On the same side: The immune regulatory protein Vista and its ligands interact in cis
VISTA, an essential immune checkpoint regulatory protein, regulates peripheral T-cell quiescence and tolerance. Despite its potential as a target for anti-tumor and autoimmune disease therapies, uncertainty regarding VISTAs binding mode and membrane orientation has hindered these developments. Contrary to the prevailing paradigm, we found using cell aggregation assays that VISTA cannot interact with its ligands in trans (between cells). Using MST and flow cytometry, we showed that soluble VISTA binds to its ligands, suggesting that VISTAs membrane orientation restricts trans interactions. In contrast, split luciferase complementation assays showed that VISTA interacts with its ligands in cis. We propose that a disulfide bond bends VISTAs Ig domain towards the membrane in an orientation that prevents trans while enabling cis interactions. 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. Our findings reveal VISTAs 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.