Immune Checkpoint Protein Vista Suppresses Toll-Like Receptor Signaling And The Production Of Inflammatory Cytokines

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. Previous studies have shown that Vsir-/- mice developed chronic inflammatory phenotypes, and Vsir-/- CD4+ and CD8+ T cells were hyper-responsive towards self- and foreign antigens. Whether VISTA regulates innate immunity is still unknown. Methods: Peritoneal macrophages from WT or Vsir-/- mice were isolated and stimulated with TLR agonists such as CpG (TLR9), R848 (TLR7), LPS (TLR4), Pam3csk4 (TLR2), and poly (I:C) (TLR3). 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. The ubiquitination status of key signaling molecules such as TRAF6, IRAK1/4 and MyD88 was examined by immunoprecipitation and Western Blotting. To examine the role of VISTA in regulating TLR-mediated inflammatory responses in the context of cancer vaccine, 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 within the tumor microenvironment (TME) was examined via quantitative RT-PCR. Results: Vsir-/- macrophages were hyper-responsive towards TLR2/4/7/9 agonists, but not TLR3 agonist, resulting in increased production of inflammatory cytokines IL-6, IL-12, and TNFa. Analysis of signaling cascade revealed that VISTA inhibited the activation of MyD88-dependent TLR signaling, via suppressing the activation of MAPKs, and the activation of transcription factors AP-1 and NF-kB. Consistent with the role of VISTA in regulating TLR-mediated innate immunity, treatment with VISTA-blocking mAb augmented levels of inflammatory cytokines and chemokines within the TME, and synergized with TLR/peptide vaccine, resulting in an optimal therapeutic outcome. Conclusions: Our study establishes that VISTA critically regulates the inflammatory responses of myeloid cells mediated by TLR signaling. In the context of cancer vaccine therapy, VISTA-blocking mAb treatment enhanced levels of inflammatory cytokine and chemokines within the TME, which is critical for the development of optimal tumor-specific T cell responses and the tumor-controlling therapeutic outcome. Citation Format: Wenwen Xu, Ying Yuan, Na Li, Yongwei Zheng, Kamal Rajasekaran, Halli Miller, Michael Olson, Demin Wang, Subramaniam Malarkannan, Li Wang. Immune checkpoint protein VISTA suppresses Toll-like receptor signaling and the production of inflammatory cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2017-2996