Figure 2.

The TIGIT/CD155 axis mediates resistance to immunotherapy from functional assays using clinical samples. (A and B) Expression of TIGIT, LAG-3, and TIM-3 ligands in tumor cells. We selected CD155 and CD112 as TIGIT ligands, galectin-3, and MHC class II (MHC-II) as LAG-3 ligands, and galectin-9 as a TIM-3 ligand. Representative flow cytometry staining (A) and summaries (B) are shown. (C) TIGIT, LAG-3, and TIM-3 expression in tumor-infiltrating T cells. Surgically resected tumors were digested, and the digested products before culture and expansion were subsequently analyzed with flow cytometry. (D) IFN-γ release assay of MEL02 (left) and MEL04 (right). Autologous tumor cells and paired TILs were cocultured with or without each immune checkpoint inhibitors for 24 hours. Supernatants were analyzed with ELISA for IFN-γ. Anti-MHC-I mAb was used for a negative control. Fold changes to samples with control mAb are shown. (E) CD155 expression in MEL04 tumor cells. The PVR gene was knocked out in the MEL04 cell line using CRISPR/Cas9 technology (PVR-KO), and the created cell line was analyzed by flow cytometry. Representative flow cytometry staining from triplicated experiments is shown. (F) IFN-γ release assay of MEL04. In vitro experiments were performed as described in (D). All in vitro experiments were performed in triplicate, and one-way analysis of variance with Bonferroni corrections were used in (B), (D), and (F) for statistical analyses. The means and SEM are shown. ****, p<0.0001; ns, not significant; IFN-γ, interferon-γ; MFI, mean fluorescent intensity; TIL, tumor-infiltrating lymphocyte.