Adaptive resistance mediated by inhibitory ligands such as PD-L1 has emerged as an important mechanism of malignant cell survival and spurred the development of new agents that disrupt the PD-L1/PD-1 immune checkpoint. 1 Analysis of patient specimens from clinical trials of novel immune checkpoint inhibitors indicates that high basal expression of PD-L1 on tumor cells may predict sensitivity to and be necessary to elicit significant clinical benefit from this drug class. 2, 3 These data suggest that strategies that increase PD-L1 levels could potentially prime malignant cells with low PD-L1 expression and render them sensitive to anti-PD-1/PD-L1 blockade. To investigate this possibility, we first conducted an analysis of basal PD-L1 transcript levels in multiple myeloma (MM) patients (n= 351) from the total therapy 2 trial. These analyses demonstrated that expression is very heterogeneous among MM patients and that MM patients as a group do not significantly overexpress PD-L1 compared with normal plasma cells (Supplementary Figure 1). Consistent with the low basal PDL1 expression we identified, significant clinical responses were not observed in any MM patients in a phase I study of nivolumab in patients with relapsed/refractory lymphoid malignancies. 4 The oncolytic reovirus-based anticancer agent Reolysin is known to have significant immunomodulatory effects and has demonstrated promising preclinical efficacy in MM models and favorable safety and tolerability in early MM clinical trials. 5–7 We hypothesized that Reolysin treatment could be used as a precision priming strategy to potentiate the anti-MM efficacy of PD-1/PD-L1 targeted therapy by promoting myeloma immune recognition and PD-L1 upregulation. To test this possibility, we infected a panel of MM cell lines and normal B, plasma and activated T cells with 30 plaque forming units per cell Reolysin for 48 h. Reolysin treatment produced significant and selective reovirus replication in MM cell lines, but not in normal B cells (Supplementary Figure 2 and Supplementary Methods). Expression profiling analyses of U266 and RPMI-8226 cells demonstrated that Reolysin treatment induced a marked increase in CD274 (PD-L1) levels (Figure 1a). Quantitative RT-PCR analyses of MM cell lines, normal B, plasma and activated T cells treated with Reolysin revealed a significant and selective induction of PD-L1 in MM cell lines (Figure 1b). The results were confirmed by immunoblotting in MM cell lines (Figure 1b). Reolysin also upregulated PD-L2 expression in a manner that mirrored PD-L1, albeit to a lesser extent than PD-L1 induction (Supplementary Figures 3A and B). Importantly, flow cytometric analysis showed that Reolysin significantly increased the cell surface expression of PD-L1 in both MM cell lines and primary CD138+ cells from MM patients (Figure 1c, Supplementary Figures 4A and B and Supplementary Table 1). Since reovirus failed to replicate and induce PD-L1 expression in normal B cells, we investigated whether active reovirus replication was essential for PD-L1 upregulation. A comparison of the anti-MM effects achieved by live reovirus versus UV-inactivated reovirus demonstrated that live reovirus is required to decrease MM cell viability (Supplementary Figure 5A) and upregulate PD-L1 expression (Supplementary Figures 5B and C). PD-L1 levels can be induced by interferon exposure. To determine if Reolysin-induced PD-L1 expression was mediated by interferon, we blocked interferon-γ using a neutralizing antibody, which significantly reduced Reolysin-mediated PD-L1 upregulation (Supplementary Figure 6). This study reveals that the interferon-γ/Janus kinase …