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.

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.

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.

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.