Repositioning baloxavir marboxil as VISTA agonist that ameliorates experimental asthma.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), a novel negative checkpoint regulator, plays an essential role in allergic pulmonary inflammation in mice. Treatment with a VISTA agonistic antibody could significantly improve asthma symptoms. Thus, for allergic asthma treatment, VISTA targeting may be a compelling approach. In this study, we examined the functional mechanism of VISTA in allergic pulmonary inflammation and screened the FDA-approved drugs for VISTA agonists. By using mass cytometry (CyTOF), we found that VISTA deficiency primarily increased lung macrophage infiltration in the OVA-induced asthma model, accompanied by an increased proportion of M1 macrophages (CD11b+F4/80+CD86+) and a decreased proportion of M2 macrophages (CD11b+F4/80+CD206+). Further in vitro studies showed that VISTA deficiency promoted M1 polarization and inhibited M2 polarization of bone marrow-derived macrophages (BMDMs). Importantly, we discovered baloxavir marboxil (BXM) as a VISTA agonist by virtual screening of FDA-approved drugs. The surface plasmon resonance (SPR) assays revealed that BXM (KD = 1.07 µM) as well as its active form, baloxavir acid (BXA) (KD = 0.21 µM), could directly bind to VISTA with high affinity. Notably, treatment with BXM significantly ameliorated asthma symptoms, including less lung inflammation, mucus secretion, and the generation of Th2 cytokines (IL-5, IL-13, and IL-4), which were dramatically attenuated by anti-VISTA monoclonal antibody treatment. BXM administration also reduced the pulmonary infiltration of M1 macrophages and raised M2 macrophages. Collectively, our study indicates that VISTA regulates pulmonary inflammation in allergic asthma by regulating macrophage polarization and baloxavir marboxil, and an old drug might be a new treatment for allergic asthma through targeting VISTA.

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.

A small molecule inhibitor of VSIG-8 prevents its binding to VISTA.

The V-region immunoglobulin-containing suppressor of T cell activation (VISTA), a unique B7 family member, is an attractive immunotherapeutic target for cancer, autoimmune and inflammatory diseases. In 2016, a patent demonstrated V-Set and Immunoglobulin domain containing 8 (VSIG-8) was the putative VISTA receptor. Antagonistic or agonistic agents can conceivably modulate VISTA and its interacting partners, which will greatly benefit the treatment of many diseases. The interaction of VISTA and VSIG-8 were measured by Enzyme-linked Immuno Sorbent Assay (ELISA), Microscale Thermophoresis (MST) and coimmunoprecipitation (Co-IP) experiments. The bioactivity of VSIG-8 inhibitor L557-0155 was evaluated in vitro and in vivo. Kd value of human VISTA binding to human VSIG-8 was 1.58 ± 0.44 µM by MST. When the related amino acid binding site of VISTA was mutated to alanine, the interaction between VISTA and VSIG-8 disappeared. VSIG-8 protein induced an decrease in the level of IL-2 in VISTA-overexpressing cells but a increase in VISTA-/- cells. Furthermore, VSIG-8 inhibitor L557-0155 promoted cytokine production and cell proliferation in PBMCs and suppressed melanoma growth. VSIG-8/VISTA coinhibitory pathway may provide a novel strategy for the treatment of human cancers, and VSIG-8 blockade may increase antitumor immunity. This study was the first time to report that VSIG-8 interacts with VISTA, and inhibited T cell function.

Identification of active small-molecule modulators targeting the novel immune checkpoint VISTA.

BACKGROUND: Cancer immunotherapy has gained increasing popularity as a novel approach to treat cancer. A member of the B7 family, V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune checkpoint that regulates a broad spectrum of immune responses. VISTA is an acidic pH-selective ligand for P-selectin glycoprotein ligand-1(PSGL-1). CA-170, a first-in-class small-molecule dual antagonist of VISTA/PD-L1, was collaboratively developed by Aurigene Discovery Technologies Limited and Curis, Inc. It is currently in Phase I clinical trial. RESULTS: In this study, we develop homology modeling for the VISTA 3D structure and subsequent virtual screening for VISTA small-molecule hit ligands. Visualization of the binding postures of docked ligands with the VISTA protein indicates that some small molecular compounds target VISTA. The ability of antagonist to disrupt immune checkpoint VISTA pathways was investigated though functional studies in vitro. CONCLUSIONS: Affinity active molecule for VISTA was obtained through virtual screening, and the antagonist compound activity to VISTA was assayed in cellular level. We reported a small molecule with high VISTA affinity as antagonist, providing ideas for development VISTA-targeted small molecule compound in cancer immunotherapy.

Safety of Human Hepatoma Cell-Line Constructing Bioartificial Liver Supporting System Treating Patients with Liver Failure.

BACKGROUND/AIMS: To observe the clinical safety of bioartificial liver supporting system constructed by human hepatoma cell line. METHODOLOGY: Seventeen patients with liver failure were treated with C3A-cell-constructed bioartificial liver supporting system, contrasting the difference of biochemical results and imaging data with 9 patients treated with non-bioartificial liver during 5-year treatment. RESULTS: 11 cases of Treatment Group survived at 3 months' follow-up, among whom 2 cases underwent hepatic transplantation. 9 cases without hepatic transplantation survived in 5-year follow-up, and 1 of them was found to occur focal liver lesion at the 5th years, and had hepatic lobectomy. Pathological prompt: hepatocellular carcinoma with moderate differentiation. Totally 4 patients in Control Group survived after 3 months' follow-up, including 1 patient of hepatic transplantation. All the 3 patients without hepatic transplantation survived the last 5-year follow-up, with basically normal biochemical indicators and no focal liver lesion were found by imaging examination. CONCLUSIONS: It was safe to use bioartificial liver constructed by tumor cell line C3A to treat liver failure.

VISTA deficiency exerts anti-tumor effects in breast cancer through regulating macrophage polarization.

Growing evidence had showed that tumor-associated macrophages (TAMs) have a tumor-promoting M2 phenotype which could drive pathological phenomena. In breast cancer, TAMs are abundantly present and may play an important role in the development of breast cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel inhibitory checkpoint and immunotherapy target for tumor through regulating immune response. However, its effects on macrophages have not been investigated, which was also the focus of this study. Here, the scRNA-seq data further revealed that VISTA was highly expressed in multiple macrophage subclusters. In vitro experiments showed that the absence of VISTA enhanced the M1 polarization of macrophages, inhibited the M2 polarization of macrophages and the proliferation and phagocytosis of 4 T1 cells induced by M2-CM. VISTA regulated the activation of STAT1 and STAT6 signaling pathways in the process of macrophage polarization. In vivo experiments demonstrated that VISTA deficient mice exhibited reduced tumor growth, possibly due to the increase of M1 macrophages and the decrease of M2 macrophages. In summary, our study is the first to reveal the effect of VISTA on macrophages in breast cancer, which showed that VISTA affects tumor growth by critically regulating the macrophage polarization through the STAT pathway.

Co-expression of immune checkpoints in glioblastoma revealed by single-nucleus RNA sequencing and spatial transcriptomics.

Glioblastoma (GBM) is one of the most malignant tumors of the central nervous system. The pattern of immune checkpoint expression in GBM remains largely unknown. We performed snRNA-Seq and spatial transcriptomic (ST) analyses on untreated GBM samples. 8 major cell types were found in both tumor and adjacent normal tissues, with variations in infiltration grade. Neoplastic cells_6 was identified in malignant cells with high expression of invasion and proliferator-related genes, and analyzed its interactions with microglia, MDM cells and T cells. Significant alterations in ligand-receptor interactions were observed, particularly between Neoplastic cells_6 and microglia, and found prominent expression of VISTA/VSIG3, suggesting a potential mechanism for evading immune system attacks. High expression of TIM-3, VISTA, PSGL-1 and VSIG-3 with similar expression patterns in GBM, may have potential as therapeutic targets. The prognostic value of VISTA expression was cross-validated in 180 glioma patients, and it was observed that patients with high VISTA expression had a poorer prognosis. In addition, multimodal cross analysis integrated SnRNA-seq and ST, revealing complex intracellular communication and mapping the GBM tumor microenvironment. This study reveals novel molecular characteristics of GBM, co-expression of immune checkpoints, and potential therapeutic targets, contributing to improving the understanding and treatment of GBM.

Transcriptome profiling reveals transcriptional regulation of VISTA in T cell activation.

PURPOSE: V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel type of immune checkpoint. This study was performed to explore the potential mechanism by which different domains of VISTA affect T-cell activation and search for potential interacting proteins. METHODS: Stably transfected Jurkat cell lines were constructed to overexpress human VISTA (VISTA-FL), cytoplasmic domain deletion mutants (VISTA-ΔECD) and extracellular domain deletion mutants (VISTA- ΔCD). Empty vector (EV) control cell lines were constructed. Four stable cell lines were subjected to transcriptome sequencing after stimulation with PMA and PHA. The differentially expressed genes (DEGs) were analysed to explore the potential pathway by which VISTA inhibits T-cell activation. Proteinprotein interaction (PPI) network analysis was used to search for potential interacting proteins of VISTA. RESULTS: In this study, 1256 DEGs were identified in Jurkat-VISTA-FL cells, 740 DEGs in Jurkat-VISTA-ΔCD cells, and 5605 DEGs in Jurkat-VISTA-ΔECD cells compared with Jurkat-EV cells. DEGs were mainly enriched in pathways related to T-cell differentiation, T-cell receptor signalling pathway and T-cell migration in Jurkat-VISTA-ΔECD cells; with cholesterol biosynthesis in Jurkat-VISTA-ΔCD cells; and with the inflammatory response in Jurkat-VISTA-FL cells. HHLA2 and CTH were identified as potential partners that interact directly with VISTA. The results also show an indirect interaction between VISTA and PSGL-1. CONCLUSIONS: This study revealed the pathways by which VISTA is involved in T-cell activation and identified the potential binding partners of VISTA through RNA-seq, providing valuable resources for developing in-depth studies of the action mechanisms of VISTA as a potential target for cancer and inflammatory diseases.

Discovery of Triazolyl Derivatives of Cucurbitacin B Targeting IGF2BP1 against Non-Small Cell Lung Cancer.

Cucurbitacin B (CuB) is a potent but toxic anticancer natural product. Herein, we designed and synthesized 2-OH- and 16-OH-modified CuB derivatives to improve their antitumor efficacy and reduce toxicity. Among them, derivative A11 had the most potent antiproliferative activity against A549 lung cancer cells (IC50 = 0.009 µM) and was approximately 10-fold more potent than CuB, while the cytotoxicity of A11 toward normal L02 cells was about 10-fold less potent, indicating a much wider therapeutic window than CuB. Derivative A11 directly binds to the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) protein with a KD value of 2.88 nM, which is about 23-fold more potent than CuB, leading to the decreased expression of downstream apoptosis- and cell cycle-related proteins. More importantly, A11 exhibited much more potent anticancer efficacy in an A549 xenograft mouse model with a TGI rate of 80% and a superior in vivo safety profile than that of CuB.

M351-0056 is a novel low MW compound modulating the actions of the immune-checkpoint protein VISTA.

BACKGROUND AND PURPOSE: The protein V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune-checkpoint molecule that belongs to the B7 family and regulates a broad spectrum of immune responses. So far, low MW compounds targeting VISTA for the treatment of autoimmune diseases or inflammation, have not been identified. EXPERIMENTAL APPROACH: We developed a homology modelling for VISTA 3D structure and subsequent virtual screening for low MW ligands binding to VISTA. Visualization of the binding postures of docked ligands with protein VISTA indicated that compound M351-0056 targeted VISTA. The biological activities of compound M351-0056 targeting VISTA were investigated in vitro using monocytes and T cells and in vivo, using mice with imiquimod-induced dermatitis. KEY RESULTS: The KD value of M351-0056 for human VISTA-extracellular domain was 12.60 ± 3.84 µM as assessed by microscale thermophoresis. M351-0056 decreased cytokine secretion from PBMCs or human CD4+ T cells, suppressed proliferation of PBMCs and enhanced expression of Foxp3+ T cells. These effects of M351-0056 modulating VISTA involved the JAK2-STAT2 pathway. Daily administration of M351-0056 ameliorated imiquimod-induced psoriasis-like dermatitis. Expression of mRNA and protein of inflammatory cytokines in psoriatic lesions was decreased after M351-0056 treatment. CONCLUSION AND IMPLICATIONS: The compound M351-0056 showed high affinity for VISTA and may modulate its immune function in vitro and in vivo. Our finding provides a lead compound for therapeutically enhancing VISTA-mediated pathways to benefit the treatment of autoimmune diseases or inflammation.

Single-cell RNA-Seq reveals the transcriptional landscape and heterogeneity of skin macrophages in Vsir-/- murine psoriasis.

Rationale: V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel inhibitory immune checkpoint molecule. Vsir-/- mice have exacerbated psoriasis-like skin inflammation. The immune cell subsets involved in inflammation in Vsir-/- psoriatic mice are largely unknown. We have used scRNA-seq as an unbiased profiling strategy to study the heterogeneity of immune cells at a single cell level in the skin of Vsir-/- psoriatic mice. Methods: In the present study, the right ear and shaved back skin of wild type and Vsir-/- mice were treated with IMQ for 5 consecutive days to induce psoriasis-like dermatitis. Then, the single-cell RNA sequencing analysis of mouse back skin lesions was performed using 10 × Genomics technique. Results: We identified 12 major cell subtypes among 23,258 cells. The major populations of the skin cells included macrophages, dendritic cells and fibroblasts. Macrophages constituted the main immune cell population in the WT (61.29%) and Vsir-/- groups (77.7%). It should be noted that DCs and fibroblasts were expanded in the Vsir-/- psoriatic mice. Furthermore, the gene expression signatures were assessed. We observed that Hspb1 and Cebpb were significantly upregulated in the Vsir-/- psoriatic mice. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these subsets and uncovered putative functions of each cell type. Date analysis resulted in the discovery of a number of novel psoriasis-associated genes in Vsir-/- mice. Conclusion: We present a comprehensive single-cell landscape of the skin immune cells in Vsir-/- psoriatic mice. These unprecedented data uncovered the transcriptional landscape and phenotypic heterogeneity of skin macrophages in psoriasis and identified their gene expression signature suggesting specialized functions in Vsir-/- mice. Our findings will open novel opportunities to investigate the role of VISTA in driving psoriasis.

The Expression Pattern and Clinical Significance of the Immune Checkpoint Regulator VISTA in Human Breast Cancer.

Background: Immunotherapies targeting CTLA-4 and PD-1 have elicited promising responses in a variety of cancers. However, the relatively low response rates warrant the identification of additional immunosuppressive pathways. V domain immunoglobulin suppressor of T cell activation (VISTA) plays a critical role in antitumor immunity and is a valuable target in cancer immunotherapy. Methods: Here, we used single-cell RNA-seq to analyze the gene expression levels of 14897 cells from a breast cancer sample and its paired 7,320 normal cells. Then, we validated the protein expression of immune checkpoint molecules (VISTA, PD-1, PD-L1, TIGIT, TIM3, and LAG3) in 324 human breast cancer samples by immunohistochemistry and quantitative immunofluorescence (QIF) approaches. Results: Single cell RNA-seq results show a higher level of immune checkpoint VISTA expression in breast cancer tissue compared to adjacent normal tissue. We also found that VISTA expressed highest in breast cancer tissue than other immune-checkpoints. Immunohistochemical results showed that VISTA was detected with a membranous/cytoplasmic staining pattern in intratumoral immune cells and breast cancer cells. Additionally, VISTA was positively correlated with pathological grade, lymph node status and the levels of PD-1 according to the chi-square test or Fisher's test. Furthermore, VISTA expression was higher in CD68+ tumor-associated macrophages (TAMs) than in CD4+ T cells, CD8+ cytotoxic T cells or CD20+ B cells. Conclusions: These findings therefore support the immunoregulatory role of VISTA in breast cancer and indicate that targeting VISTA may benefit breast cancer immunotherapy.