Fig. 2. PD-L1 inhibitors secreted from VV-iPDL1/GM-infected cells bind to PD-L1 on tumor cells and immune cells.

a MC38 tumor cells were infected with VV-RFP, VV-iPDL1/GM at an MOI = 0.5, or PBS for 24 h. The percentage of IgG Fc⁺ population representing iPDL1 (soluble PD-1-IgG Fc)-bound VV-infected (RFP⁺) or uninfected (RFP⁻) PD-L1-expressing tumor cells was measured by flow cytometry. b MC38 tumor cells that were stimulated with IFN-γ (20 ng/mL) for 48 h were infected with the indicated VVs. PD-L1 expression of infected (RFP⁺) or uninfected (RFP⁻) cells was determined by flow cytometry. c–g MC38 cells were subcutaneously inoculated into the left (1 × 10⁶) and right (5 × 10⁵) flanks of C57BL/6 mice. When left flank tumor sizes reached ~100 mm³ (counted as day 0), the tumors of the left flank were intratumorally injected with 50 μL of PBS, VV-RFP, VV-GM, or VV-iPDL1/GM (5 × 10⁷ pfu per tumor), or 200 μg of anti-PD-L1 antibody (clone 10F.9G2) intravenously on days 0 and 3. Two days post-second VV treatment, VV-treated (primary tumor) and untreated, distant tumors were collected, weighed and digested with collagenase type I and DNase. Tumor cell suspensions were blocked with anti-CD16/32 antibody and then stained with antibodies against CD45, CD3, CD8, CD4, CD11c, CD11b, Gr-1, FoxP3, PD-L1, and IgG Fc to assess PD-L1 expression or IgG Fc⁺ frequency on infected (RFP⁺) or uninfected (RFP⁻) tumor cells from the treated primary tumors c or untreated distant tumors (CD45⁻ cells) d, and PD-L1 expression on infiltrating immune cells from treated e or untreated distant tumors f, IgG Fc⁺ frequency on infiltrating immune cells g. Infiltrating immune cells include cytotoxic T cells (CD45⁺CD3⁺CD8⁺), DCs (CD45⁺CD11c⁺), myeloid-derived suppressor cell MDSCs (CD45⁺CD11c⁻CD11b⁺Gr-1⁺), and Treg (CD45⁺CD3⁺CD4⁺FoxP3⁺); n = 5 mice. Significant differences are indicated as **P < 0.01, ***P < 0.001, or ****P < 0.0001 determined by two-tailed Student’s t-test.