Homology modeling of the VISTA 3D structure
The human VISTA protein extracellular domain without the signal peptide (162 amino acids, UniProt: Q9H7M9) and the murine VISTA protein extracellular domain without the signal peptide (159 amino acids, UniProt: Q9D659) were submitted to the I-TASSER online server[35-37] for 3D structure homology modeling.
Virtual screening for VISTA small-molecule hit ligands
The 3D models of the human/murine VISTA protein extracellular domains as well as their ligand-binding pockets were predicted by COACH . Approximately 130,000 SDF files of structurally diverse small molecules from chemdiv library（Molecular Weight:300-500, XLogP:3~5,）were converted to PDBQT format as ligands using Open Babel (version 2.4)[38-39].The grid (ligand docking search space) was maximized based on the VISTA protein extracellular domain. Then, AutodockVina 1.1.2 was used for the subsequent molecular docking.
Docked ligands located in the predicted ligand-binding pockets with a binding energy lower than -6.0 kcal/mol were considered the candidate murine VISTA ligands. The best 20 candidate ligands were selected for the subsequent experimental protein-ligand interaction verification (ELISA)(Suppl. Table 1). The hit ligand verified by the above virtual screening with the lowest Kd values (ELISA) was applied for the VISTA-targeted in vitro experiment.
Visualization of docked ligands with the VISTA protein
Protein-ligand interactions were visualized using Pymol version 184.108.40.206. The amino acid residues of the human/murine VISTA proteins close to the hit ligands (≤1 Å) were highlighted as potential interactive residues involved in the protein-ligand interaction.
Microscale thermophoresis (MST) experiment
Murine VISTA-ECD was labeled following the protocol provided in the Monolith NTTM Protein Labeling Kit RED-NHS (NanoTemper Technologies GmbH). Compound 6809-0223 was prepared in up to 16 serial dilutions and mixed with the labeled protein in the same volume. Then, the mixtures were incubated at room temperature for 30 min in the dark.
Mice (C57BL/6) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were performed in accordance with the Laboratory Animal Management Committee of Jiangsu Province and approved by the ethics committee of China Pharmaceutical University. To ameliorate any suffering of mice observed throughout these experimental studies, mice were euthanized by CO2 inhalation.
Murine CD4+ and CD8+ T cell activation assays.
CD4+ or CD8+ T cells were isolated from C57BL/6 mice using an EasySep murine CD4+ or CD8+ T cell isolation kit (Stem Cell). The cells were cultured in complete RPMI 1640 medium supplemented with 10% FBS, 10 mM Hepes, 50 µM 2-mercaptoethanol, 1% penicillin/streptomycin and 1% L-glutamine. Purified CD4+ or CD8+ T cells (100,000 cells per well) were cultured in 96-well flat-bottom plates in the presence of anti-CD3 (clone 2C11) and either VISTA-Ig or control-Ig at the indicated concentration ratios. The cell supernatants were harvested at 48 h for the cytokine secretion assay. For the proliferation assay, the cells were stained with a 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) kit following the manufacturer’s protocol (Biolegend). Cell proliferation was analyzed at 72 h by CFSE dilution.
Cytometric bead assay (CBA) for CD8+ T cells
First, 96-well flat-bottom plates were coated with anti-mouse CD3 (clone OKT3, Biolegend) at 2.5 μg/mL together with 5 μg/mL of murine VISTA-ECD protein in PBS at 4°C overnight. CD8+ T cells were plated at a density of 1 × 105 cells/well in complete RPMI medium (RPMI 1640, 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 50 µM 2-mercaptoethanol, 10mM HEPEs and 1 mM GlutaMAX™; all Biological Industries). A titrated amount of compound 6809-0223 was added to the culture medium. The cultured supernatants were collected at 48 h, and the cytokine levels in the supernatant were analyzed using a BD Cytometric Bead Array (CBA) kit according to the manufacturer’s instructions.
The data are expressed as the mean ± SD unless indicated otherwise. An unpaired Student’s t-test was used to determine statistically significant differences. A value of P < 0.05 was considered significant at the 95% confidence level.