PD-L1 binds with B7-1 only in cis on the same cell surface

Abstract

Abstract Programmed death ligand 1 (PD-L1)-mediated immune suppression is a major regulator of peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but in addition binds to B7-1 (CD80) to regulate T cell function. The binding interactions of the PD-L1-B7-1 pathway and its functional role need further investigation in order to understand differences between PD-1 and PD-L1 mediated tumor immunotherapy. We examined the molecular orientation for PD-L1 binding to B7-1 using cell to cell binding assays, ELISA, and flow cytometry. Surprisingly, we found that PD-L1 transfected cells did not bind to B7-1 transfected cells, whereas PD-L1 transfected cells bound to PD-1 transfected cells. As expected, B7-1 transfected cells bound to CD28 or cytotoxic T-lymphocyte antigen 4 (CTLA-4) transfected cells. By ELISA and flow cytometry with purified proteins, we found that PD-L1 and B7-1 had a strong binding interaction only when the PD-L1 molecule was accessible and flexible. On decreasing its accessibility, the binding was greatly reduced. We observed the PD-L1-B7-1 interaction competed for the binding of PD-L1 to PD-1. Further, we co-transfected PD-L1 and B7-1 on the same cell surface and confirmed the cis binding using a Nanobit Assay system. These results indicate that the PD-L1-B7-1 interaction can occur in cis when the molecules are on the same cell but not in trans when the molecules are on different cells. This binding orientation emphasizes the functional importance of co-expression of B7-1 and PD-L1 on the same cell. Interaction in cis competes for binding to other receptors and modulates their function. Our findings help in further understanding PD-L1 mediated immune suppression.