Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling.

IMPORTANCE: Multiple pattern recognition receptors sense vRNAs and initiate downstream innate signaling: endosomal Toll-like receptors (TLRs) 3, 7, and 8 and cytoplasmic RIG-I-like receptors (RLRs) RIG-I, and MDA5. They engage distinct signaling scaffolds: mitochondrial antiviral signaling protein (RLR), MyD88, and TLR-adaptor interacting with SLC15A4 on the lysosome (TLR7 and TLR8) and toll/IL-1R domain-containing adaptor inducing IFN (TLR3). By virtue of their unusual vRNA structure and direct host cell entry path, the innate response to EVs uniquely is orchestrated by MDA5. We reported that PVSRIPO's profound attenuation and loss of cytopathogenicity triggers MDA5-directed polar TBK1-IRF3 signaling that generates priming of polyfunctional antitumor CD8+ T-cell responses and durable antitumor surveillance in vivo. Here we unraveled EV-host relations that control suppression of host type-I IFN responses and show that PVSRIPO's deficient immediate host eIF4G cleavage generates unopposed MDA5-directed downstream signaling cascades resulting in sustained type-I IFN release.

Intratumor childhood vaccine-specific CD4+ T-cell recall coordinates antitumor CD8+ T cells and eosinophils.

BACKGROUND: Antitumor mechanisms of CD4+ T cells remain crudely defined, and means to effectively harness CD4+ T-cell help for cancer immunotherapy are lacking. Pre-existing memory CD4+ T cells hold potential to be leveraged for this purpose. Moreover, the role of pre-existing immunity in virotherapy, particularly recombinant poliovirus immunotherapy where childhood polio vaccine specific immunity is ubiquitous, remains unclear. Here we tested the hypothesis that childhood vaccine-specific memory T cells mediate antitumor immunotherapy and contribute to the antitumor efficacy of polio virotherapy. METHODS: The impact of polio immunization on polio virotherapy, and the antitumor effects of polio and tetanus recall were tested in syngeneic murine melanoma and breast cancer models. CD8+ T-cell and B-cell knockout, CD4+ T-cell depletion, CD4+ T-cell adoptive transfer, CD40L blockade, assessments of antitumor T-cell immunity, and eosinophil depletion defined antitumor mechanisms of recall antigens. Pan-cancer transcriptome data sets and polio virotherapy clinical trial correlates were used to assess the relevance of these findings in humans. RESULTS: Prior vaccination against poliovirus substantially bolstered the antitumor efficacy of polio virotherapy in mice, and intratumor recall of poliovirus or tetanus immunity delayed tumor growth. Intratumor recall antigens augmented antitumor T-cell function, caused marked tumor infiltration of type 2 innate lymphoid cells and eosinophils, and decreased proportions of regulatory T cells (Tregs). Antitumor effects of recall antigens were mediated by CD4+ T cells, limited by B cells, independent of CD40L, and dependent on eosinophils and CD8+ T cells. An inverse relationship between eosinophil and Treg signatures was observed across The Cancer Genome Atlas (TCGA) cancer types, and eosinophil depletion prevented Treg reductions after polio recall. Pretreatment polio neutralizing antibody titers were higher in patients living longer, and eosinophil levels increased in the majority of patients, after polio virotherapy. CONCLUSION: Pre-existing anti-polio immunity contributes to the antitumor efficacy of polio virotherapy. This work defines cancer immunotherapy potential of childhood vaccines, reveals their utility to engage CD4+ T-cell help for antitumor CD8+ T cells, and implicates eosinophils as antitumor effectors of CD4+ T cells.

PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling.

For RNA virus families except Picornaviridae, viral RNA sensing includes Toll-like receptors and/or RIG-I. Picornavirus RNAs, whose 5' termini are shielded by a genome-linked protein, are predominately recognized by MDA5. This has important ramifications for adaptive immunity, as MDA5-specific patterns of type-I interferon (IFN) release are optimal for CD4+T cell TH1 polarization and CD8+T cell priming. We are exploiting this principle for cancer immunotherapy with recombinant poliovirus (PV), PVSRIPO, the type 1 (Sabin) PV vaccine containing a rhinovirus type 2 internal ribosomal entry site (IRES). Here we show that PVSRIPO-elicited MDA5 signaling is preceded by early sensing of the IRES by the double-stranded (ds)RNA-activated protein kinase (PKR). PKR binding to IRES stem-loop domains 5-6 led to dimerization and autoactivation, displaced host translation initiation factors, and suppressed viral protein synthesis. Early PKR-mediated antiviral responses tempered incipient viral translation and the activity of cytopathogenic viral proteinases, setting up accentuated MDA5 innate inflammation in response to PVSRIPO infection. IMPORTANCE Among the RIG-I-like pattern recognition receptors, MDA5 stands out because it senses long dsRNA duplexes independent of their 5' features (RIG-I recognizes viral [v]RNA 5'-ppp blunt ends). Uniquely among RNA viruses, the innate defense against picornaviruses is controlled by MDA5. We show that prior to engaging MDA5, recombinant PV RNA is sensed upon PKR binding to the viral IRES at a site that overlaps with the footprint for host translation factors mediating 40S subunit recruitment. Our study demonstrates that innate antiviral type-I IFN responses orchestrated by MDA5 involve separate innate modules that recognize distinct vRNA features and interfere with viral functions at multiple levels.

Viral infection of cells within the tumor microenvironment mediates antitumor immunotherapy via selective TBK1-IRF3 signaling.

Activating intra-tumor innate immunity might enhance tumor immune surveillance. Virotherapy is proposed to achieve tumor cell killing, while indirectly activating innate immunity. Here, we report that recombinant poliovirus therapy primarily mediates antitumor immunotherapy via direct infection of non-malignant tumor microenvironment (TME) cells, independent of malignant cell lysis. Relative to other innate immune agonists, virotherapy provokes selective, TBK1-IRF3 driven innate inflammation that is associated with sustained type-I/III interferon (IFN) release. Despite priming equivalent antitumor T cell quantities, MDA5-orchestrated TBK1-IRF3 signaling, but not NFκB-polarized TLR activation, culminates in polyfunctional and Th1-differentiated antitumor T cell phenotypes. Recombinant type-I IFN increases tumor-localized T cell function, but does not mediate durable antitumor immunotherapy without concomitant pattern recognition receptor (PRR) signaling. Thus, virus-induced MDA5-TBK1-IRF3 signaling in the TME provides PRR-contextualized IFN responses that elicit functional antitumor T cell immunity. TBK1-IRF3 innate signal transduction stimulates eventual function and differentiation of tumor-infiltrating T cells.

Aryl Hydrocarbon Receptor Signaling Controls CD155 Expression on Macrophages and Mediates Tumor Immunosuppression.

Crosstalk between costimulatory and coinhibitory ligands are a prominent node of immune cell regulation. Mounting evidence points toward a critical role for CD155, the poliovirus receptor, in suppressing T cell function, particularly in cancer. However, relative to other known costimulatory/coinhibitory ligands (e.g., CD86, CD80, PD-L1), the physiological functions of CD155 and the mechanisms controlling its expression remain unclear. We discovered that CD155 expression is coregulated with PD-L1 on tumor-associated macrophages, is transcriptionally regulated by persistently active aryl hydrocarbon receptor (AhR), and can be targeted for suppression via AhR inhibition in vivo. Therapeutic inhibition of AhR reversed tumor immunosuppression in an immune competent murine tumor model, and markers of AhR activity were highly correlated with tumor-associated macrophage markers in human glioblastomas. Thus, CD155 functions within a broader, AhR-controlled macrophage activation phenotype that can be targeted to reverse tumor immunosuppression.