*5.2. Adaptive Anti-Tumour Immunity*

In addition to PAMPs and DAMPs, tumour-associated antigens (TAAs) are also released into the TME during oncolysis. TAAs are phagocytosed by antigen presenting cells (APCs), such as DCs, and the cytokine-rich milieu stimulates DC maturation [102]. Reovirus-activated DCs cross-present TAAs via major histocompatibility complex (MHC) class I to naive CD8+ve T cells [102,103]. These processes facilitate the priming of tumour-specific cytotoxic T lymphocytes (CTLs) [89,102,103]. Interestingly, direct reovirus oncolysis is not essential to generate adaptive anti-tumour immunity, as tumour-specific CTLs have been successfully generated against reovirus-resistant melanoma cells in vivo [104]. Thus, even if a particular cancer is not killed directly by the lytic effects of reovirus, reovirus treatment may offer immunotherapeutic value for patients. By contrast, a recent study by Martin et al., [105] suggested that reovirus was ineffective at priming a systemic immune response compared to alternative OVs, despite effective eradication of the primary tumour. These conflicting data are difficult to interpret; however, the discrepancies observed could be due to the different mouse strains; previous studies [103,104] have utilized TH1-dominant C57BL/6 mouse models, whilst this later study used TH2 dominant Balb/c mice. Of note, Martin et al., did not examine the induction of tumour specific CTLs but eradication of a secondary tumour. Therefore, it is possible that reovirus did prime effector CTLs which were inhibited due to the upregulation of immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1), or the induction of regulatory T cells (Tregs) within the TME. Indeed, it is important to note that reovirus can promote the accumulation of Tregs and MDSCs [106–108] and also upregulates immune checkpoint molecules [108–110], which could impede both effector NK cell and CTL responses. Figure 2 (the inner circle) provides an overview of known reovirus mechanisms of action, including oncolysis and the induction of innate and adaptive anti-tumour immunity.

"switched on" **Figure 2.** Overview of reovirus mechanisms of action and the developments required. The **inner circle** illustrates what is currently known about reovirus. **1.** In healthy cells, anti-viral immune responses limit reovirus replication and prevent lytic killing. By contrast, oncogenic signalling pathways render tumour cells susceptible to reovirus replication and direct oncolysis. **2.** Reovirus replicates in the tumour vasculature and stroma due to reciprocal cell:cell interactions which alter anti-viral signalling. **3.** Infection of tumour cells leads to the release of viral progeny, cytokines and tumour-associated antigens (TAAs), which initiates innate anti-tumour immunity including cytokine-mediated bystander killing and natural killer (NK) cell-mediated cytotoxicity. **4.** Adaptive anti-tumour immunity is generated following the phagocytosis of TAAs by dendritic cells (DCs) and presentation of TAAs to CD4 <sup>+</sup>ve and CD8 <sup>+</sup>ve T cells, which facilitates priming of tumour-specific cytotoxic T lymphocytes (CTLs). **5.** In addition to innate and adaptive anti-tumour immune responses, humoral anti-viral immunity is induced, leading to the production of reovirus-specific neutralising antibodies (NAbs). **6.** Following induction of anti-tumour/anti-viral immune responses, regulatory immune mechanisms are "switched-on" to control ongoing immune responses, including upregulation of immune checkpoints and increased levels of regulatory T cells (Tregs) and/or myeloid-derived suppressor cells (MDSCs). The **outer circle** highlights priority research areas to improve reovirus efficacy. These include gaining a greater understanding of: (i) the consequence of humoral and/or cell-mediated anti-viral immunity on reovirus efficacy which would inform the development of, or the requirement for, cellular chaperones; (ii) the tumour microenvironment (TME) and how it influences reovirus oncolysis and anti-tumour immunity; (iii) the cellular determinants utilized by reovirus for direct oncolysis, including mechanisms of reovirus resistance; (iv) the potential benefits of genetically-modified reovirus platforms; (v) reovirus scheduling to maximize virus delivery and efficacy including the best route of virus administration; and (vi) combinatorial approaches that are designed to boost both direct oncolysis and anti-tumour immune responses. PAMPs: pathogen-associated molecular patterns; DAMPs: damage-associated molecular patterns; GM-CSF: granulocyte-macrophage colony stimulating factor. Figure created using Biorender.com.

In a recent phase I study of intravenous (i.v.) reovirus there was an increase in transcripts of the pro-recruitment chemokines macrophage inflammatory protein (MIP)-1α and MIP-1β in tumour RNA and in the expression of the intracellular adhesion molecule 1 (ICAM-1) by T cells 48–72 h after infusion [109]. Along with CD68+ve myeloid cells, tumours of reovirus-treated vs. control patients appeared to contain a higher number of CD8+ve T cells [109], whose presence is strongly associated with superior outcomes [111]. Moreover, pro-inflammatory cytokines and IFN were upregulated in the serum of reovirus-treated patients [109,112], which can promote APC maturation and activate NK and T cells, as evidenced by the increased expression of CD69 [113]. Collectively, the evidence suggests that, as an immune adjuvant, reovirus can promote leukocyte infiltration into tumours and support tumour immune surveillance. However, to promote and sustain reovirus-induced anti-tumour immunity it is essential that long-term characterisation of the TME after reovirus treatment is carried out and that combination strategies are developed to counteract any inhibitory/regulatory mechanisms that develop.
