V-domain Immunoglobulin Suppressor Of T Cell Activation Research Articles

VISTA-mediated immune evasion in cancer.

Over the past decade, V-domain immunoglobulin suppressor of T-cell activation (VISTA) has been established as a negative immune checkpoint molecule. Since the role of VISTA in inhibiting T-cell activation was described, studies have demonstrated other diverse regulatory functions in multiple immune cell populations. Furthermore, its relevance has been identified in human cancers. The role of VISTA in cancer immune evasion has been determined, but its mechanisms in the tumor microenvironment remain to be further elucidated. Understanding its contributions to cancer initiation, progression, and resistance to current treatments will be critical to its utility as a target for novel immunotherapies. Here, we summarize the current understanding of VISTA biology in cancer.

Immune Checkpoint VISTA Negatively Regulates Microglia Glycolysis and Activation via TRIM28-Mediated Ubiquitination of HK2 in Sepsis-Associated Encephalopathy.

V-domain immunoglobulin suppressor of T cell activation (VISTA) has emerged as a crucial player in the pathogenesis of neurological disorders. However, the specific mechanism by which VISTA regulates microglial activation remains unclear. Septic mice were intracerebroventricularly injected with an agonistic anti-VISTA antibody or isotype control. To investigate the differential gene expression profiles, RNA sequencing was conducted on brain tissues from these mice. In vitro, VISTA was silenced in BV2 microglial cells using shRNA. Co-immunoprecipitation assays were performed to identify protein-protein interactions involving hexokinase 2 (HK2), and ubiquitination assays were used to examine the ubiquitination status of HK2. Additionally, BV2 cells were transfected with either tripartite motif-containing 28 overexpression plasmids (TRIM28-PcDNA3.1( +)) or TRIM28-specific siRNA to assess the impact of TRIM28 on VISTA-mediated microglial activation. The cellular glycolytic activity was measured using extracellular acidification rate assays, and proinflammatory cytokine and chemokines were quantified. Treatment with VISTA antibodies significantly alleviated microglial activation and prevented cognitive impairment in septic mice. In contrast, VISTA silencing in BV2 microglia led to the overexpression of proinflammatory cytokines and enhanced glycolysis in an HK2-dependent manner. Mechanistically, HK2 expression was regulated by the E3 ubiquitin ligase TRIM28 through K63-linked ubiquitination, which targeted HK2 for proteasomal degradation. Furthermore, knockdown of TRIM28 reduced the elevated glycolysis and proinflammatory response observed in VISTA-silenced microglia. VISTA modulates microglial activation in sepsis-associated encephalopathy by regulating HK2 expression through TRIM28-mediated K63-linked ubiquitination. These findings highlight VISTA as a potential therapeutic target for modulating microglial activation in sepsis.

Small molecules from antibody pharmacophores (SMAbPs) as a hit identification workflow for immune checkpoints.

Small-molecule modulators of immune checkpoints are poised to revolutionize cancer immunotherapy. However, efficient strategies for hit identification are lacking. We introduce small molecules from antibody pharmacophores (SMAbPs), a workflow leveraging cocrystal structures of checkpoints with antibodies to create pharmacophore maps for virtual screening. Applying SMAbPs to five immune checkpoints yielded hits with submicromolar potency in both cell-free and cellular assays. Notably, SMAbPs identified the most potent T cell immunoglobulin and mucin-domain containing-3 and V-domain immunoglobulin suppressor of T cell activation (VISTA) inhibitors reported to date and first-in-class modulators of B and T lymphocyte attenuator, 4-IBB, and CD27. Targeting inhibitory and costimulatory checkpoints with hits identified through SMAbPs demonstrated remarkable in vivo antitumor activity, exemplified by MG-V-53 (VISTA inhibitor) and MG-C-30 (CD27 agonist), which significantly reduced tumor volumes in MC38 and EG7-OVA mouse models, respectively.

Discovery of Benzo[d]oxazoles as Novel Dual Small-Molecule Inhibitors Targeting PD-1/PD-L1 and VISTA Pathway.

The blockers of programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway have achieved great clinical success. However, the limited efficacy and low tumor response rate of anti-PD-1/PD-L1 monotherapy limit the clinical application of PD-1/PD-L1 inhibitors. V-domain immunoglobulin suppressor of T-cell activation (VISTA), a novel checkpoint regulator, exhibits potential synergy with PD-1/PD-L1 in enhancing antitumor immunity. Herein, we report the discovery of benzo[d]oxazole B3 as novel dual small-molecule inhibitors targeting PD-1/PD-L1 and VISTA with high PD-1/PD-L1 inhibitory activity and VISTA binding affinity. B3 rescues the immunosuppression of T-cells mediated by PD-L1 and VISTA and activates antitumor immunity effectively. Moreover, B3 could induce degradation of PD-L1 and VISTA in tumor cell. Furthermore, B3 displays significant in vivo antitumor efficacy in a CT26 mouse model. Our results discover B3 as a promising dual PD-1/PD-L1 and VISTA inhibitor, providing a novel therapeutic strategy to overcome the limitations of current anti-PD-1/PD-L1 therapy.

TAM-tastic: from resistance to resilience in cancer

Overcoming resistance to immunotherapy in cancer is challenging due, in part, to tumor-associated macrophages (TAMs) co-expressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA) in tumor microenvironments (TME) with sparse T cell infiltration. In a recent article, Vanmeerbeek et al. found that blocking TIM3 or VISTA on IL-4-supported TAMs, in combination with paclitaxel (PTX), reprogrammed TAMs to attack cancer cells, highlighting a potential new therapeutic strategy.

Small-Molecule Radiotracers for Visualization of V-Domain Immunoglobulin Suppressor of T Cell Activation.

V-domain immunoglobulin suppressor of T cell activation (VISTA) plays a critical role in regulating innate and adaptive immune responses within the tumor immune microenvironment. Quantifying VISTA expression is necessary to determine whether patients respond to a related combination immunotherapy. This study developed two 68Ga-labeled small-molecule probes ([68Ga]Ga-DCA and [68Ga]Ga-DNCA) for visualizing and differentiating VISTA expression. These probes exhibited excellent targeting capabilities for multiple tumor types (including B16-F10, 4T1, MC38, and CT26 tumors), consistent with the levels of VISTA expression determined by immunoblotting. Co-injection of inhibitor CA-170 led to decreased tumor uptake of both [68Ga]Ga-DCA and [68Ga]Ga-DNCA. [68Ga]Ga-DCA was used to verify the feasibility of monitoring VISTA expression in lung metastasis models. In summary, this study describes the use of 68Ga-labeled CA-170 analogues as small-molecule probes for imaging VISTA. This could provide a visual method and enable personalized immunotherapy in patients.

Nanoparticles targeting immune checkpoint protein VISTA induce potent antitumor immunity

BackgroundImmune checkpoint protein V-domain immunoglobulin suppressor of T cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. Previous mechanistic studies have indicated that VISTA impairs...

Targeting conserved TIM3+VISTA+ tumor-associated macrophages overcomes resistance to cancer immunotherapy.

Despite the success of immunotherapy, overcoming immunoresistance in cancer remains challenging. We identified a unique niche of tumor-associated macrophages (TAMs), coexpressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA), that dominated human and mouse tumors resistant to most of the currently used immunotherapies. TIM3+VISTA+ TAMs were sustained by IL-4-enriching tumors with low (neo)antigenic and T cell-depleted features. TIM3+VISTA+ TAMs showed an anti-inflammatory and protumorigenic phenotype coupled with inability to sense type I interferon (IFN). This was established with cancer cells succumbing to immunogenic cell death (ICD). Dying cancer cells not only triggered autocrine type I IFNs but also exposed HMGB1/VISTA that engaged TIM3/VISTA on TAMs to suppress paracrine IFN-responses. Accordingly, TIM3/VISTA blockade synergized with paclitaxel, an ICD-inducing chemotherapy, to repolarize TIM3+VISTA+ TAMs to proinflammatory TAMs that killed cancer cells via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. We propose targeting TIM3+VISTA+ TAMs to overcome immunoresistant tumors.

VISTA Deficiency Exacerbates the Development of Pulmonary Fibrosis by Promoting Th17 Differentiation.

Interstitial lung disease (ILD), characterized by pulmonary fibrosis (PF), represents the end-stage of various ILDs. The immune system plays an important role in the pathogenesis of PF. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an immune checkpoint with immune suppressive functions. However, its specific role in the development of PF and the underlying mechanisms remain to be elucidated. We assessed the expression of VISTA in CD4 T cells from patients with connective tissue disease-related interstitial lung disease (CTD-ILD). Spleen cells from wild-type (WT) or Vsir -/- mice were isolated and induced for cell differentiation in vitro. Additionally, primary lung fibroblasts were isolated and treated with interleukin-17A (IL-17A). Mice were challenged with bleomycin (BLM) following VISTA blockade or Vsir knockout. Moreover, WT or Vsir -/- CD4 T cells were transferred into Rag1 -/- mice, which were then challenged with BLM. VISTA expression was decreased in CD4 T cells from patients with CTD-ILD. Vsir deficiency augmented T-helper 17 (Th17) cell differentiation in vitro. Furthermore, IL-17A enhanced the production of inflammatory cytokines, as well as the differentiation and migration of lung fibroblasts. Both VISTA blockade and knockout of Vsir increased the percentage of IL-17A-producing Th17 cells and promoted BLM-induced PF. In addition, mice receiving Vsir -/- CD4 T cells exhibited a higher percentage of Th17 cells and more severe PF compared to those receiving WT CD4 T cells. These findings demonstrate the significant role of VISTA in modulating the development of PF by controlling Th17 cell differentiation. These insights suggest that targeting VISTA could be a promising therapeutic strategy for PF.

Immune checkpoint inhibitors: breakthroughs in cancer treatment.

Over the past two decades, immunotherapies have increasingly been considered as first-line treatments for most cancers. One such treatment is immune checkpoint blockade (ICB), which has demonstrated promising results against various solid tumors in clinical trials. Monoclonal antibodies (mAbs) are currently available as immune checkpoint inhibitors (ICIs). These ICIs target specific immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1). Clinical trial results strongly support the feasibility of this immunotherapeutic approach. However, a substantial proportion of patients with cancer develop resistance or tolerance to treatment, owing to tumor immune evasion mechanisms that counteract the host immune response. Consequently, substantial research focus has been aimed at identifying additional ICIs or synergistic inhibitory receptors to enhance the effectiveness of anti-PD-1, anti-programmed cell death ligand 1 (anti-PD-L1), and anti-CTLA-4 treatments. Recently, several immune checkpoint molecular targets have been identified, such as T cell immunoreceptor with Ig and ITIM domains (TIGIT), mucin domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), V-domain immunoglobulin suppressor of T cell activation (VISTA), B and T lymphocyte attenuator (BTLA), and signal-regulatory protein α (SIRPα). Functional mAbs targeting these molecules are under development. CTLA-4, PD-1/PD-L1, and other recently discovered immune checkpoint proteins with distinct structures are at the forefront of research. This review discusses these structures, as well as clinical progress in mAbs targeting these immune checkpoint molecules and their potential applications.

LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses.

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell-specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1+ CD62Lhi PD-1low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1+ PD-1+). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8+ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.

Abstract CT181: First-in-human phase I of anti-VISTA monoclonal antibody W0180 with and without anti-PD-1 pembrolizumab in patients with locally advanced or metastatic solid tumors

Abstract Background: V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) is an immune checkpoint regulator highly expressed on myeloid cells. VISTA has been shown to maintain tumor microenvironment (TME) in an immunosuppressive state. Its expression is reported as a resistance mechanism to immune checkpoint blockers, making it an attractive target for cancer treatment. Preclinical data showed that VISTA blockade mediates antitumor activity by activating the antitumor immune response and is enhanced in combination with anti-PD-1. W0180 is a humanized, Fc competent, IgG1 antibody targeting VISTA with high affinity. This study investigates W0180 with/without pembrolizumab (pembro) in patients (pts) with solid tumors. Methods: Pts initially received W0180 monotherapy in dose escalation (3.5-600 mg weekly (QW)). Following evaluation of the first 3 dose levels in monotherapy, dose escalation of W0180 (60 to 300 mg QW) in combination with pembro (200 mg Q3W) started in pts treated with prior anti-PD-(L)1. Objectives were to characterize safety and tolerability of W0180, determine RDE and PK/PD on blood and paired tumor biopsies. Results: By October 2023, 33 pts were treated in dose escalation, 24 pts with W0180 monotherapy and 9 pts with W0180 + pembro. Two dose-limiting toxicities were observed, Grade 2 subacute cerebral infarction and Grade 3 infusion related reaction (IRR). Overall, 33 pts experienced ≥1 treatment-emergent AE (TEAE). Most common TEAEs were IRR (32/33), fatigue (14/33), and anemia (11/33). Grade 3/4 TEAEs occurred in 23 pts, neutropenia (6/23) and anemia (6/23) were the most common. There was no treatment-related death. 6 (18%) pts discontinued due to AEs. Following the observation of Grade 3 arterial thromboembolism (n=1) and Grade 2 subacute cerebral infarction (n=1), eligibility criteria were amended to require adequate lipid profile, troponin and exclude pts with history of arterial thrombotic events and current smoking. W0180 PK was nonlinear with a rapid clearance at 3.5-600 mg doses. No objective response was observed but 5 pts had stable disease (SD) lasting for >16 weeks. One pt with appendix cancer treated at 600mg monotherapy had SD for >15 months. One pt with head and neck cancer treated at 300mg in combination had SD for 13 months. Biomarkers data suggested evidence of PD effect with a trend for dose response through sustained increase of proliferative CD8+ T-cells in blood at the highest W0180 exposures and increased density of tumor-infiltrating lymphocytes in 10 out of 16 pts. These changes were independent of tumor VISTA expression level and correlated to high PD-L1 expression (CPS>5) for 4 pts at baseline. Conclusions: Analysis is ongoing to assess the RDE of W0180 + pembro and collect additional evidence of proof of mechanism in pts with solid tumors. NCT04564417 Citation Format: Carlos Gomez-Roca, Stéphane Champiat, Philippe Cassier, David Jegou, Marie Primard, Aurélie Pétain, Geneviève Gueguen-Dorbes, Claire Fabre, Ignacio Melero, Aurélien Marabelle. First-in-human phase I of anti-VISTA monoclonal antibody W0180 with and without anti-PD-1 pembrolizumab in patients with locally advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT181.

Abstract 1358: Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity

Abstract Immune checkpoint receptors (ICRs) negatively regulate antitumor immune responses. While significant advances have been made in understanding the mechanisms of well-established ICRs such as PD-1 and CTLA-4, other checkpoint proteins remain incompletely characterized and may be targeted to synergistically boost antitumor immunity. One such protein is V-domain immunoglobulin suppressor of T-cell activation (VISTA), an ICR whose expression curtails the function of several lymphoid and myeloid lineages, including tumor-reactive cytotoxic T cells. Globally blocking VISTA elicits potent T cell-mediated antitumor immunity and systemic myeloid cell-mediated inflammatory responses. Mechanistically, VISTA has been shown to inhibit T cell activation by acting as an inhibitory ligand on antigen-presenting cells. However, the impact of T cell-intrinsic VISTA on modulating tumor-reactive T cell responses has not been thoroughly investigated, and it remains unclear whether blocking T cell-specific VISTA is adequate for revitalizing T cell-mediated tumor control. In this study, we utilized VISTA conditional knockout mice models to examine the relative contribution of T cell-intrinsic VISTA versus myeloid-derived VISTA in the regulation of antitumor immunity. We present evidence that blocking VISTA on T cells alone is insufficient to induce strong antitumor immune responses, whereas blocking VISTA on myeloid cells counteracts its dominant role in orchestrating T cell suppression. Citation Format: Sachin Patnaik, Elizabeth Delaney, Cassandra Gilmour, Keman Zhang, Amin Zakeri, Hieu M. Ta, Prerana B. Parthasarathy, Tyler Alban, Timothy Chan, Li Lily Wang. Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1358.

Viewing the immune checkpoint VISTA: landscape and outcomes across cancers

Optimizing immune checkpoint inhibitor (ICI) therapy may require identification of co-targetable checkpoint pathways via immune profiling. Herein, we analyzed the transcriptomic expression and clinical correlates of V-domain immunoglobulin suppressor of T-cell activation (VISTA), a promising targetable checkpoint. RNA sequencing was carried out on 514 tissues reflecting diverse advanced/metastatic cancers. Expression of eight immune checkpoint markers [lymphocyte-activation gene 3 (LAG-3), tumor necrosis factor receptor superfamily 14 (TNFRSF14), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), cytotoxic T-lymphocyte antigen 4 (CTLA-4)], in addition to VISTA, was analyzed, along with clinical outcomes. High VISTA RNA expression was observed in 32% of tumors (66/514) and was the most common highly expressed checkpoint among the nine assessed. High VISTA expression was independently correlated with high BTLA, TIM-3, and TNFRSF14, and with a diagnosis of pancreatic, small intestine, and stomach cancer. VISTA transcript levels did not correlate with overall survival (OS) from metastatic/advanced disease in the pan-cancer cohort or with immunotherapy outcome (progression-free survival and OS from the start of ICI) in 217 ICI-treated patients. However, in ICI-treated pancreatic cancer patients (n = 16), median OS was significantly shorter (from immunotherapy initiation) for the high- versus not-high-VISTA groups (0.28 versus 1.21 years) (P= 0.047); in contrast, VISTA levels were not correlated with OS in 36 pancreatic cancer patients who did not receive ICI. High VISTA expression correlates with high BTLA, TIM-3, and TNFRSF14 checkpoint-related molecules and with poorer post-immunotherapy survival in pancreatic cancer, consistent with prior literature indicating that VISTA is prominently expressed on CD68+ macrophages in pancreatic cancers and requiring validation in larger prospective studies. Immunomic analysis may be important for individualized precision immunotherapy.

Blockade of V-domain immunoglobulin suppressor of T-cell activation reprograms tumour-associated macrophages and improves efficacy of PD-1 inhibitor in gastric cancer.

In gastric cancer, the response rate of programmed cell death protein-1 (PD-1) inhibitor is far from satisfactory, indicating additional nonredundant pathways might hamper antitumour immunity. V-domain immunoglobulin suppressor of T-cell activation (VISTA) has been reported in several malignancies as a novel immune-checkpoint. Nevertheless, the role of VISTA in gastric cancer still remains obscure. Our purpose is to explore the clinical significance and potential mechanism of VISTA in affecting gastric cancer patients' survival and immunotherapeutic responsiveness. Our study recruited eight independent cohorts with a total of 1403 gastric cancer patients. Immunohistochemistry, multiplex immunofluorescence, flow cytometry or intracellular flow cytometry, quantitative polymerase chain reaction, western blotting, fluorescence-activated cell sorting, magnetic-activated cell sorting, smart-seq2, in vitro cell co-culture and ex vivo tumour inhibition assays were applied to investigate the clinical significance and potential mechanism of VISTA in gastric cancer. VISTA was predominantly expressed on tumour-associated macrophages (TAMs), and indicated poor clinical outcomes and inferior immunotherapeutic responsiveness. VISTA+ TAMs showed a mixed phenotype. Co-culture of TAMs and CD8+ T cells indicated that VISTA+ TAMs attenuated effective function of CD8+ T cells. Blockade of VISTA reprogrammed TAMs to a proinflammatory phenotype, reactivated CD8+ T cells and promoted apoptosis of tumour cells. Moreover, blockade of VISTA could also enhance the efficacy of PD-1 inhibitor, suggesting that blockade of VISTA might synergise with PD-1 inhibitor in gastric cancer. Our data revealed that VISTA was an immune-checkpoint associated with immunotherapeutic resistance. Blockade of VISTA reprogrammed TAMs, promoted T-cell-mediated antitumour immunity, and enhanced efficacy of PD-1 inhibitor, which might have implications in the treatment of gastric cancer.

Identification of novel immuno-oncology compounds as VISTA-inhibitors for cancer therapy: A computational approach integrating virtual screening and molecular dynamics simulation

The landscape of cancer treatment has witnessed a paradigm shift with the emergence of immunotherapy, particularly the inhibition of immune checkpoints like programmed cell death protein 1 (PD-1) and its ligand PD-L1. While these therapies have revolutionized cancer treatment, a substantial proportion of patients still fail to respond, highlighting the need to explore alternative immune checkpoints. V-domain immunoglobulin suppressor of T cell activation (VISTA), a member of the B7 family, has orchestrated significant attention. Abundantly expressed in tumor-infiltrating lymphocytes, VISTA's role in regulating immune responses, especially in breast cancer, hints at its potential as a linchpin in breast cancer immunotherapy. This research focuses on molecular docking simulations of a diverse array of immuno-oncology compounds to identify potential VISTA inhibitors. Three promising candidates (Compound_6784, Compound_14718, and Compound_34839) exhibited favorable binding affinities with VISTA, forming key interactions with critical amino acid residues. ADME-Tox profiling revealed these compounds to be highly druggable, displaying minimal toxicity risks, making them suitable for clinical trials and drug development. Additionally, molecular dynamics simulations provided insights into the structural dynamics of these compounds. The metrics underline the superior stability of Compound_14718, making it the most promising candidate for further research and drug development. This research lays a robust foundation for the development of novel immunotherapies targeting VISTA in cancer treatment. The identified compounds present promising candidates for further investigation and drug development, offering hope for improved cancer immunotherapy strategies.

VISTA nonredundantly regulates proliferation and CD69low γδ T cell accumulation in the intestine in murine sepsis.

Sepsis is a dysregulated systemic immune response to infection i.e. responsible for ∼35% of in-hospital deaths at a significant fiscal healthcare cost. Our laboratory, among others, has demonstrated the efficacy of targeting negative checkpoint regulators (NCRs) to improve survival in a murine model of sepsis, cecal ligation and puncture (CLP). B7-CD28 superfamily member, V-domain immunoglobulin suppressor of T cell activation (VISTA), is an ideal candidate for strategic targeting in sepsis. VISTA is a 35 to 45 kDa type 1 transmembrane protein with unique biology that sets it apart from all other NCRs. We recently reported that VISTA-/- mice had a significant survival deficit post-CLP, which was rescued upon adoptive transfer of a VISTA-expressing pMSCV-mouse Foxp3-EF1α-GFP-T2A-puro stable Jurkat cell line (Jurkatfoxp3 T cells). Based on our prior study, we investigated the effector cell target of Jurkatfoxp3 T cells in VISTA-/- mice. γδ T cells are a powerful lymphoid subpopulation that require regulatory fine-tuning by regulatory T cells to prevent overt inflammation/pathology. In this study, we hypothesized that Jurkatfoxp3 T cells nonredundantly modulate the γδ T cell population post-CLP. We found that VISTA-/- mice have an increased accumulation of intestinal CD69low γδ T cells, which are not protective in murine sepsis. Adoptive transfer of Jurkatfoxp3 T cells decreased the intestinal γδ T cell population, suppressed proliferation, skewed remaining γδ T cells toward a CD69high phenotype, and increased soluble CD40L in VISTA-/- mice post-CLP. These results support a potential regulatory mechanism by which VISTA skews intestinal γδ T cell lineage representation in murine sepsis.

Immune Checkpoint Expression Patterns on T Cell Subsets in Light-Chain Amyloidosis: VISTA, PD-1, and Tigit As Potential Therapeutic Targets

Abstract Background: Systemic immunoglobulin light chain amyloidosis (AL amyloidosis) is a rare plasma cell dyscrasia with a poor prognosis. T-cell exhaustion can lead to impaired T cell-mediated antitumor immunity, but there is limited information available regarding the T-cell immunodeficiency status in AL amyloidosis. This study aims to investigate the T-cell immune checkpoint expression patterns in AL amyloidosis and its relationship with clinicobiological characteristics. Methods: Using multi-color fluorescent flow cytometry, we examined the expression levels of V-domain immunoglobulin suppressor of T cell activation (VISTA), programmed death 1 (PD-1), T cell immunoglobulin and mucin-domain-containing-3 (Tim-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT) on CD3+, CD4+, CD8+ T-cell, and regulatory T cell (Treg) in peripheral blood (PB) and bone marrow (BM) from 19 patients with newly diagnosed AL amyloidosis. We used 36 PB and BM samples from patients with newly diagnosed multiple myeloma (MM) and 19 PB and 10 BM samples from healthy individuals (HIs) as controls. Results: Our results showed that PB from patients with AL amyloidosis had significantly higher percentages of VISTA+ and PD-1+ subsets within CD3+, CD4+, CD8+ T cells and Tregs when compared to HIs. No statistical differences for frequency of VISTA+, PD-1+, Tim-3+ and TIGIT+ T-cell in BM were observed between the two groups. The percentages of double-positive VISTA+ PD-1+, VISTA+ Tim-3+, VISTA+ TIGIT+, PD-1+ Tim-3+ and PD-1+ TIGIT+ T cells in PB from the patients with AL amyloidosis were also considerably higher than those in the HIs. Moreover, we discovered that the patients with renal involvement had greater percentages of PD-1+ or TIGIT+ subsets within CD3+, CD4+ T cells and Tregs than the individuals without renal involvement. Additionally, PD-1+CD3+%, PD-1+CD4+%, and PD-1+Treg% were found to be positively correlated with 24-hour proteinuria levels. Furthermore, we observed that the AL amyloidosis patients had significantly higher counts of PD-1+ Treg in PB than the MM patients, while the MM patients had significantly higher counts of TIGIT+ subsets within CD3+, CD4+ and CD8+ T cells than the AL amyloidosis patients. Conclusions: Our findings suggest that there are elevated proportions of VISTA+ and PD-1+ T cells in PB of AL amyloidosis patients, which are indicators of an immunosuppressive milieu. The rise in PD-1+ or TIGIT+ T cells was associated with renal damage. Notably, MM and AL amyloidosis have very distinct immunosuppressive patterns based on immune checkpoint alteration. Overall, VISTA, PD-1, and TIGIT may be considered as potential targets for reversing T cell exhaustion and enhancing T cell activity in AL amyloidosis.

Effective Antitumor Immunity Can Be Triggered by Targeting VISTA in Combination with a TLR3-Specific Adjuvant.

Resistance to anti-PD-1/PD-L1 treatment is often associated with accumulation of intratumoral inhibitory macrophages. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a nonredundant immune checkpoint that can induce both T-cell and myeloid-cell immunosuppression. In this study, we found that high levels of VISTA+ immune cells were associated with advanced stage bladder cancer and predicted poor survival in patients. A combination of high infiltration of VISTA+ immune cells and PD-L1+ immune cells or PD-1+ T cells predicted the worst survival. Flow cytometry and multiplex immunofluorescence analyses confirmed that VISTA expression was higher in macrophages than in T cells or neutrophils, and only VISTA+CD163+ macrophage density predicted poor prognosis in patients with bladder cancer. Toll-like receptor (TLR) agonists are known to trigger the innate immune response in macrophages. We found that the VISTA-specific mAb 13F3 augmented the ability of a TLR3-specific adjuvant to induce macrophage activation in vitro. In the MB49 syngeneic mouse model of bladder cancer, treatment with 13F3 curbed tumor growth and prolonged survival when combined with a TLR3-specific adjuvant. The combination treatment reduced the intratumoral frequency of CD206+ anti-inflammatory macrophages and levels of the immunosuppressive molecule TGFβ1, but it upregulated expression of immunostimulatory molecules (Ifna, Ifnb, and Trail) and increased the CD8+ T cell/regulatory T-cell ratio. These findings indicate that elevated VISTA expression in immune cells, particularly macrophages, is associated with an unfavorable prognosis in patients with bladder cancer and suggest that targeting VISTA in combination with a TLR3-specific adjuvant has translational potential.

Imatinib and M351-0056 enhance the function of VISTA and ameliorate the development of SLE via IFN-I and noncanonical NF-κB pathway.

V-domain immunoglobulin suppressor of T-cell activation (VISTA), an important negative checkpoint protein, participates in immunoregulation. Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients exhibit high levels of autoantibodies and multi-organ tissue injury, primarily involving the kidney and skin. In wild-type (WT) mice and Vsir-/- mice with pristane-induced lupus-like disease, we found that VISTA deficiency exacerbated the lupus-like disease in mice, possibly through aberrant activation of type I interferon (IFN-I) signaling, CD4+ T cell, and noncanonical nuclear factor-κB (NF-κB) pathway. Surface plasmon resonance results showed that imatinib, an FDA-approved tyrosine kinase inhibitor, may have a high affinity for human VISTA-ECD with a KD value of 0.2009 μM. The biological activities of imatinib and VISTA agonist M351-0056 were studied in monocytes and T cells and in lupus-like disease murine model of chronic graft-versus-host disease (cGVHD) and lupus-prone MRL/lpr mice. VISTA small-molecule agonist reduced the cytokine production of peripheral blood mononuclear cells (PBMCs) and Jurkat cells and inhibited PBMCs proliferation. Moreover, they attenuated the levels of autoantibodies, renal injury, inflammatory cytokines, chemokines, and immune cell expansion in the cGVHD mouse model and MRL/lpr mice. Our findings also demonstrated that VISTA small-molecule agonist ameliorated the development of SLE through improving aberrantly activated IFN-I signaling and noncanonical NF-κB pathway. In conclusion, VISTA has a protective effect on the development and progression of SLE. VISTA agonist M351-0056 and imatinib have been firstly demonstrated to attenuate SLE, suggesting interventions to enhance VISTA function may be effective in treating SLE. VISTA deficiency exacerbates pristane-induced lupus-like disease in mice by promoting activation of the IFN-I and noncanonical NF-κB pathway. Imatinib was screened as a small-molecule VISTA agonist by molecular docking, SPR, and cellular level experiments. VISTA agonists (M351-0056 and imatinib) alleviated lupus-like disease progression in the cGVHD mouse model and MRL/lpr mice by inhibiting activation of IFN-I and noncanonical NF-κB pathway.