Fig 2: Intrathecal injection of PD-L1 promotes recovery and attenuate inflammation response. (A–C) Statistical analysis of the Louisville Swim Scale and BMS score of mice in different groups after SCI (n = 5 mice per group at each time point, values are the mean ± SD, *p < 0.05, **p < 0.01 vs. PD-L1 Protein, #p < 0.05, ##p < 0.01 vs. Anti-PD-L1 mAb, repeated measurement analysis of variance). (D, E) Statistical analysis of the mechanical and thermal threshold of mice in different groups after SCI. (n = 5 mice per group at each time point, values are the mean ± SD, *p < 0.05, **p < 0.01 vs. PD-L1 Protein, #p < 0.05, ##p < 0.01 vs. Anti-PD-L1 mAb, repeated measurement analysis of variance). (F–I) Flow cytometry analysis of CD11b+ iNOS+ and CD11b+ CD206+ cells in different groups at day 14 post-SCI (n = 6 per group, values are the mean ± SD, *p < 0.05, **p < 0.01, one-way ANOVA).

Fig 3: scFv PD-L1 trans-binding activity to PD-L1 on the cell surface of bystander cellsIFN?-treated EMT6 cells maximally infected with RRV-scFv-HF PDL1 (HA-tagged scFv-PD-L1) or RRV-GFP at ratios indicated in parenthesis were split into 2 sets. (A) One set of cells was stained with Alexa Fluor 647-conjugated anti-HA antibody and (B) the second set of cells was stained with PE-conjugated anti-mouse PD-L1 antibody. HA-positive, PD-L1-positive, and GFP-positive cell populations were measured by flow cytometric analysis.

Fig 4: Pre-transduced tumor cells expressing scFv PD-L1 demonstrate a dose-dependent anti-tumor activity and enhanced tumor infiltrating lymphocytes(A) Subcutaneous tumor model using Tu-2449SC cells maximally infected with RRV-scFv PDL1 or RRV-GFP at indicated ratios were subcutaneously implanted on the right flank of 8-week-old female B6C3F1 mice and assigned to indicated groups (n = 10 per group). Control groups are mice implanted with 100% Tu-2449SC/RRVGFP cells that received PBS (0% scFv) or anti-mouse PD-1 antibody (0% scFv + a-PD-1). Anti-PD-1 antibody (Clone J43) was i. p. administered on day 0 (300 µg per mouse), day 3, day 6 and day 9 (200 µg per mouse). Statistical significance was determined by 2-way ANOVA of the following data sets: *p < 0.0001 for 0% vs 30% scFv; **p < 0.0001 for 0% vs 100% scFv. (B) Naïve mice (n = 5) and mice that cleared their initial tumor implant from RRV-scFv-PDL1 treated group (n = 15) were challenged with 2 × 106 Tu-2449SC on the left side of the flank. Tumor growth and measurement were monitored over time. *p < 0.0001 (0% scFv vs. 2%, 30% and 100% scFv). Error bars indicate the standard error measurement (SEM) of the dataset. (C) and (D) Frequency of tumor-infiltrating CD4+ (C) and CD8+ (D) lymphocytes among CD45+ cells by flow cytometry, 39 days post-tumor implantation. Statistical analyses were performed using Mann-Whitney test at 95% CI (*P < 0.05, **P < 0.01). Values represent means ± SD of the dataset.

Fig 5: scFv PD-L1 serum levels in tumor-bearing mice(A, B) Sera from Tu2449 subcutaneous tumor pre-transduced with 30% RRV-scFv-PDL1 and (C, D) Tu-2449 intracranial tumor treated with isotype, pre-transduced with 100% RRV-scFv-PDL1 or injected with 1 × 106 TU of RRV-scFv-PDL1 were collected at indicated time point post tumor implant. scFv PD-L1 serum concentrations were measured by surface plasmon resonance with standard curves at concentrations ranging between 0.1 nM–200 nM (0.0028 µg/mL-5.6 µg/mL) using naïve serum spiked-in with purified scFv PD-L1 and serve as positive controls. Sera from RRV-GFP pre-transduced tumor or isotype antibody treated tumor-bearing mice were included for baseline levels. Each symbol represents one mouse serum sample. Each colored symbol except black represents one mouse serum sample. Black, standard curves generated for every 20 cycles of regeneration. (B) and (D) Graphical presentation of the data conversion from responsive unit shown in (A) and (C) to µg/mL. The lower limit of quantification (LLOQ) is determined to be 0.011 µg/mL (0.39 nM) and the dashed horizontal line indicates LLOQ of the assay. For graphical presentation, samples measured below LLOQ were entered with a fixed value of 0.010 µg/mL. The data represent one data set from two separate experiments.