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VISTA: An Emerging Immune Checkpoint Target

VISTA/B7-H5 is a T Cell Co-inhibitory Checkpoint Protein

VISTA/B7-H5 is T cell inhibitory checkpoint protein that is primarily expressed on hematopoietic cells, with the highest expression in humans being on myeloid cells, CD4+ T cells, and FoxP3+ regulatory T cells.1-3 In contrast to other B7 family ligands which contain both IgV-like and IgC-like domains in their extracellular regions, the extracellular domain of VISTA contains a single IgV-like domain similar to the B7 family receptors, CD28, CTLA-4,and ICOS. Unlike other B7 family members, VISTA has been suggested to function as both a T cell co-inhibitory ligand and a co-inhibitory receptor.1, 2, 4, 5 As a co-inhibitory ligand, VISTA acts through an unidentified T cell-expressed receptor to directly inhibit T cell functions. This conclusion is supported by the observation that a recombinant VISTA Ig fusion protein inhibited CD4and CD8+ T cell proliferation, IL-2 and IFN-gamma production, and promoted the conversion of naïve T cells into FoxP3+ regulatory T cells in vitro.1, 2 In agreement with these studies, overexpression of VISTA on bone marrow-derived dendritic cells reduced T cell proliferation and cytokine production, and this effect was neutralized by a VISTA-specific monoclonal antibody.2

As a co-inhibitory T cell receptor, VISTA has been shown to function independently of antigen-presenting cells (APCs) to suppress T cell functions. VISTA deficient mice accumulated spontaneously activated T cells, secreted high levels of pro-inflammatory cytokines, and developed more severe experimental autoimmune encephalomyelitis than control mice, with VISTA on both T cells and APCs contributing to disease severity.4, 6 Additionally, treatment of CD4+ T cells with a VISTA-specific agonistic antibody was shown to inhibit CD4+ T cell activation both in vitro and in vivo, demonstrating that VISTA is a CD4+ T cell inhibitory receptor.4 This T cell intrinsic effect of VISTA is thought to occur by VISTA interacting with either itself or another receptor through T cell-T cell interactions.7 The ligand for VISTA may also be expressed on tumor cells, allowing tumors to directly inhibit T cell activity through VISTA. Our own in-house data demonstrates that VSIG-3/IGSF11 is a ligand of VISTA and that this interaction can inhibit human T cell proliferation and cytokine production, although the significance of this interaction in vivo is still unknown.8

Multiple studies suggest that VISTA is involved in regulating anti-tumor immunity. In the original study that identified VISTA, the authors showed that its overexpression on tumor cells inhibited the protective anti-tumor immune response.2 A subsequent study demonstrated that in mouse tumor models, VISTA expression was up-regulated on both CD11b+Gr1+ myeloid cells and regulatory T cells present in the tumor microenvironment, as well as on tumor cells under hypoxic conditions.9 This study demonstrated that blockade of VISTA using a monoclonal antibody enhanced the proliferation, infiltration, and effector functions of tumor-specific T cells, decreased the number of myeloid-derived suppressor cells present in the tumor microenvironment, inhibited the development and activity of regulatory T cells, and suppressed tumor growth. Expression of VISTA in human tumors has typically been found to increase with tumor progression and correlate with poor survival.10 Recent studies have also shown that VISTA expressed on tumor cells decreases the number of tumor-infiltrating CD8+ T cells in syngeneic mouse tumor models and that an anti-VISTA antibody prolongs the survival of tumor-bearing mice.11 Furthermore, it was recently suggested that VISTA expressed on tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), or tumor epithelial cells may interact with T cell-expressed PSGL-1 under acidic conditions, enabling it to inhibit immune activation specifically in acidic microenvironments such as the tumor microenvironment.12 As a result of these and multiple other studies demonstrating the immunosuppressive activity of VISTA, researchers are now looking at targeting this protein either alone or in combination with other immune checkpoint therapies,depending on the tumor phenotype.13

VISTA Functions as a T Cell Co-inhibitory Ligand and Receptor

VISTA inhibits T cell proliferation and effector functions acting as both a T cell co-inhibitory ligand and receptor.

VISTA/B7-H5 functions as both a T cell inhibitory ligand and receptor. VISTA is expressed on both antigen-presenting cells (APCs) and T cells and has been shown to function as both a T cell co-inhibitory ligand and receptor. As a ligand, VISTA on APCs or tumor cells binds to an unidentified receptor expressed on activated T cells and inhibits T cell proliferation and cytokine production. Recently, it has been suggested that expression of VISTA on tumor cells or myeloid cells present in the tumor microenvironment may inhibit T cell functions by interacting with T cell-expressed PSGL-1 under acidic conditions. In addition to functioning as a T cell co-inhibitory ligand, T cell-expressed VISTA functions as a co-inhibitory receptor to suppress T cell functions. This interaction has been proposed to occur by VISTA interacting with either itself or a currently unidentified protein through T cell-T cell interactions, or through tumor cell-T cell interactions, if the ligand for VISTA is expressed on tumor cells.

Assessment of the Bioactivity of R&D Systems Recombinant Human VISTA Protein

Analysis of the effect of R&D Systems Recombinant Human VISTA Fc Chimera protein on IL-2 secretion by activated T cells.

VISTA Inhibits Anti-CD3-Induced IL-2 Secretion by Human T Cells. Human T cells were incubated with an immobilized Mouse Anti-Human CD3 epsilon Monoclonal Antibody (R&D Systems, Catalog # MAB100) and the indicated concentrations of Recombinant Human VISTA/B7-H5 Fc Chimera (R&D Systems, Catalog # 7126-B7). IL-2 secretion was measured in cell culture supernatants using the Human IL-2 QuantikineTM ELISA Kit (R&D Systems, Catalog # D2050). The ED50 for this effect is typically 1-6 μg/mL.

Binding of VISTA to VSIG-3 and Neutralization Using Anti-Human VISTA Antibodies

Analysis of the binding response between R&D Systems Recombinant Human VISTA and Recombinant Human VSIG-3 proteins.

VSIG-3 Binds to VISTA. Recombinant Human VISTA/B7-H5 Fc Chimera (R&D Systems, Catalog # 7126-B7) was immobilized at 2 μg/mL (100 μL/well), and the indicated concentrations of Recombinant Human VSIG-3 Fc Chimera (R&D Systems, Catalog # 9229-VS) were added. The concentration of Recombinant Human VSIG-3 Fc Chimera that produced 50% of the optimal binding response was approximately 0.25 μg/mL (3.4 nM).

Analysis of the ability of Anti-Human VISTA antibodies to neutralize the binding of Recombinant Human VISTA and VSIG-3.

Anti-Human VISTA Antibodies Neutralize the Binding of VSIG-3 and VISTA. Biotinylated Recombinant Human VISTA Fc Chimera (R&D Systems, Catalog # BT7126; 1 μg/mL) was pretreated with the indicated concentrations of Sheep Anti-Human VISTA/B7-H5 Polyclonal Antibody or Mouse Anti-Human VISTA/B7-H5 Monoclonal Antibody (R&D Systems, Catalog # AF7126 or Catalog # MAB71261) or Normal Sheep IgG Control or Mouse IgG2B Isotype Control (R&D Systems, Catalog # 5-001-A or Catalog # MAB0041) and then added to ELISA plates containing immobilized Recombinant Human VSIG-3 Fc Chimera (R&D Systems, Catalog # 9229-VS ; 2 μg/ mL). After washing away any unbound proteins, Streptavidin-HRP (R&D Systems, Catalog # DY998) and substrate solution (R&D Systems, Catalog # DY999) were added to the wells. The color development was stopped and the intensity of the color was measured using an ELISA plate reader.

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  2. Wang, L. et al. (2011) VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses. J. Exp. Med. 208:577. PMID: 21383057.

  3. Flies, D.B. et al. (2014) Coinhibitory receptor PD-1H preferentially suppresses CD4+ T cell-mediated immunity. J. Clin. Invest. 124:1966. PMID: 24743150.

  4. Schildberg, F.A. et al. (2016) Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity 44:955. PMID: 27192563.

  5. Wang, J. et al. (2019) VSIG-3 as a ligand of VISTA inhibits human T-cell function. Immunology 156:74. PMID: 30220083.

  6. Le Mercier, I. et al. (2014) VISTA regulates the development of protective anti-tumor immunity. Cancer Res. 74:1933. PMID: 24691994.

  7. EITanbouly, M.A. et al. (2019) VISTA: a novel immunotherapy target for normalizing innate and adaptive immunity. Semin. Immunol. 42:101308. PMID: 31604531.

  8. Mulati, K. et al. (2019) VISTA expressed in tumour cells regulates T cell function. Br. J. Cancer 120:115. PMID: 30382166.

  9. Johnston, R.J. et al. (2019) VISTA is an acidic pH-selective ligand for PSGL-1. Nature 574:565. PMID: 31645726.

  10. EITanbouly, M.A. et al. (2020) VISTA: Coming of age as a multi-lineage immune checkpoint. Clin. Exp. Immunol. 200:120. PMID: 31930484.