On the same side: VISTA and its ligands interact in <i>cis</i> on the cell surface

VISTA, an essential immune checkpoint protein, regulates peripheral T-cell quiescence and tolerance. Despite its potential as a target for anti-tumor and autoimmune disease therapies, uncertainty regarding VISTA's binding mode and membrane orientation has hindered the development of these therapies. Contrary to the prevailing view, we found, using cell aggregation assays, that VISTA does not interact with its ligands in a trans (between-cell) manner. Using MST and flow cytometry, we showed that when in soluble form, VISTA binds to its ligands, suggesting that VISTA's membrane orientation restricts trans interactions. In contrast, split luciferase complementation assays demonstrated that membrane-tethered VISTA interacts with its ligands in a cis manner (i.e., on the same cell). Co-expression data analysis from the Cancer Genome Atlas showed a strong correlation between VISTA and its ligand, PSGL-1, consistent with our in vitro cis interaction data. We propose that VISTA's Ig domain bends toward the membrane in an orientation that prevents trans while enabling cis interactions. Our findings reveal VISTA's binding mechanism and suggest an intrinsic inhibition signaling pathway independent of additional cells. Importantly, our experimental framework provides a platform for identifying novel VISTA-targeted therapeutics.