Effective Antitumor Immunity Can be Triggered by Targeting VISTA in Combination with a TLR3-specific Adjuvant.

T cell control could not be unleashed in tumors resistant to anti-PD-1/PD-L1 treatment, which appears to be associated with inhibitory macrophages. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a non-redundant immune checkpoint that can induce both T cell and myeloid cell immunosuppression. We determined high levels of VISTA+ immune cells were associated with advanced stage, and predicted poor survival in bladder cancer patients with or without bacillus Calmette-Guerin immunotherapy. Combination of high infiltration of VISTA+ immune cells and PD-L1+ immune cells or PD-1+ T cells predicted the worst prognosis. Flow cytometry and multiplex immunofluorescence staining confirmed that VISTA expression was higher in macrophages than that in T cells or neutrophils, and only VISTA+CD163+ macrophage density predicted poor prognosis in bladder cancer patients. Toll-like receptor (TLR) agonists can trigger the innate immune response in macrophages. VISTA mAb 13F3 augmented the ability of a TLR3-specific adjuvant to induce macrophage activation in vitro. In a MB49 syngeneic mouse model, treatment with VISTA mAb 13F3 curbed tumor growth and prolonged mice survival when combined with TLR3-specific adjuvant. The combination treatment reduced CD206+ anti-inflammatory macrophages and immunosuppressive molecule TGF-β1, but it up-regulated immunostimulatory molecules (Ifna, Ifnb and Trail) and increased CD8+ T cell/Treg ratio in the tumor microenvironment. These findings indicated that elevated VISTA expression in immune cells, particularly within macrophages, is closely associated with an unfavorable prognosis in patients with bladder cancer. Targeting VISTA with TLR3-specific adjuvant synergy triggered an effective anti-tumor immunity and delayed tumor growth in mice with bladder cancer.