNA delivery is contact-dependent, BMDCs were separated from irradiated-tumor cells via a transwell screen that only allows particles un" @default.
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• W1994826332 date "2014-11-01" @default.
• W1994826332 modified "2023-10-18" @default.
• W1994826332 title "STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors" @default.
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