Th eJournal of Brief Reviews Immunology

Critical Interactions between Immunogenic Cell , Oncolytic , and the Immune System Define the Rational Design of Combination ,1 †,‡,x,1 Jacob P. van Vloten,* x Samuel T. Workenhe, Sarah K. Wootton,* Karen L. Mossman,†,‡, ,2 and Byram W. Bridle*,2 Oncolytic viruses (OVs) are multimodal cancer thera- emphasis of cancer research is therapies that overcome im- peutics, with one of their dominant mechanisms munosuppression and tolerance to reawaken the immune being in situ vaccination. There is a growing consensus system to kill cancer cells. Immunogenic (ICD) that optimal cancer therapies should generate robust engages multiple conserved cell death pathways, triggering tumor-specific immune responses. Immunogenic cell an against tumor Ags and culminating in death (ICD) is a paradigm of cellular demise culminat- antitumor immunity (4). ing in the spatiotemporal release of danger-associated The engagement of overlapping, phylogenetically conserved molecular patterns that induce potent anticancer cell death pathways in ICD results in the spatiotemporal release immunity. Alongside traditional ICD inducers like of danger-associated molecular patterns (DAMPs) from dying chemotherapeutics and radiation, OVs cells (5) (Fig. 1A). Critical DAMPs initiate responses by have emerged as novel members of this class of thera- attracting innate cells, particularly dendritic cells (DCs), to the tumor. DAMPs mature Ag-carrying DCs, endowing them peutics. OVs replicate in and release tumor with the ability to activate tumor-specific T cells. Thus, ICD Ags, which are perceived as dangerous because of simul- is tied to the danger hypothesis (6), wherein factors produced taneous expression of pathogen-associated molecular by stressed and dying cells alert the immune system without patterns that activate APCs. Therefore, OVs provide an obligatory role for foreign inflammatory stimuli. the target Ags and danger signals required to induce Several therapies can elicit ICD, including select chemo- adaptive immune responses. This review discusses therapies, radiation, high hydrostatic pressure, photodynamic why OVs are attractive candidates for generating therapy, small molecules, and oncolytic viruses (OVs) (4, 7–10). ICD, biological barriers limiting their success in the Theoretically, potentiating ICD has the advantage of stimu- clinic, and groundbreaking strategies to potentiate lating a patient’s immune system in an Ag agnostic manner, ICD and antitumor immunity with rationally designed making it personalized, yet broadly applicable. It is likely OV-based combination therapies. The Journal of that ICD inducers will be optimal when used in combination Immunology, 2018, 200: 450–458. with other therapies by involving multiple overlapping death pathways.

he immune system is a critical factor in cancer de- DAMP profiles and molecular mechanisms of translocation velopment and resolution. In 2004, Dunn, Old, and The critical DAMPs in ICD are commonly referred to as the T Schreiber (1) described the three E’s of cancer hallmarks of ICD. These DAMPs are endogenous factors that immunoediting: elimination, equilibrium, and escape. By the translocate to abnormal cellular compartments in a temporal time cancer patients reach the clinic, tumors have evaded cascade. Translocation of (CRT) from the endo- immune recognition and engaged in complex cell-to-cell (2) plasmic reticulum to the cell surface (forming surface-expressed and systemic (3) signaling to drive immunosuppression. An CRT [ecto-CRT]), secretion of ATP and nuclear high mobility

*Department of Pathobiology, Ontario VeterinaryCollege,UniversityofGuelph,Guelph, stipend funding to J.P.v.V.), and a Highly Qualified Personnel Scholarship (Ontario Ontario N1G 2W1, Canada; †Department of Pathology and Molecular Medicine, McMaster Ministry of Agriculture, Food and Rural Affairs; stipend funding to J.P.v.V.). University, Hamilton, Ontario L8S 4L8, Canada; ‡McMaster Immunology Research Centre, x Address correspondence and reprint requests to Dr. Byram W. Bridle, Room 4834, McMaster University, Hamilton, Ontario L8S 4L8, Canada; and Institute for Infectious Building 89, Department of Pathobiology, Ontario Veterinary College, University of DiseaseResearch,McMasterUniversity,Hamilton,OntarioL8S4L8,Canada Guelph, 50 Stone Road E, Guelph, ON N1G 2W1, Canada. E-mail address: 1J.P.v.V. and S.T.W. contributed equally to this work. [email protected] 2K.L.M. and B.W.B. contributed equally to this work. Abbreviations used in this article: Ad, adenovirus; CRT, calreticulin; DAMP, danger- associated molecular pattern; DC, ; ecto-CRT, surface-expressed CRT; ORCIDs: 0000-0001-9521-3903 (S.T.W.); 0000-0002-5985-2406 (S.K.W.); 0000- HMGB1, high mobility group box 1; HSP, ; ICD, immunogenic 0002-8335-1118 (B.W.B.). cell death; LRP1, lipoprotein receptor-related protein 1; OV, oncolytic ; Oxa-Cyc, Received for publication July 17, 2017. Accepted for publication August 23, 2017. -cyclophosphamide; PAMP, pathogen-associated molecular pattern; PRR, pattern recognition receptor; TAA, tumor-associated Ag; TME, tumor microenviron- This work was supported by the Terry Fox Research Institute (Project 1041; to B.W.B.), ment; TMZ, temozolomide; T , regulatory ; VACV, vaccinia virus. the Canadian Breast Cancer Foundation and the Canadian Cancer Society (to K.L.M.), Reg the National Sciences and Engineering Research Council (to S.K.W.), a Canadian Ó Graduate Scholarship (Natural Sciences and Engineering Research Council of Canada; Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701021 The Journal of Immunology 451

FIGURE 1. (A) ICD causes the re- lease of DAMPs (HMGB1, ATP, ecto-CRT and type I IFNs) from dy- ing tumor cells. These DAMPs attract innate effector cells, like DCs that acquire tumor Ags and receive matu- ration signals. Extracellular ATP binds to the purigenic receptors P2Y2 and P2X7, which promote recruitment and maturation of DCs, respectively. Ecto-CRT binds to low-density LRP1 and promotes phagocytosis and in- duction of proinflammatory . HMGB1 binds to TLR-4 and receptor for advanced glycation endproducts, which promote production of cyto- kines and Ag cross-presentation. Type I IFNs bind to their cognate receptor (IFNAR) and drive the expression of a large array of IFN-stimulated genes that support the induction of acquired immune responses. Mature DCs can present tumor Ags to cancer-specific T cells leading to antitumor immunity via production of the effector IFN-g and cytolysis mediated by the release of granzyme B and perforin. (B) OVs infect and preferentially kill cancer cells. Tumors facilitate the spread of OVs, and tumor cell lysis releases TAAs. During the virus life cycle, PAMPs are released, which stimulate PRRs that in turn induce antiviral type I IFNs and inflamma- tory cytokines. Different viruses en- gage or inhibit cell death pathways leading to an array of cell death phenotypes that are immunogenic, including , , im- munogenic , and autophagic cell death.

group box 1 (HMGB1), and the production of type I IFNs all phagocytosis and NF-kB pathway activation (16, 17). Heat precede ICD (11, 12) (Fig. 1A). Once translocated, DAMPs shock proteins (HSPs) also translocate to the cell surface interact with receptors and stimulate cellular and cytokine re- during ICD and similarly stimulate tumor Ag uptake (18). sponses against cancer-derived Ags. Extracellular ATP is a potent chemoattractant and is inti- The prototypic framework of DAMP translocation was mately linked to , which helps attract DCs (19). largely delineated from studies using anthracycline chemo- Intensive research identified a multiplex pathway for ATP therapies in murine cancer models (8). Upon therapy-induced release (20). Activated -3 cleaves and activates Pan- ER stress, Ca2+ efflux, and accumulation of reactive oxygen nexin 1, which translocates to the cell surface to mediate ATP species, CRT complexes with ERp57 during transport from secretion (21). DCs follow extracellular ATP gradients via the ER and docks on CD91/low-density lipoprotein receptor- binding to purinergic receptors; P2Y2 facilitates recruitment related protein 1 (LRP1) (13) (Fig. 1A). Ecto-CRT/ERp57 of DCs, whereas P2X7 stimulates maturation (Fig. 1A). signals through CD91 and scavenger receptors SR-A and HMGB1, a primarily nuclear nonhistone DNA-binding SREC-1 (14, 15) on innate immune cells, promoting protein, is also a pleiotropic cytokine and chemokine that is 452 BRIEF REVIEWS: POTENTIATING WITH OVs released by leukocytes in response to inflammation (22). As such, the addition of immune checkpoint inhibitors During viral infection, type I IFN signaling causes hyper- could be particularly effective at improving the function acetylation of HMGB1, promoting its secretion (23, 24). of suppressed tumor-infiltrating T cells (43). Oxaliplatin- Once secreted, the redox form of internal cysteine residues cyclophosphamide (Oxa-Cyc) treatment recruited CD103+ determines its cytokine or chemokine function (25). HMGB1 DCs to the TME of murine lung adenocarcinomas harboring is passively released by dying cancer cells during ICD and low T cell numbers. CD103+ DC priming of CD8+ T cells in secondary necrosis (26). HMGB1 signals through TLR-4 and the presence of immune checkpoint blockade increased the receptor for advanced glycation endproducts on DCs, eliciting ratio of effector T cells to regulatory T cells (TRegs), a positive proinflammatory cytokine production and cross-presentation biomarker for solid tumors (44). Although CD103+ DCs are of tumor Ags to T cells (27–30) (Fig. 1A). As a prognostic a rare myeloid subset within the TME (45), they are the biomarker, elevated tumor or serum levels correlate with poor primary cells responsible for carrying tumor Ags to draining outcomes (31–33) and drug resistance (34), whereas low se- lymph nodes. Delivery of FLT3L with the TLR3 agonist rum levels may stratify patients for novel immunotherapies polyinosinic:polycytidylic acid increased recruitment, differ- (35). Paradoxically, chronic release of HMGB1 by tumors entiation, and maturation of CD103+ DCs via the type I IFN may aid in survival and metastasis (36–38), whereas acute axis, improving tumor-specific T cell responses and making release after cell death may induce tumor-specific immunity. tumors sensitive to immune checkpoint blockade. One could A myriad of other molecules are implicated in ICD (11, 39), perceive a therapeutic regimen wherein cancer-specific OV but they are not considered critical for bona fide ICD, as therapy replaces poly(I:C) to generate potent type I IFN re- demonstrated by prophylactic vaccination of mice with killed sponses and an immunogenic TME to overcome resistance to cancer cells that protects against subsequent tumor challenge immune checkpoint inhibition. (40). The contribution of other DAMPs to ICD may be NK cells. NK cells express activating and inhibitory receptors, revealed as research continues. For example, type I IFNs with the net balance of their ligation discriminating between signal autocrinely and paracrinely to stimulate expression of dangerous and normal cells. Upon activation, NK cells in the an immense array of IFN-stimulated genes, driving inflam- TME produce proinflammatory cytokines and influence matory responses against Ags (12, 41) (Fig. 1A). Type I IFNs adaptive immunity through interactions with DCs and di- are important when considering ICD induced by infectious rect effects on T cells. NK cell–derived IFN-g and TNF-a agents such as OVs, because viruses are classic agonists of the promote upregulation of costimulatory and migratory IFN response. As preclinical research into ICD continues, markers and production of IL-12 and IFN-g by DCs, adherence to certain standards to define ICD inducers will be enhancing their ability to prime cytotoxic T cells (46–48). critical (11). Further, IFN-g from NK cells can promote cytotoxic T cell responses (49). NK cell–mediated cancer cell lysis increases Immunological effector mechanisms of ICD the availability of tumor Ags for cross-presentation to T cells The dogma of cancer has T cells driving an by DCs (50), whereas antitumor NK cell functions rely on the anticancer immune response. Because induction of cytotoxic production of type I IFNs by DCs (51, 52). T cells is dependent on the activation and maturation of DCs, Many cause severe DNA damage, leading to research on ICD has focused primarily on the DC–T cell axis. upregulation of ligands for NK activating receptors, particu- However, other effector subsets undoubtedly play a role in larly NKG2D and DNAM-1 (53). Ligation of activating re- ICD. Although NK and B cells play important roles in re- ceptors and dysregulation of inhibitory signals result in sponses against cancers, and many immunotherapies attempt contact-depending killing of targets and cytokine produc- to amplify their efficacy, only NK cells have been considered tion. Stressed tumor cells can increase production of HSPs, in the context of ICD. including CRT and HSP70, which are critical signaling ef- Dendritic cells. DCs bridge innate and adaptive immunity by fectors in ICD (11). In multiple tumor models, NK cells presenting Ags to T cells. They express a large repertoire of recognize HSP70 on the surface of target cells (54). In a xe- pattern recognition receptors (PRRs), which enable them to nograft model of human melanoma, HSP70 was found to interact with a broad range of stimuli. In ICD, DCs are increase the antitumor activity of NK cells against tumor cells recruited to the tumor microenvironment (TME) by expressing NKG2D ligands MICA/B (55). HSP danger sig- ligation of TLR4 and P2Y2 with HMGB1 and ATP, nals may regulate recruitment and maturation of APCs and respectively. Dying cancer cells are rapidly phagocytosed if also drive antitumor NK responses, a concept that deserves they express ecto-CRT, which binds LRP1, SR-A, and further investigation. As with many other cancer treatments, SREC-1 on DCs (14, 15). In murine cancer models treated the contribution of NK cells may be a powerful tool for en- with immunogenic , recruitment of myeloid hancing ICD, especially in the context of OV-based therapies, cells orchestrated by ATP secretion from dying tumor cells because viruses can be potent NK cell activators. promoted in situ differentiation into mainly CD11c+CD11b+ T cell dysfunction in cancers. A major clinical problem is the rapid Ly6CHi inflammatory DCs (42). These DCs surrounded development of T cell suppression by tumors. Most cancer Ags caspase-3+ tumor cells and locally primed T cells, indicated are derived from self, and the corresponding TCR repertoire by the preservation of antitumor immunity upon removal of relies on T cells that have escaped negative selection during tumor-draining lymph nodes and pharmacological inhibition central tolerance (56). Most patients have T cell repertoires of tertiary lymphoid structures. Thus, immunogenic with a low-to-moderate number of clones with low- to chemotherapy can promote DC recruitment and maturation moderate-affinity TCRs, limiting overall avidity of anticancer in an ATP-dependent manner, leading to activation of tumor- responses. T cells with low/moderate-affinity TCRs risk becoming infiltrated T cells. anergic, where weak MHC:TCR binding or costimulatory The Journal of Immunology 453 signaling results in T cells that are unable to develop effector Adenosine signaling in the TME can impede cytotoxicity and function (57) and have a limited expansion (58). T cell IFN-g secretion by NK and CD8+ T cells (81, 82), stimulate impairment can also occur through exhaustion, a result of T cell anergy, and increase infiltration of TRegs (83). More- chronic Ag stimulation coupled with poor costimulation. over, adenosine is a critical immunosuppressive effector Exhausted T cells in the TME express high levels of mechanism for TRegs (84), myeloid-derived suppressor cells, inhibitory receptors, cease to produce cytokines, and have and suppressive type 2 tumor-associated (85, altered transcriptional and epigenetic profiles (59, 60). 86). Single nucleotide polymorphisms in purinergic receptors The mutational progression of cancer leads to development could impair recruitment of leukocytes into tumors by virtue of neoantigens that are not found in the normal human ge- of their inability to properly navigate ATP gradients as seen nome and are not represented during thymic selection. The with polymorphisms in HMGB1 and TLR4, which can limit accumulation of neoantigens is variable among cancers with the immunological effects of ICD (4). melanomas containing high numbers (61). Theoretically, During infection with viruses (87) and treatment with neoantigens represent ideal targets for cancer immunotherapy, chemotherapies (12), cancer cells secrete type I IFNs. The assuming that T cells capable of engaging them are superior to antitumor effects of are, in part, mediated by self-–specific T cells (62). Intriguingly, T cells with the induction of type I IFNs after TLR3-mediated sensing of higher-affinity TCRs tend to be preferential targets for ex- self-RNA secreted after drug-induced damage (12). Tumor haustion by tumor-derived factors (63). In practice, high cells lacking type I IFN receptors fail to respond properly to mutational and neoantigen burden correlates with improved anthracyclines (12). In humans, defects or altered expression responses to cancer immunotherapies, including immune of type I IFN signaling components influences disease pro- checkpoint blockade (64–67). Indeed, deficiencies in mis- gression and prognosis (12). match repair machinery, which facilitate the accruement of Necroptosis is a form of triggered by somatic mutations, are a predictor for response to checkpoint stimuli that engage the TNF superfamily, ligation of various blockade (68, 69). It is tempting to extrapolate the correlates PRRs, and/or genotoxic and oxidative stresses induced by demonstrated during checkpoint blockade to ICD-driven anticancer drugs (88). Unlike apoptosis, necroptosis requires therapies, which also rely on reactivation of the T cell rep- inactivation of caspase-8 and subsequent activation of serine/ ertoire, especially because immunogenic chemotherapy sen- threonine receptor–interacting protein kinases RIPK1 and sitizes cancers to immune checkpoint blockade (43). RIPK3 (88). Necroptosis activates anticancer immunity and The reliance on a pool of T cells with low-affinity TCRs for contributes to the of transplantable tumors anticancer vaccination is a roadblock for in situ vaccination by (89, 90). In contrast, necroptosis is described as immuno- OVs, because they engage both virus- and tumor-specific suppressive in pancreatic cancers and in the context of T cells, with the nontolerized antiviral response being domi- autochthonous murine cancer models (91). The effect of nant. Historically, researchers attempted to diminish antiviral necroptosis in spontaneous cancers in mice has yet to be T cell responses through immunosuppression or Ag masking demonstrated. Therefore, the clinical relevance of necroptosis via cell carriers to maximize the replication of OVs in tumors to induction of tumor-specific immunity is debatable. More (70–72). However, immunosuppression reduces the efficacy research is needed to clarify the patterns of inflammatory of OVs by simultaneously dampening the anticancer immune cytokines and DAMPs released during necroptosis that bias response. This issue can be addressed through prime-boost the outcome toward tumor promotion versus immune sup- vaccination, wherein the anticancer repertoire is primed pression. Strategies to promote necroptotic ICD will need to against a tumor-associated Ag (TAA) and subsequently consider that some cancers inactivate or lose expression of boosted with a heterologous OV encoding the same TAA RIPK1, RIPK3, and the executioner MLKL (92), key com- (73). By using OVs as booster vaccines, the primary response ponents that drive necroptosis (88). against viral backbone proteins can be overwhelmed by the secondary response against the TAA expressed by the virus. OVs: anticancer vaccines and ICD inducers OVs are targeted biological therapeutics that infect and kill Mutations and immunological defects limiting ICD in tumors cancer cells, triggering systemic antitumor immune responses Several immunogenic therapies lead to activation of ER stress (93). OVs have demonstrated preclinical and clinical success and expression of ecto-CRT (74–76). Although ecto-CRT has culminating in the U.S. Food and Drug Administration ap- a prophagocytic effect, it is countered by CD47. The balance proving the herpesvirus T-Vec for advanced melanomas (94). between CRT and CD47 is a determinant of phagocytic OVs have the implicit capacity to generate inflammatory re- uptake of stressed and dying cells, and manipulating it is an sponses through production of pathogen-associated molecular important early step in promoting the development of anti- patterns (PAMPs) and release of tumor Ags, thus acting as in tumor adaptive immunity (77). Loss of LRP1 receptors on situ cancer vaccines (Fig. 1B). Engineering expression of im- DCs in tumor-bearing hosts impairs phagocytosis induced by munomodulatory transgenes, such as cytokines (e.g., GM- ecto-CRT (4). Notably, the balance of ecto-CRT:CD47 li- CSF for T-Vec), or immune checkpoint inhibitors enhances gation can potentially be tipped in favor of prophagocytic OV-driven antitumor immunity (95, 96). signaling through the use of CD47-blocking Abs (16, 78). Interactions between viruses and hosts have shaped a Tumor cells further limit ICD by defective production or complex network of cell death and antiviral signaling pathways active degrading of ATP. For example, overexpression of the and viral escape mechanisms (97, 98). Cancer represents an ectonucleotidases CD39 or CD73 promotes degradation of aberration to virus–host interactions, because many cancer extracellular ATP (79). The primary degradation product mutations (99) come at a fitness cost to the antiviral response is adenosine, which is potently immunosuppressive (80). or allow viruses to usurp upregulated growth pathways. Sev- 454 BRIEF REVIEWS: POTENTIATING IMMUNOGENIC CELL DEATH WITH OVs eral OVs elicit ICD through engagement of cell death path- OV-based therapies to enhance ICD: strategies to overcome suboptimal ways resulting in diverse expression of DAMPs. The anti- induction of ICD cancer effects of OVs may be potentiated by combination Three key DAMPs (ecto-CRT, extracellular HMGB1, and ATP) with other ICD inducers (100). are often used as biochemical markers of ICD (112). Indeed, for Adenovirus (Ad)-based OVs have been in clinical devel- ICD-inducing chemotherapeutics, these DAMPs appear nec- opment for decades and are approved in China for head and essary to efficiently induce immune responses (4). However, neck cancers (101). Ad5 containing a D-24-RGD cancer- they are not absolute indicators of ICD (11): drugs such as targeting mutation kills glioma cells through autophagic cell cardiac glycosides show the stereotypical in vitro biochemical death and activation of caspase-8, both hallmarks of ICD features of ICD but fail to induce immunological memory in (102), and induces tumor Ag-specific adaptive immune re- vaccination assays, suggesting a requirement for additional un- sponses (103). The mechanistic understanding of Ad-driven identified DAMPs in induction of bona fide ICD (113). Thus, autophagic cell death led to preclinical testing of Ad combined strategies to enhance ICD should increase the quantity and with the alkylating agent temozolomide (TMZ) (104). TMZ broaden the diversity of DAMPs, while simultaneously upreg- also induces autophagy, and in combination with Ad5 leads to ulating expression of cognate receptors on innate immune cells. induction of ICD in breast and prostate cancer cell lines. This OVs cause premortem stress leading to secretion of one or combination was applied to a cohort of 17 patients with ad- more DAMPs that are typically released during chemotherapy vanced solid tumors, where it was well tolerated and improved (100). However, the immunogenicity of cell death induced by anticancer T cell responses and overall survival (104). In- OVs, especially the signaling cascades preceding secretion of triguingly, there was a correlation between high levels of DAMPs, is not well characterized. Future studies should focus HMGB1 in patient serum after treatment and improved sur- on dissecting the relevance of DAMPs released during OV vival. A phase 1 trial is under way to evaluate the combination infection with therapeutic outcomes. Because viruses are of Ad and TMZ in glioblastoma patients (DNAtrix, amenable to genetic modification, OVs can be engineered to NCT01956734), showing how an evidence-based combinato- express ICD-associated DAMPs (100) or components of ICD rial approach with OVs and approved drugs to promote ICD signaling pathways that are mutated or missing in tumor cells, can be translated to the clinic. However, each combination of a thus increasing their immunotherapeutic potential. Large chemotherapeutic and OV should be carefully investigated to DNA OVs such as HSV-1 encode many proteins that counter ensure that there are no reciprocal deleterious effects. ICD and innate and adaptive immunity (114). Characterizing Vaccinia virus (VACV) is a poxvirus progressing toward the ICD-evading proteins of OVs would provide the rationale regulatory approval (105). VACV induces a combined form of for engineering mutant viruses lacking these proteins. cell death, involving immunogenic apoptosis and necrosis Although ICD-inducing therapies are promising, mono- (106). Given that poxviruses carry genes to antagonize cell therapies often promote the emergence of resistant tumor var- death pathways, VACV causes an incomplete emission of iants (40). As a result, combination therapies will likely be ICD-inducing DAMPs (107). Removing the viral anti- required to provide cures. There is some evidence to suggest that apoptotic genes SPI-1 and SPI-2 enhanced necrosis and re- broadening the induction of DAMPs potentiates anticancer lease of HMGB1, extending survival in immunocompetent immunity. For some chemotherapies, failure to initiate one or tumor-bearing mice (108). Unfortunately, VACV does not more of the hallmark DAMPs results in an inability to mediate increase extracellular ATP (106, 109). This may be a common immunogenic effects (4). For example, cisplatin-treated tumors feature among OVs because ATP is a highly used resource release ATP and HMGB1, but fail to cause sufficient ER stress during production of viral progeny. to expose ecto-CRT. Incorporating ER stressors such as OVs The potential of combining VACV with ICD inducers has can promote the immunogenicity of cisplatin (115). In a dif- been explored for drug-resistant ovarian cancers. Komorowski ferent approach, inhibitors of nucleotidases were administered et al. (110) combined recombinant VACV with doxorubicin, along with ICD-inducing therapies in patient tumor types that a standard of care anthracycline. In vitro treatment of ovarian express high levels of CD39 and CD73 (115). Also, subversion cancer cells with VACV enhanced susceptibility to doxoru- of HMGB1 release by cancer cells reduces the potential for bicin, likely mediated through type I IFNs, and increased DC signaling through TLR4, which can be countered by adminis- tumor Ag uptake. A bona fide ICD vaccination experiment tering synthetic TLR4 agonists such as dendrophilin (115). In a showed that tumor cells killed with VACV and doxorubicin particularly promising combination approach, we showed that limited engraftment compared with either treatment alone. treating tumor-bearing mice with immunogenic HSV-1 OV VACV-induced ICD has been exploited to shape the TME and immunogenic chemotherapy effectively potentiated ICD to favor antitumor immunity. VACV infection can release (116, 117). Identifying ideal combinations of ICD-inducing tumor Ags into an inflamed microenvironment consisting of agents to minimize tumor escape variants represents an im- proinflammatory cytokines, DAMPs and PAMPs, but si- portant future research direction. The use of OVs with other multaneously increased expression of PDL-1 on recruited ICD inducers is particularly promising. leukocytes, limiting cancer-specific immune responses (111). Treatment with anti–PDL-1 following VACV prevented the Improving research models to predict success of ICD-inducing therapies shutdown of cancer-specific T cells and reduced infiltration of in the clinic TRegs and immunosuppressive myeloid-derived suppressor Checkpoint blockade immunotherapies exert durable anti- cells, resulting in improved efficacy (111). Thus, combination cancer effects in patients who already have T cell infiltrates of immunomodulatory checkpoint inhibitors with ICD- (118). However, only a fraction of patients respond to inducing therapies like OVs deserves further preclinical and checkpoint blockade immunotherapy (118). From preclinical clinical investigation. studies (43) it is plausible to speculate that ICD induction The Journal of Immunology 455 will activate T cell responses with broad antigenic coverage in yet-to-be-identified DAMPs that can drive optimal ICD. human patients and increase the proportion of patients who Notably, the primary emphasis in this field has been on un- respond to checkpoint blockade immunotherapies. Two lines derstanding the role of ICD in promoting tumor-specific T cell of evidence suggest that the type of cancer cell death has an responses. But ideal tumor-specific immunity will be under- immunomodulatory effect: 1) T cell infiltration of human pinned by multimechanistic effector mechanisms. There is a breast cancers at diagnosis (a high ratio of CD8+ T cells to small but growing body of evidence that provides the rationale TRegs) predicts efficacy in patients receiving some chemo- and some mechanistic insight required to start designing ICD therapies (119), and 2) treatment of cancer patients with inducers to engage NK cells, along with T lymphocytes. A gaping adoptive transfer of DCs loaded with dying cancer cells hole exists in terms of understanding the role of ICD inducers in showing features of ICD leads to development of potent tumor-specific Ab responses. This may be due to the historical antitumor immunity (120). Although these studies suggest demonstration that Abs fail to play a role in ICD caused by that ICD may influence cancer patient outcomes, from its anthracyclines (126). However, Abs represent an important arm early conception to experimental validation, the field of ICD of the immune system for targeting surface-expressed tumor Ags, has relied heavily on the use of immunogenic transplantable promoting Ag presentation to T cells via Ab-mediated uptake of tumor models. Although these models provide tools to study proteins from dying cells and facilitating Ab-dependent cellular ICD, they do not capture the immunosuppressive nature of cytotoxicity. As such, the identification of nonanthracycline ICD spontaneous human tumors. inducers that can promote Ab responses may be a way to tap Recently, it was shown that immunogenic chemotherapy into this currently unharnessed effector mechanism. using Oxa-Cyc induces T cell infiltration in a KRAS/Trp53- We contend that combining OVs with classical ICD- mutated lung adenocarcinoma model and sensitizes tumors to inducing chemotherapeutics may represent a particularly checkpoint blockade immunotherapy (43). Although oxali- promising way to optimize ICD-focused therapies for clinical platin has been well studied for its direct cell death–mediated use. OVs and drugs overlap in their ability to induce the key immunogenic potential, cyclophosphamide is not well char- hallmarks of ICD (exogenous ATP and HMGB1, and ecto- acterized (121, 122). Nonetheless, prophylactic vaccination CRT) in dying cancer cells. Moreover, viruses are potent in- with Oxa-Cyc–killed tumor cells provided protection against ducers of type I IFNs, which have recently been defined as the spontaneous tumor development (43). In sharp contrast, a fourth hallmark of ICD. They also induce the expression of previous report used spontaneously arising mammary tumors broad arrays of DAMPs, which may include at least some of to conclude that antitumor effects of chemotherapy are in- those that are currently undefined but likely required for dependent of the immune system (123). This study used optimal ICD. OVs are also capable of engaging multiple chemotherapies that were well characterized for their immu- pathways of cell death, which could minimize the risk of nogenic effect in transplantable tumor models. However, the selecting ICD-escape variants. Finally, viruses potently activate chemotherapies were administered systemically at a high dose, NK and B cells, thereby providing the potential to recruit these which could cause general suppression of the immune system, additional immunological effector mechanisms. Using OVs to leading to loss of therapeutic efficacy. potentiate ICD-inducing drugs has excellent potential for In short, murine studies show that different types of che- reactivating the tumor-killing potential of the immune systems motherapies and dosing schedules exert different immuno- of cancer patients. This could provide a relatively cost-effective, genic and therapeutic effects in a model-dependent fashion. As short-duration, personalized treatment for cancers. a result, it remains a question what type of nonimmunogenic tumor model will best predict the inherent immunogenic properties of chemotherapies. Moreover, translating preclinical Disclosures ICD observations into the clinic requires understanding the The authors have no financial conflicts of interest. inherent immunological differences between humans and mice (124). A comparative study of inflammatory stressors in hu- References mans and mice showed remarkably different genomic re- 1. Dunn, G. P., L. J. Old, and R. D. Schreiber. 2004. The three Es of cancer immunoediting. Annu. Rev. Immunol. 22: 329–360. sponses (125). Although in vitro biochemical assays to detect 2. Fearon, D. T. 2017. Immune-suppressing cellular elements of the tumor micro- ecto-CRT, extracellular ATP, and HMGB1 can be used to environment. Annu. Rev. Cancer Biol. 1: 241–255. 3. Lippitz, B. E. 2013. 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