Introduction

With the advent of immunotherapy, cancer treatment modalities have undergone a revolution heralding a new era of specialized treatments that are supposed to improve the chances of successful therapies. Indeed, immunotherapy aims to activate or suppress the immune system to boost anti-tumor responses or attenuate the specific adaptive immune response against self-antigens. Since lymphocytes are the most critical players in immune responses, activatory and inhibitory receptors within them are the center of attention during immunotherapy [1]. Immune checkpoint receptors (ICRs) regulate the balance between the immune system’s stimulatory and inhibitory pathways and help maintain immunosurveillance. With the introduction of the inhibitory ICRs, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell death protein 1 (PD-1) by Alison et al., more attention was paid to this field in cancer patients, and the initial immune checkpoint inhibitors (ICIs) showed remarkable therapeutic effects. In addition to CTLA-4 and PD-1, several ICRs have been identified over the years, and they are in various stages of clinical trials. Moreover, the number of cases receiving United States Food and Drug Administration (FDA) approval is on the rise. Their brilliant results in creating dramatic and long-term clinical outcomes, especially in refractory cancers such as non-small cell lung cancer (NSCLC) and melanoma, have raised hopes for cancer therapy [2,3,4].

Among the novel ICRs, VISTA, also known as B7-H5, PDCD-1 homolog (PD-1H), stress-induced secreted protein 1 (SISP1), death domain1alpha (DD1a), Gi24, and differentiation of embryonic stem cells 1 (Dies1) plays a central role in immune system functions, and its association with several human disorders, including autoimmune disease, inflammatory diseases, infection, and cancer was confirmed [5]. Although there has been considerable research regarding the function and role of VISTA and its potential therapeutic target, little information is available regarding the ligands of VISTA and their interactions. This article will provide a comprehensive review of VISTA, its related ligands' roles and functions, the impact of thier interactions, and the newest targeting agents for them to evaluate their potential as therapeutic targets in the future.

VISTA structure

VISTA, as a type I transmembrane protein (55–65 kDa), is encoded by a gene called VSIR and is located on 10q22.1 within an intron of the CDH23 gene. VISTA protein structure (without signal peptide: 32 amino acids) contains 279-aa; 130-aa in the hyperglycosylated extracellular Ig-V domain, 33-aa in the stalk region, 20-aa in the transmembrane domain, and 96-aa in the cytoplasmic domain [6].

The extracellular domain (ECD) of VISTA adopts a canonical β-sandwich formation with the front face representing H-, A-, G-, F-, C-, and C′ β-strands, while the back face represents A′-, B-, E-, D-, and C′′ β-strands. Moreover, three disulfide bonds exist between the strands of B-F (Cys22–Cys114), A′-H (Cys12–Cys146), and CC′ loop-F (Cys51–Cys113) as a result of the presence of six cysteines. The ECD of VISTA, mainly the C–C′ loop, contains a considerable number of histidine (H) residues with a positive charge. These residues are responsible for forming pH-dependent binding sites, which enhance the binding of VISTA to its ligands in acidic environments such as tumor microenvironment (TME) [5, 7]. The cytoplasmic region of VISTA does not contain tyrosine-based signaling motifs, including immunoreceptor tyrosine-based activation motif (ITAM), immunoreceptor tyrosine-based inhibitory motif (ITIM), and immunoreceptor tyrosine-based switch motif (ITSM). Additionally, the presence of proline-rich motifs, including three C-terminal Src homology domain 3 (SH3) binding motifs (PxxP) and a Src homology domain 2 (SH2) binding motif (YxxQ), as well as specific sequences for phosphokinase C (PKC) and casein kinase 2 (CK2) binding sites enable VISTA to alter some cellular functions within the cell and act both as a receptor and a ligand. The cytoplasmic region of VISTA, which is very important in terms of intracellular functions, is similar to members of the CD28 family, such as PD-1 and CTLA-4. Then, VISTA seems to share common functional properties with the CD28 family members [6, 7].

Phylogenetic analysis showed that VISTA has a highly conserved sequence, especially between humans and mice with 76% similarity. In light of the sequence similarity between VISTA and the other members of the B7 family, especially in the ECD, it has been classified as a member of this group. Analysis of the Ig-V domain within ECD showed that the highest homology rate between VISTA and B7 family members belongs to programmed death-ligand 1 (PD-L1), where 23% of the sequences are identical. Nevertheless, VISTA still exhibits some unique characteristics that make it stand unique from other members of the B7 family: (1) chromosomal locus in which VSIR gene is located apart from others, (2) conformational differences in the ECD domain; containing ten β-strands compared to nine in the B7 family fold, having an extra helix sequence (FQDL) and unique C–C′ unstructured loop of 21 residues, 3) Two extra disulfide bonds in IgV-like domain with unique cysteine residues (Cys44, Cys83, Cys144, and Cys177) that are absent in others, 4) While B7 family members have both IgC and IgV-like domains, VISTA lacks an IgC-like domain and exhibits extremely large IgV-like, and 5) lacking any ITAM, ITIM, ITSM motifs, which are existed in other B7 family members [5, 7].

VISTA expression

At a steady state, VISTA is expressed in a wide range of human tissues, particularly hematopoietic compartments and tissues containing infiltrating leukocytes, e.g., bone marrow (BM), thymus, spleen, and lymph node [8, 9]. Evidence also indicates the presence of VISTA in secretory form [10, 11].

In terms of hematopoietic tissues, peripheral blood mononuclear cells (PBMCs), mainly myeloid lineages, including monocytes, myeloid dendritic cells (DCs), macrophages, neutrophils, and basophils have the highest expression level of VISTA [8, 9]. Concerning lymphoid cells with lower expression of VISTA than myeloid cells, while subsets of CD4 + T cells, exceedingly naïve CD4 + T cells, and forkhead box P3 (FoxP3 +) regulatory T cells (Tregs) exhibit higher levels of VISTA, CD8 + T cells, plasma cells, lymphoid DCs, CD56low natural killer (NK) cells, and thymocytes express it at lower expression levels. However, its expression level on CD19 + B cells and CD56high NK cells has not yet been observed. Regarding the expression of VISTA in mice, it has almost the same expression pattern as humans, mainly confined to hematopoietic tissues [8, 9].

Activation of immune cells could increase the expression level of VISTA depending on each cell type [6]. Nevertheless, this upregulation in CD4 + and CD8 + T cells decreases over time [5]. Hu et al. showed that activated CD4 + T cells showed higher expression levels of VISTA than activated CD8 + T cells; however, the intensity of increased expression was higher in activated CD8 + T cells than in activated CD4 + T cells [65]. Gao et al. [66] showed that following ipilimumab therapy in prostate cancer patients, the expression level of VISTA and PD-L1 and infiltration of immune cells, especially CD68 + macrophages (VISTA + and PD-L1 +) that have M2 phenotype (suppressive phenotype) within the TME increased. It was shown that blocking VISTA in combination with CTLA-4 and PD-1 showed a synergistic effect and prevented the development of resistance to therapy in cancer patients [67]. Accordingly, it is suggested that inhibiting VISTA simultaneously with other ICs may not only result in more effective therapeutic outcomes but also prevent drug resistance.

The important side effect to consider in using ICIs in cancer treatment is the possibility of immune-related adverse events (irAEs), resulting in complications similar to those of autoimmune diseases caused by a disruption of self-tolerance. As a matter of fact, blocking a non-redundant NCR can result in a less suppressed immune system and a higher likelihood of over-reacted immune responses. Depending on the organ involved, these side effects include colitis, dermatitis, pneumonitis, myalgias, arthralgias, etc. [68,69,70]. According to a large meta-analysis, the incidence of irAE is 83% for CTLA-4 inhibitors, 72% for PD-1 inhibitors, and 60% for PD-L1 inhibitors, which are high rates [71]. In terms of the number of studies conducted on irAEs associated with VISTA, there are only a few. There was evidence that mice lacking the VISTA gene produced more IFN-γ-secreting T cells and showed chronic inflammation. Nevertheless, no organ-specific autoimmune disease developed [27]. Consequently, identifying alternative targets for ICIs, such as ligands of ICRs that suppress the immune checkpoint pathways, may increase the possibility of treating cancer patients while reducing the possibility of develo** subsequent irAEs.

Ligands of VISTA

As mentioned before, VISTA acts as both a ligand and a receptor. VISTA expressed within cells other than T cells (e.g., APCs and tumor cells) acts as a ligand via binding to unknown receptors on T cells. VISTA expressed on T cells acts as a receptor that interacts with ligands and inhibits T cells' activity via transducing downstream inhibitory pathways related to TCR [9, 13, 30]. On the other hand, VISTA shows homophilic interactions that facilitate its correlation with other VISTA proteins expressed in other cells. A confirmatory study of this issue showed that VISTA homophilic interactions intermediate macrophages’ efferocytosis and inhibition of T cell activation [14].

Although VISTA co-inhibitory ligands have not been fully elucidated, recent studies have suggested VSIG-3 and PSGL-1 as prominent and Galectin-9 (Gal-9), VSIG-8, matrix metalloproteinase-13 (MMP-13), syndecan-2 (Sdc2), and leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as less well-confirmed receptors (Fig. 2).

Fig. 2
figure 2

VISTA and its related ligands. VISTA interacts with its associated ligands through the ECD region, where histidine and cysteine residues play important roles. ECD (extracellular domain), VISTA (V-domain immunoglobulin suppressor of T cell activation), PSGL-1 (P-selectin glycoprotein ligand-1), VSIG-3 (V-Set and Immunoglobulin domain containing 3), Gal-9 (Galectin-9), VSIG-8 (V-Set and Immunoglobulin domain containing 8), MMP-13 (matrix metalloproteinase-13), Sdc2 (syndecan-2), LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1), NH2 (N-terminus), COOH (C-terminus), PKC (phosphokinase C), CK2 (casein kinase 2), SH2 (Src homology domain 2), SH3 (Src homology domain 3). Created with BioRender.com

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V-set and immunoglobulin domain containing 3 (VSIG-3)

VSIG-3, also mainly known as BT-IgSF and IGSF11, is a single-pass (type I) transmembrane protein belonging to the Ig superfamily and consists of several domains, including N-terminal (Ig-like V type and Ig-like C2 type domains), transmembrane, and C-terminal (PDZ domain). VSIG-3 gene is located on chromosome 3q13.32 and contains 431 amino acids in humans [72, 134]. Mehta et al. [7] compared binding affinities of VSIG-3 and PSGL-1 for VISTA at different pH. They showed that at pH 7.4 (physiological pH), VISTA has a binding affinity of 20 nM for VSIG-3, while no binding was detected for PSGL-1. Conversely, at pH 6.0, while VSIG-3 showed an apparent binding affinity of 80 nM (fourfold decrease), PSGL-1 showed 4 nM (significant increase). Considering that limited information is available regarding VISTA interaction details with other ligands, we are not able to discuss more about them and the signaling pathways involved. Furthermore, it should be noted that most studies on the effects of VISTA binding to its ligands have been conducted in the context of cancer; hence, our knowledge about the impact of VISTA interaction with ligands has mostly been derived from research related to cancer, which mostly dampens anti-cancer responses and inflammation within the TME. Little is known about the effect of their interactions on the pathogenesis of autoimmune or inflammatory diseases (Fig. 3).

Fig. 3
figure 3

The effect of VISTA interaction with ligands and their binding effects. a The interaction between VSIG-3 and VSIG-8 expressed within tumor cells with VISTA on T cells causes inhibition in T cell activation and proliferation, reduction in IFN-γ, IL-2, IL-17, CCL3, CCL5, CXCL11 production, and suppression of immune cell infiltration to the TME. b The binding of Gal-9 secreted from the AML cells to VISTA expressed on T cells induces apoptosis in activated T cells and inhibits immune responses in the TME. c The interaction between PSGL-1 expressed on T cells with VISTA expressed on tumor cells, TILs, and TAMs/MDSCs not only suppresses T cell activation (blocking NF-κB pathway and reduction in IFN-γ production) and proliferation but also decreases the production of anti-inflammatory mediators in TME. d The MMP-13 produced by MM tumor cells binds to the VISTA expressed on the osteoclasts and T cells, causing bone resorption and T cell suppression, respectively. e The binding of VISTA to its unidentified ligand on monocyte surfaces is associated with Sdc-2 interactions, which have an impact on monocyte biological functions. VISTA (V-domain immunoglobulin suppressor of T cell activation), VSIG-3 (V-Set and Immunoglobulin domain containing 3, VSIG-8 (V-Set and Immunoglobulin domain containing 8), PSGL-1 (P-selectin glycoprotein ligand-1), Gal-9 (Galectin-9), MMP-13 (matrix metalloproteinase-13), Sdc2 (syndecan-2), IFN-γ (interferon gamma), TNF (tumor necrosis factor), NF-κB (nuclear factor kappa B), IL (interleukin), CCL3 (chemokine (C–C motif) ligand 3), CCL5 (chemokine (C–C motif) ligand 5), CXCL11 (C-X-C motif chemokine 11), Cas-3 (caspase 3), TAMs (tumor-associated macrophages), MDSCs (myeloid-derived suppressor cells), TME (tumor microenvironment), AML (acute myeloid leukemia), MM (multiple myeloma). Created with BioRender.com

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VISTA and its ligands in clinical trials

As mentioned before, VISTA occupies unique features that make it stand out among others: distinct signaling pathway from CTLA-4 and PD-1/PD-L1, synergistic effects with anti-CTLA-4 and –PD-1, and involvement in resistance to anti-CTLA-4/PD-1 therapies. Therefore, targeting VISTA in TME suppresses the tumor-promoting effects and induces anti-tumoral responses. In terms of combination therapy, no information has been available regarding the simultaneous inhibition of VISTA and the use of other standard treatments, such as chemotherapy or radiotherapy. VISTA also plays a crucial role in maintaining self-tolerance, and its agonists have also been shown to be valuable in treating autoimmune and inflammatory diseases. According to the most recent multiple preclinical and clinical studies, VISTA appears to have tremendous therapeutic potential, either as an agonist or antagonist (Table 1).

Table 1 Current ongoing VISTA therapeutics (small molecule inhibitors and mAbs) in preclinical and clinical development
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Agonists

VISTA agonistic mAbs promote the activity of VISTA; induction of activation-induced cell death (AICD) and enhancement of peripheral T cell tolerance, inhibit myeloid chemotaxis, and reprogramming macrophages towards an anti-inflammatory profile [16, 135]. Hence, they can be a suitable treatment option for inflammatory and autoimmune disorders. Recent animal studies showed that using VISTA agonist antibodies results in immunomodulatory effects, such as suppressing NF-κB signaling pathway and production of pro-inflammatory cytokines [16]. In GVHD, targeting VISTA in donor CD4 + T cells with agonistic antibody before the transfer led to the deletion of donor alloreactive T cells via the T cell-intrinsic pathway and prevented disease [30]. As mentioned before, the VISTA agonist antibody (4C11) suppressed lung inflammation and reduced the severity of the disease [41]. Five agonistic anti-VISTA mAbs are under investigation in autoimmune disorders, 8G8, INX803, 7G1, 7G5, and 7E12, which target VISTA in mice, humans, or both. Studies indicate that these antibodies induce VISTA signaling and suppress immunity, although little information about their effects is available [136, 137]. It should be noted that only VISTA is currently being developed as a checkpoint agonist in clinical studies.

Antagonists

Small molecule inhibitors

CA-170

A small molecule inhibitor named CA-170, taken orally, targets VISTA (H strand) and PD-L1/L2 pathways without interrupting PD-1/PD-L1 interaction. Since 2015, Curis has licensed the technology from Aurigene, and in vitro studies showed that CA-170 promotes cell proliferation and IFN-γ production in T cells suppressed by VISTA or PD-1/PD-L1 [138, 139]. In syngeneic mouse models of melanoma and colon cancer (B16, CT26, and MC38), CA-170 suppressed tumor growth, promoted the activation of peripheral T cells, and activation of TILs [140]. Phase I trial (NCT02812875) showed its safety and effectiveness in solid tumors and lymphoma. Patients involved presented increased activated CD4 + and CD8 + T cells in the periphery [141]. Phase II studies in lung cancer, Hodgkin lymphoma, head and neck/oral cavity, and MSI-high cancers are currently underway by Aurigene in India [142]. Despite all these findings, the binding of human VISTA to CA-170 was not confirmed [143].

AUPM-493

Small molecule developed by Aurigene and acts as PD-L1 and VISTA antagonist. In a preclinical study, AUPM-493 suppressed the interaction between VISTA and VSIG-8, which led to the activation of T cells and IFN-γ production. It also showed anti-tumoral effects in syngeneic models of melanoma and colon cancer [117].

Blocking antibodies

VSTB112

Regarding mAbs blocking VISTA, JNJ-61610588 or VSTB112 is the first humanized IgG1κ antibody developed by ImmuNext/Janssen, targeting human VISTA through C–C′ loops (H121 and H122 residues) and adjacent Helix. There is no pH dependence on the interaction between VSTB and VISTA [144]. It was found that VSTB112 suppressed VISTA signaling in vitro and also tumor regression in a mouse model of bladder cancer (human VISTA knock-in mice) as a result [145]. In 2016, Janssen Biotech started a phase I trial (NCT02671955) to assess its safety, tolerability, and pharmacokinetics in advanced tumors, including lung, pancreatic, head and neck, colorectal, and cervical cancers [146]. The study was prematurely terminated after 2 years for unknown reasons in a situation where one of 12 patients experienced cytokine release syndrome.

CI-8993

Curis is currently pursuing VSTB112 (JNJ-61610588) as CI-8993 (Onvatilimab) in the phase I trial (NCT04475523) in relapsed/refractory solid tumors [147]. It is important to note that even at subtherapeutic doses, CI-8993 triggers a significant release of cytokines that can cause neurotoxicity, maybe due to its cell-depleting IgG1 backbone.

W018 (K01401-020)

The newest anti-VISTA mAb with IgG1 subtype, developed by Pierre Fabre, inhibits PSGL-1 binding to VISTA at pH 6–7.4 [148]. W018 phase I trial (NCT04564417) has recently commenced (149).

BMS767 (P1-068767)

Anti-VISTA human mAb investigated by Bristol-Myers Squibb. It is the only VISTA pH-sensitive antibody interacting with VISTA (H121, H122, and some C–C' loop residues) at pH 6.0 (not physiological pH) [144].

SG7

Developed by yeast surface display technology and has inhibitory effects against VISTA. It has overlap** epitopes with VSTB112 and BMS767, binding only to human VISTA. However, it has unique regions for binding to VISTA expressed in murine and cynomolgus monkey, which makes SG7 species cross-reactive antibody with high affinity. Jurkat T cell activation assay showed that the activation level of T cells was restored by SG7, and blocking VISTA via SG7 reduced tumor growth in syngeneic tumor models. SG7 was also found to reduce the number of polymorphonuclear MDSCs (PMN-MDSCs) in a TME from 4T1-bearing mice and increased the number of CD4 + and CD8 + T cells. PMN-MDSCs are cells with a high expression of VISTA and suppress anti-tumoral responses in TME. Nevertheless, no effect was observed on other examined myeloid cells such as CD11c + DCs, CD11b + macrophages, and monocytic myeloid-derived suppressor cells (M-MDSCs). SG7 inhibited VISTA's interaction with VSIG-3 at pH 7.4 and PSGL-1 at pH 6.0, mainly via H122 and E125 residues [144].

HMBD-002

An IgG4 anti-VISTA mAb developed by Hummingbird Biosciences, which primarily interacts with the C–C' loop of VISTA, where VISTA interacts with VSIG-3 and LRIG1. It neutralizes VISTA functions without depleting VISTA + cells by acting via an Fc-independent mechanism. Regarding VISTA/VSIG-3 interactions, HMBD-002 resolved the suppressory effects of VSIG-3, and anti-CD3-activated T cells produced IFN-γ. Additionally, it has been shown that HMBD-002 reverses the inhibitory effects of MDSCs on T cells, suppresses tumor cell invasion, and, most importantly, enables T cells to shift toward Th1/Th17 [150]. In several humanized and syngeneic murine models of breast, colorectal, and lung cancer, HMBD-002 showed therapeutical effects and suppressed tumor growth without apparent toxicity. A preclinical study showed that in combination with pembrolizumab (anti-PD-L1), HMBD-002 demonstrated superior efficacy, particularly in tumors with high infiltration of MDSCs [151,152,153]. It is now under investigation in the phase 1/2 trial (NCT05082610) as a single agent and combined with pembrolizumab in advanced solid tumors expressing VISTA [154].

Both CI-8993 and W018 induce anti-tumor effects through Fc-dependent activities of their IgG1, which frequently result in ADCC or complement-dependent cytotoxicity (CDC)-mediated cell death [155]. It is important to note that VISTA is expressed in a wide range of healthy cells, which means that this activity can cause the death of a large number of cells that are not targeted. HMBD-002 epitope is distinct from CI-8993 and W018 and exhibits high binding specificity for VISTA in various species (human, rat, monkey, and murine orthologs). VSTB112, SG7, and BMS767 all interact with the H122 residue within VISTA to effectively inhibit the binding of both VSIG-3 and PSGL-1. SG7 showed more affinity binding around 25 to 50 fold compared with VSTB112 or BMS767. While the interaction between BMS767 and VISTA is pH-dependent, the binding of SG7 and VSTB112 is not, which makes BMS767 a potential anti-VISTA targeting mAb homing TME. Furthermore, VSTB112 and BMS767 show depletion in VISTA-expressing cells (active Fc), but SG7 does not (dead Fc) [144, 156]. Currently, some mAbs are in the preclinical stage of development, such as KVA 12.1 [157], PMC-309 [158], APX-201, VTX-0811, SNS-101 [159], IMT-18, and IGN-381 for which only a few publications have been published so far, and some are still in the development process [160, 161].

Conclusions and future perspectives

There is no doubt that VISTA has attracted attention in immunotherapy thanks to its distinguishing features compared with other NCRs, where it shows more profound immunoregulatory effects as a result of these options. The impressive results that have been published from treatments based on VISTA targeting highlight the importance and highest value of examining VISTA in more detail [67, 135]. Regarding therapy efficacy, VISTA agonists appear to be more effective in autoimmune and inflammatory disorders than antagonists in cancer because of VISTA-associated irAEs and bi-directional role. However, it should be kept in mind that in MS and lupus cases, various elements such as genetics and inflammatory factors could affect the expression of VISTA. In this regard, investigating the expression pattern of VISTA in autoimmune disorders may be helpful before starting any therapy based on this gene.

Based on the information discussed above, it is clear that all VISTA ligands, apart from VISTA, play an important role in tumor development and growth. In some cases, their importance is so great that they have even been proposed as targets for immunotherapy. Among the VISTA ligands, PSGL-1 and VSIG-3 are valuable options to be considered due to their unique expression and functions, which may reduce side effects related to VISTA targeting. Nevertheless, newly identified ligands, such as Gal-9 and MMP-13, can also be investigated as potential targets blocking the VISTA pathway in the future. Because blocking their interaction with VISTA showed that their inhibition has the potential not only to activate immune responses but also suppress tumor growth. However, in proposing VISTA ligands as alternative targets for blocking the VISTA inhibitory pathway, consideration should be given to their functions in other parts of the body as well as the immune system. Because even if its inhibition suppresses tumor cell growth, it can result in serious secondary complications. Therefore, in determining which of the identified ligands for VISTA should be targeted, choosing the most effective option in suppressing VISTA signaling with fewer irAEs is advisable.

Regarding designing mAbs, it would be advantageous to choose specific residues shared between multiple VISTA ligands to inhibit all relevant VISTA pathways and/or the choice of non-overlap** regions to block a particular path. This case requires identifying the details of the regions through which the ligands are attached to VISTA. Moreover, the Fc activity of the antibodies is another critical factor to consider. Fc-independent antibodies have a high priority in the effort to eliminate ADCC and CDC. It is also possible for bispecific antibodies to be used as a combination therapy in order to inhibit VISTA and its related pathways, as well as other ICs.

On the other hand, environmental factors are also important for improving therapy efficacy. As discussed before, the environment’s pH is a critical factor in VISTA’s performance and binding to its ligands [162]. Hence, some strategies could be used to optimize this factor. For example, PSGL-1 interacts with VISTA in an acidic pH environment and, therefore, has the highest priority to inhibit the VISTA pathway in TME and would be a suitable therapeutic option for cancer therapy. BMS767 is the only VISTA-targeting antibody developed based on this point.

Altogether, it is clear that VISTA's ligands are just as important as VISTA itself, and their interactions play a significant role in many diseases related to the immune system, especially cancer. Therefore, VISTA and its ligands can be quite promising candidates when it comes to considering new immunotherapeutic targets. Nevertheless, VISTA's interaction with ligands, especially other than PSGL-1 and VSIG-3, their related effects, and intracellular pathways remain a work in progress. As a result, it is imperative that more studies be conducted in vitro, particularly in vivo, to fill these knowledge gaps and to maximize the potential of targeting VISTA through new therapeutic approaches.