Anti–PD-L1 treatment decreases intrahepatic tumor burden and improves antitumor immune responses against C1498.GFP. (A) Cohorts of C57BL/6 mice were inoculated with 10⁶ C1498.GFP cells IV, and received 200 μg of anti–PD-L1 or isotype control antibody IP on days 0, 3, 6, and 9. On day 12, livers were removed, and single-cell suspensions were generated, pooled between mice, and analyzed by flow cytometry for the percentage of GFP⁺ cells present. (B) Liver sections from C57BL/6 mice treated with isotype control (left) or PD-L1 blocking antibody (right) were stained with hematoxylin and eosin and imaged with a Zeiss Axioshop histology microscope using bright-field objective lenses. Top panels, 4× magnification; bottom panels, 20× magnification. Images were acquired with Zeiss Imaging Software, Version 4.7.1. (C) Spleens were harvested from tumor-challenged C57BL/6 mice treated with isotype control or PD-L1 blocking antibody 12 days after injection with C1498.GFP. A total of 10⁶ splenocytes from individual mice were restimulated overnight with either complete DMEM, 5 × 10⁴ irradiated C1498.GFP cells (10 000 rad), or PMA plus ionomycin in an IFN-γ ELISPOT assay. Bars represent the mean number of spot-forming cells (± SD) in each group. P = .001 for the comparison between mice treated with PD-L1 blocking antibody versus isotype control antibody. (D) Livers were harvested from C57BL/6 mice 12 days after C1498.GFP injection. Sections from various mice were stained with anti-CD4 and -CD8 antibodies, and the numbers of CD4⁺ and CD8⁺ cells from each liver were enumerated per high-power field (hpf; 40×). Similar results were obtained in 3 independent experiments.