Fig 1: Blockade of V‐domain immunoglobulin suppressor of T‐cell activation (VISTA) enhances effective function of CD8+ T cells and promotes efficacy of programmed cell death protein‐1 (PD‐1) inhibitor. (A) An ex vivo tumour inhibition assay was established to investigate the potential impact of VISTA and/or PD‐1 blockade in gastric cancer (GC). Single‐cell suspensions derived from freshly resected GC tissues (Zhongshan Hospital Fudan University [FDU‐ZSH] Experimental Cohort [EXPC] Arm G) were randomly divided into four treatment groups: isotype control (IgG2B, 15 μg/mL, R&D Systems; IgG4, 10 μg/mL, BioLegend); VISTA blockade subgroup (α‐VISTA antibody, 15 μg/mL, R&D Systems; IgG4, 10 μg/mL, BioLegend); PD‐1 blockade subgroup (camrelizumab, 10 μg/mL, Suncadia Biopharmaceuticals; IgG2B, 15 μg/mL, R&D Systems); dual blockade subgroup (α‐VISTA antibody, 15 μg/mL, R&D Systems; camrelizumab, 10 μg/mL, Suncadia Biopharmaceuticals). Each treatment group was cultured in RPMI 1640 medium (Gibco) containing 10% foetal bovine serum (FBS; Gibco) for 12 h at 37°C. FDU‐ZSH EXPC (Arm G) was our own dataset and contained 10 GC patients (Figure S1). (B) In another CD8+ T‐cell‐deprived ex vivo tumour inhibition assay, CD8+ T cells were depleted from single‐cell suspensions (FDU‐ZSH EXPC Arm H) by human CD8 nanobeads (MojoSort, BioLegend). Single‐cell suspensions were cultured in 1 mL RPMI 1640 medium (Gibco) containing 10% FBS (Gibco). Then, the cells were cultured with α‐VISTA antibody (15 μg/mL, R&D Systems) or IgG2B (15 μg/mL, R&D Systems) for 12 h at 37°C. After overnight culture, the cells were harvested for phenotype analysis by flow cytometry (FC)/intracellular flow cytometry (ICFC). FDU‐ZSH EXPC (Arm H) was our own dataset, including 5 GC patients (Figure S1). (C and D) In FDU‐ZSH EXPC (Arm G), blockade of PD‐1 showed increased apoptosis of tumour cells in the tumours infiltrated with VISTAlow TAMs, rather than VISTAhigh TAMs. Similarly, elevated expression of interferon‐γ (IFN‐γ) and granzyme B (GzmB) within CD8+ T cells was observed in the tumours with VISTAlow TAMs rather than VISTAhigh TAMs, after application of camrelizumab. (E) In FDU‐ZSH EXPC (Arm G), blockade of VISTA showed increased apoptosis of tumour cells. Combination of VISTA blockade and PD‐1 inhibitor camrelizumab showed more significant apoptosis of tumour cells, compared with VISTA blockade or camrelizumab alone. Data are expressed as mean ± standard deviation. (F–H) In FDU‐ZSH EXPC (Arm G), blockade of VISTA showed increased expression of IFN‐γ, GzmB and Perforin within CD8+ T cells. Combination of VISTA blockade and PD‐1 inhibitor camrelizumab showed more significant reactivation of CD8+ T‐cell‐effective function, compared with VISTA blockade or camrelizumab alone. Data are expressed as mean ± standard deviation. (I) In FDU‐ZSH EXPC (Arm H), the antitumour effect of VISTA blockade was abolished when CD8+ T cells were depleted from single‐cell suspensions, while the single‐cell suspensions with CD8+ T cells depletion alone did not show significant decrease in tumour cell apoptosis compared with those without CD8+ T cells depletion. Data are expressed as mean ± standard deviation. Significance values were determined by paired t test (D), RM one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (E–H), or Sidak's multiple comparisons test (I). * p < .05, ** p < .01, *** p < .001 and n.s. refers to not significant. EpCAM, epithelial cell adhesion molecule.