Fig 1: Combining anti-PD-L1 with non-replicative Ad5-CMV-mTNFa/mIL-2 improves tumor-infiltrating T cell bioenergetics and addition of virus induces a tertiary lymphoid structure gene signature in MOC1 ICI refractory tumors. (A) Bioenergetics map of MOC1 anti-PD-L1 refractory tumor-infiltrating lymphocytes (CD3+) isolated by magnetic bead (negative selection) approach and analyzed by Seahorse XF analyzer. Data is presented by basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). (B) Corresponding real-time ATP production rate by glycolysis and aerobic respiration as quantified by Seahorse XF analyzer software. (C–E) Bulk RNA-Seq of CD45+/- populations isolated by from MOC1 anti-PD-L1 refractory tumors by magnetic bead approach. Heat maps for significantly differentially expressed genes between anti-PD-L1 refractory tumors and anti-PD-L1 refractory tumors treated with Ad5-CMV-mTNFa/mIL-2. Heat maps are organized into (C) T cell activation genes, immune checkpoints and MHC genes (D) B-cell activation genes, (E) typical tertiary lymphoid structure genes. (F) Fold change increase of significantly differentially expressed genes related to immunoglobulin synthesis between anti-PD-L1 refractory tumors and anti-PD-L1 refractory tumors treated with Ad5-CMV-mTNFa/mIL-2. (G, H) Immunohistochemistry of tertiary lymphoid structure related markers (HE, Ki67, CD19, CD3 and CD45r) from anti-PD-1 refractory tumors treated with (H) or without (G) non-replicative Ad5-CMV-mTNFa/mIL-2. Red boxes highlight positively stained areas (brown) for respective marker indicated in the bottom left of each panel. DESeq2 was used to compare gene expression between groups and the Walkd test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 were determined as differentially expressed genes. Statistical significance for ATP production rate was evaluated using one-way ANOVA and presented as means ± SEM with significance represented as *p < 0.05, and ****p < 0.0001.