VISTA Ligation Reduces Antitumor T-Cell Activity in Pancreatic Cancer

**Background:** Pancreatic ductal adenocarcinoma (PDAC) has a devastating 5-year survival rate of only 11% and has been largely refractory to current immunotherapies. The V-domain Ig suppressor of T-cell activation (VISTA) is an immune checkpoint protein that inhibits T-cell effector function and maintains peripheral tolerance. While VISTA has been investigated in several cancers, its role in PDAC was largely unknown. This study aimed to characterize VISTA expression patterns in PDAC and evaluate its potential as a therapeutic target.

**Methods:** The authors assessed VISTA expression in 76 PDAC tissue sections and 5 adjacent nontumorous pancreatic tissue samples using immunohistochemistry, and in 67 PDAC samples using multiplex immunofluorescence staining. VISTA expression on tumor-infiltrating immune cells and matched blood samples from 13 PDAC patients was measured via multicolor flow cytometry. In vitro, T cells were cocultured with the AsPC-1 pancreatic cancer cell line and treated with recombinant VISTA protein to assess effects on TNFα and IFNγ production. In vivo, an orthotopic PDAC mouse model using TB32048 cells in C57BL/6 mice was treated with a blocking VISTA antibody (200 µg) or control protein, with tumor weights measured on Day 18. TCGA and GTEx RNA-seq data (179 PDAC, 171 normal pancreas) were also analyzed.

**Key Results:** PDAC tissue showed significantly higher VISTA expression compared to nontumorous pancreas. Patients with a high density of VISTA-expressing tumor cells (VISTA+ PanCK+) had reduced overall survival (trend, p not explicitly stated for survival analysis). VISTA expression on CD4+ and CD8+ T cells was increased after stimulation and particularly after coculture with tumor cells. Recombinant VISTA protein reversed the stimulation-induced increase in TNFα and IFNγ expression by both CD4+ and CD8+ T cells. In the orthotopic mouse model, VISTA blockade resulted in significantly reduced tumor weights compared to control. At the mRNA level, VSIR (VISTA gene) showed the highest expression among all tested immune checkpoint modulators in PDAC. High VSIR expression was associated with high levels of leukocyte marker PTPRC (CD45), T-cell marker TBX21, and APC marker HLA-DRA.

**Clinical Implications:** This study provides evidence that VISTA is highly expressed in PDAC and that its expression on tumor cells, rather than on immune cells, is associated with worse survival. The ability of VISTA blockade to reduce tumor growth in a mouse model supports the potential of anti-VISTA immunotherapy for PDAC, a cancer type that has not benefited from existing checkpoint inhibitors. These findings align with ongoing Phase I clinical trials testing anti-VISTA antibodies in advanced solid tumors, including PDAC. Limitations include the use of a single cell line (AsPC-1) for in vitro experiments, the small number of normal pancreas samples (n=5), and the inherent limitations of mouse models in predicting human responses.