VISTA: A Mediator of Quiescence and a Promising Target in Cancer Immunotherapy

Abstract

V-domain Ig suppressor of T cell activation (VISTA) is a B7 family member that maintains T cell and myeloid quiescence and is a promising target for combination cancer immunotherapy. During inflammatory challenges, VISTA activity reprograms macrophages towards reduced production of proinflammatory cytokines and increased production of interleukin (IL)-10 and other anti-inflammatory mediators. The interaction of VISTA with its ligands is regulated by pH, and the acidic pH ~6.0 in the tumor microenvironment (TME) facilitates VISTA binding to P-selectin glycoprotein ligand 1 (PSGL-1). Targeting intratumoral pH might be a way to reduce the immunoinhibitory activity of the VISTA pathway and enhance antitumor immune responses. We review differences among VISTA therapeutics under development as candidate immunotherapies, focusing on VISTA binding partners and the unique structural features of this interaction.

Keywords: Acidic tumor microenvironment; Cancer immunotherapy; Immunosuppression; PSGL-1; VISTA; VSIG3.

Figure 1.. Structure of Human V-Domain Ig Suppressor of T Cell Activation (VISTA) and Its Ligands.

(A) VISTA contains a ‘clamped’ stalk region of nine amino acids, in contrast to the longer stalk of programmed cell death protein 1 (PD-1) (~20 amino acids) [29]. The PD-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) (V/I/LxYxxL) and an immunoreceptor tyrosine-based switch motif (ITSM) (TxYxxL); cytotoxic T lymphocyte-associated protein 4 (CTLA-4) contains one SH2- (YxxM) and one SH3-binding (PxxP) motif [67]. The cytoplasmic domain of VISTA does not contain any immunoreceptor tyrosine-based signaling motifs [10]. However, it does have a conserved SH2-binding motif (YxxQ) in the middle of the cytoplasmic tail, but it remains to be tested which motifs/kinases are important for signaling [9]. (B) P-selectin glycoprotein ligand 1 (PSGL-1) is a homodimeric 240 kDa adhesion molecule that mediates leukocyte trafficking by binding to selectins, which involves adaptor proteins DNAX-activating protein of 12 kDa (DAP12) and Fc receptor γ (FcRγ), ezrin, radixin, and moesin (ERM) proteins, and Src and Syk kinases [68]. The V-set and Ig domain-containing 3 (VSIG3) extracellular domain (ECD) contains an N-terminal IgV-like domain and an IgC-like domain. VSIG3 can function as an adhesion molecule that regulates synaptic transmission and plasticity by binding to postsynaptic scaffolding protein PSD-95 [12]. (C) Summary of root mean square deviation (RMSD) of superimpositions of VISTA (PDB 6U6V) and other B7 family members shows that VISTA most closely resembles PD-L1. (D) The ECD of VISTA (PDB 6U6V) generated with Pymol (C–C′ loop and H strand, yellow). Inset 1, histidine rim; inset 2, VSTB mAb epitope; inset 3, disulfide bond joining strands A and H; inset 4, Ig disulfide bond; inset 5, disulfide bond joining the C–C′ loop to the F strand. (E) Epitope targeted by pH-selective VISTA.18 monoclonal antibody (mAb) (PDB 6MVL). Putative hydrogen bonds calculated by Pymol [69]. Numbering of all amino acid positions is from Met = 1.

Figure 2.. The Complexities of V-Domain Ig Suppressor of T Cell Activation (VISTA) Interactions.

In humans and mice, VISTA is highly expressed in the myeloid compartment, particularly in myeloid-derived suppressor cells (MDSCs), and in low amounts on tumor-infiltrating lymphocytes (TILs) [9]. VISTA can be expressed on cancer cells, although this is less common [33]. (A) Appropriately glycosylated and tyrosine sulfated P-selectin glycoprotein ligand 1 (PSGL-1) expressed on leukocytes binds to P-selectin molecules on the endothelium at physiological pH to facilitate leukocyte extravasation [70]. (B) In mice, VISTA signaling on myeloid cells can inhibit myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptor (TLR) signaling and cytokine production, and thereby induce a more immunosuppressive phenotype [50]. VISTA activity decreases proinflammatory cytokines and increases anti-inflammatory cytokines and mediators [50,51]. (C) In humans, the V-set and Ig domain-containing 3 (VSIG3) interaction with VISTA on T cells suppresses T cell activation and proliferation. This interaction is functional at neutral pH and the affinity declines fourfold at pH 6.0 [4]. (D–F) VISTA expressed on cancer cells, MDSCs, or tumor-associated macrophages (TAMs) can bind to PSGL-1 on T cells at acidic pH but not at neutral pH. The signaling pathways of VISTA and PSGL-1 after ligation at pH 6.0 remain uncharacterized. (G) In mice, PSGL-1 ligation with anti-mouse PSGL-1 (clone 4RA10) leads to inhibition of T cell receptor (TCR) signaling and cytokine production via reduced phospho-ERK (extracellular signal-regulated kinase), protein kinase B (AKT), and signal transducer and activator of transcription 5 (STAT5), thereby promoting T cell exhaustion [23]. Abbreviations: CCL3, chemokine (C-C motif) ligand 3; CCL5, chemokine (C-C motif) ligand 5; CXCL11, C-X-C motif chemokine 11; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; PD-1, programmed cell death protein 1; TNF, tumor necrosis factor.

Figure 3.. The Binding of Human V-Domain Ig Suppressor of T Cell Activation (VISTA) to P-selectin glycoprotein ligand 1 (PSGL-1) is pH-Dependent.

(Upper left) In acidic conditions, the VISTA histidines are protonated, allowing ionic interaction with negatively charged glutamic acid residues and sulfated tyrosines in PSGL-1 [3]. (Lower left) At pH 7.4, the histidine rim of VISTA is unprotonated and does not bind to PSGL-1. (Upper right) VISTA is highly expressed on myeloid-derived suppressor cells (MDSCs) and other myeloid cells. In the acidic tumor microenvironment (TME), PSGL-1 can interact with VISTA and mediate T cell and myeloid dysfunction. (Lower right) PSGL-1 is highly expressed on leukocytes such as T cells, and binding to selectins on endothelial cells at neutral pH mediates rolling of leukocytes along the vasculature and extravasation into tissues [3]. Abbreviation: TAM, tumor-associate macrophage.

Figure 4.. Mechanisms of pH Regulation in the Tumor Microenvironment (TME).

Monocarboxylate transporters 1 and 4 (MCT1/MCT4) mediate the efflux into the TME of lactate and H⁺ generated by glycolysis [75]. Sodium bicarbonate transport channels (NBCs) maintain the alkaline intracellular pH (pHi) through the transport of HCO⁻₃[26]. Carbonic anhydrases (CAIX/CAII) play a key role in two pathways (MCT and NBC), facilitating H⁺ distribution and HCO⁻₃ production, respectively [26]. Na⁺/H⁺ exchangers release H⁺ into the TME through the influx of Na⁺ [26]. Cancer cells express vacuolar-ATPase (V-ATPase) on the cell surface through utilization of the a3 isoform, and this mediates the transport of cathepsins and H⁺ from lysosomes to the TME [60]. In cancer cells, inhibition of cellular respiration by pyruvate dehydrogenase kinase (PDK) and increased activity of glucose transporter 1 (GLUT1), that transports glucose into the cell, promote glycolysis (the Warburg effect) [79]. Forkhead box protein M1 (FOXM1) binds to the promoter of the mitochondrial D-lactate dehydrogenase (LDHD) gene and enhances the expression of LDHD that converts pyruvate to lactate [80]. Glucose-6-phosphate generates CO₂ via glucose-6-phosphate dehydrogenase (G6PD) and the pentose-phosphate pathway (PPP) [81]. Mammalian target of rapamycin (mTOR) activation increases hypoxia-inducible factor (HIF)-1α translation [82]. During normoxia, HIF-1α binds to von Hippel–Lindau (VHL) and undergoes ubiquitination, resulting in proteasomal degradation of HIF-1 [83]. Abbreviations: HRE, hypoxia response element; OXPHOS, oxidative phosphorylation; pHe, extracellular pH; TCA, tricarboxylic acid cycle; Ub, ubiquitin.