**2. Activation of the Innate Anti-Tumor Immune Response by NDV**

The type I IFN pathway plays a central role in mediating antiviral immunity in mammals [36]. Type I IFNs have antiviral, proapoptotic, and immunomodulatory effects, all of which contribute in large part to the mechanism by which NDV induces antitumor response [36,37]. Type I IFN production in response to viral infection within the tumor microenvironment may have direct antiproliferative effects in some tumors [38]. More significantly, type I IFN signaling activates both innate and adaptive immunity through recruitment of innate cells including natural killer (NK) cells and antigen-presenting cells (APCs), upregulation of cell adhesion, major histocompatibility complex (MHC) and costimulatory molecules, and priming of antigen-specific T cells [37,39–42]. Thus, activation of type I IFN signaling is one of the key pathways being explored for cancer immunotherapy, and this is supported by the findings that tumors with high CD8+ T cell proliferation and responsiveness to immune checkpoint inhibitors are enriched for genes associated with type I IFN signaling [43].

Upon NDV infection, pathogen-associated molecular patterns (PAMPs) inherent to the virus and danger-associated molecular patterns (DAMPs) released by dying cells are recognized by pattern recognition receptors (PRRs) including extracellular Toll-like receptors (TLRs) 3, 7, 8, and 9; intracellular nucleotide-binding oligomerization domain (NOD) proteins; and intracellular RNA helicases such as RIG-1 or MDA5 [44,45] (Figure 1). Recognition of PAMPs and DAMPs by PRRs leads to the activation of transcription factors including IFN regulatory factor (IRF)3, IRF7, and nuclear factor kappa B via the adaptors interferon β stimulator-1 and stimulator of interferon genes (STING) [44]. This signaling cascade results in the transcription and expression of genes encoding proinflammatory cytokines and type I and type III IFN proteins [19,44]. In the case of NDV, cytosolic RNA generated by NDV infection is sensed by RIG-1, and reduction of RIG-1 protein levels has been shown to correlate with decreased intensity of type I IFN response to NDV in vitro [23,46] (Figure 1).

Tumor cells often have impaired type I IFN signaling, which is one of the principal mechanisms resulting in increased tumor cell sensitivity to NDV infection. Despite these deficiencies, the impairment in type I IFN production is typically not absolute, especially as NDV is capable of infecting normal cells in the tumor microenvironment, which have preserved type I IFN response [46–48]. Transcriptional profiling of mouse tumors injected with NDV reveals upregulation of type I IFN response-related genes and a range of cytokines and chemokines that mediate recruitment and proliferation of innate and adaptive immune cells [47,49]. Interestingly, this signature was shown to be independent of NDV-mediated replicative or lytic potential in a study utilizing the lentogenic NDV LaSota strain, indicating that type I IFN signaling activated to even a limited virus infection is sufficient to drive the inflammatory response [49].

While a strong type I IFN response to NDV results in a proinflammatory tumor microenvironment that contributes to the antitumor response, it may, on the other hand, limit therapeutic efficacy by suppressing NDV replication and virus-mediated lysis. Indeed, pretreatment with type I IFN has been shown to limit NDV replication in some tumor cell lines [20,23,46,48]. Therefore, a key unanswered question in the field concerns the timing of type I IFN induction, whereby a balance should be achieved between adequate virus replication and tumor lysis and induction of innate immune response to promote further adaptive immunity. A recombinant lentogenic NDV strain (Hitchner B1) expressing the influenza A virus IFN antagonist protein NS1, which suppresses RIG-1 receptor signaling, IRF3 dimerization, and expression of IFN-β, potently reduced IFN signaling across a panel of cancer cell lines and resulted in increased NDV replication and cytolysis [50]. In vivo, this virus was more effective in controlling tumor growth and prolonging survival in a syngeneic melanoma mouse model [50]. Similar results were demonstrated using the recombinant mesogenic Beaudette C NDV strain expressing an

IFN-antagonist protein which showed higher efficiency in tumor regression in a xenotransplanted fibrosarcoma mouse model [47]. Despite these findings, type I IFN has been shown to be essential for antitumor activity of NDV, and in mice lacking type I IFN receptor, the virus exhibited no ability to control tumor growth [51]. did not, suggesting that while NK cells are important early responders to NDV infection, their role appears to be essential only for the initial inflammatory response [53,54]. Last, NDV infection also results in the recruitment of myeloid cells, which have important roles in phagocytosis and antigen presentation [54,55].

model abrogated antitumor efficacy, while depletion of NK cells concomitantly with NDV treatment

*Cancers* **2020**, *12*, x 4 of 16

showed higher efficiency in tumor regression in a xenotransplanted fibrosarcoma mouse model [47]. Despite these findings, type I IFN has been shown to be essential for antitumor activity of NDV, and in mice lacking type I IFN receptor, the virus exhibited no ability to control tumor growth [51].

In addition to activation of tumor cell-inherent type I IFN signaling, the inflammatory environment generated by NDV results in the recruitment of innate effector cells and adaptive immune cells (discussed below) that contribute to antitumor immunity (Figure 1). In particular,

**Figure 1.** Newcastle disease virus (NDV) activates innate and adaptive anti-tumor immune responses. NDV selectively infects tumor cells that have defective anti-viral defenses. Extracellular and intracellular signaling mediated by sensors such as the RNA helicase RIG-1 leads to expression of type I IFN and related genes. Autocrine and paracrine IFN signaling upregulates MHC class I and II presentation, co-stimulatory molecules, and immune checkpoints on the cell surface. The release of cytokines and chemokines in addition results in the recruitment of innate effector cells such as NK cells and macrophages and antigen-presenting cells (APCs). Virus-mediated direct oncolysis leads to release of tumor antigens, PAMPs, and DAMPs that activate APCs including dendritic cells capable of antigen cross-presentation. Activated APCs prime T cells, resulting in generation of cytolytic T cells directed toward tumor and viral antigens; however, effector function of the activated T cells can be inhibited by upregulation of PD-L1 on tumor cells and APCs, and PD-1 and CTLA-4 on T cells. Upregulation of these negative feedback mechanisms provide the rationale for combining NDV with immune checkpoint inhibitors. **Figure 1.** Newcastle disease virus (NDV) activates innate and adaptive anti-tumor immune responses. NDV selectively infects tumor cells that have defective anti-viral defenses. Extracellular and intracellular signaling mediated by sensors such as the RNA helicase RIG-1 leads to expression of type I IFN and related genes. Autocrine and paracrine IFN signaling upregulates MHC class I and II presentation, co-stimulatory molecules, and immune checkpoints on the cell surface. The release of cytokines and chemokines in addition results in the recruitment of innate effector cells such as NK cells and macrophages and antigen-presenting cells (APCs). Virus-mediated direct oncolysis leads to release of tumor antigens, PAMPs, and DAMPs that activate APCs including dendritic cells capable of antigen cross-presentation. Activated APCs prime T cells, resulting in generation of cytolytic T cells directed toward tumor and viral antigens; however, effector function of the activated T cells can be inhibited by upregulation of PD-L1 on tumor cells and APCs, and PD-1 and CTLA-4 on T cells. Upregulation of these negative feedback mechanisms provide the rationale for combining NDV with immune checkpoint inhibitors.

In addition to activation of tumor cell-inherent type I IFN signaling, the inflammatory environment generated by NDV results in the recruitment of innate effector cells and adaptive immune cells (discussed below) that contribute to antitumor immunity (Figure 1). In particular, intratumoral NDV injection leads to a significant tumor infiltration with natural killer (NK) cells [42,52,53]. Interestingly, depletion of NK cells prior to NDV treatment in a syngeneic mouse tumor model abrogated antitumor efficacy, while depletion of NK cells concomitantly with NDV treatment did not, suggesting that while NK cells are important early responders to NDV infection, their role appears to be essential only for the initial

inflammatory response [53,54]. Last, NDV infection also results in the recruitment of myeloid cells, which have important roles in phagocytosis and antigen presentation [54,55].
