Introduction: V-domain Ig suppressor of T-cell activation (VISTA, gene Vsir) is an inhibitory immune-checkpoint molecule that suppresses CD4+ and CD8+ T-cell activation. Vsir-/- mice developed chronic inflammatory phenotypes, and Vsir-/- CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Our recent study (Li et al., Sci Rep 2017) has identified a novel role of VISTA as a critical regulator of IL-23/IL-17 inflammatory axis induced by Toll-like receptor (TLR) stimulation. The molecular mechanisms by which VISTA inhibits TLR signaling remain to be elucidated.

Methods: Peritoneal macrophages from WT or Vsir-/- mice were isolated and stimulated with TLR agonists. Alternatively, human monocyte THP-1 cells overexpressing VISTA were stimulated by TLR2 agonist Pam3CSK4. The activation of TLR signaling pathways and the production of inflammatory cytokines were examined by Western blotting, gel shift assay, or ELISA. Tumor-bearing mice were treated with VISTA-specific monoclonal antibody (mAb) and a peptide vaccine containing TLR agonists. The production of inflammatory cytokines and chemokines was examined via RT-PCR and ELISA.

Results: VISTA downregulates Toll-like receptor (TLR)/TRAF6/TAK1-mediated signaling pathway via promoting K48-linked polyubiquitination and proteasomal degradation of TRAF6 and inhibiting K63-linked polyubiquitination and activation of TRAF6. VISTA blockade by an antibody or genetic deletion augments the activation of MAPKs/AP-1 and IKK/NF-kB signaling cascades in myeloid cells and induces the accumulation of inflammatory cytokines and chemokines within tumor tissues. Inflamed tumor tissues promote the infiltration and effector function of tumor-reactive CD8+ T cells. TLR/TRAF6-mediated inflammatory responses promote the antitumor efficacy of VISTA-blocking antibodies and contribute to a synergistic outcome when VISTA blockade is combined with a TLR agonistic vaccine.

Conclusions: Our study establishes that VISTA critically regulates the inflammatory responses of myeloid cells mediated by TLR signaling. Unlike targeting other immune checkpoint proteins, the therapeutic efficacy of VISTA inhibition benefits from the activation of myeloid cells and early induction of inflammatory cytokines may predict positive clinical responses.

Citation Format: Wenwen Xu, Yongwei Zheng, Juan Zhou, Ying Yuan, Hieu Minh Ta, Jun Dong, Halli E. Miller, Michael Olson, Kamalakannan Rajasekaran, Marc S. Ernstoff, Demin Wang, Subramaniam Malarkannan, Li Wang. Immune checkpoint protein VISTA controls antitumor immunity via regulating Toll-like receptor signaling and myeloid cells-mediated inflammation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A82.