biostudies-literatureRobinson MAUKRI Medical Research CouncilMedical Research CouncilAstraZenecaWellcome Trust114768https://www.ebi.ac.uk/biostudies/studies/S-EPMC7618755biostudies-literatureUnknown43(9)The CTLA-4 and PD-1 checkpoints control immune responses and are key targets in immunotherapy. Both pathways are connected via a cis interaction between CD80 and PD-L1, the ligands for CTLA-4 and PD-1, respectively. This cis interaction prevents PD-1-PD-L1 binding but is reversed by CTLA-4 trans-endocytosis of CD80. However, how CTLA-4 selectively removes CD80, but not PD-L1, is unclear. Here, we show CTLA-4-CD80 interactions are unimpeded by PD-L1 and that CTLA-4 binding with CD80 does not displace PD-L1 per se. Rather, both rigidity and bivalency of CTLA-4 molecules are required to orientate CD80 such that PD-L1 interactions are no longer permissible. Moreover, soluble CTLA-4 released PD-L1 only at specific expression levels of CD80 and PD-L1, whereas CTLA-4 trans-endocytosis released PDCell reportsRigid, bivalent CTLA-4 binding to CD80 is required to disrupt the cis CD80/PD-L1 interaction.PMC7618755MR/R015759/1227384/Z/23/Z204798/Z/16/ZMR/W006774/1MR/Y001273/1Filer LDovedi SJSansom DMLloyd CKennedy AYeung KChen HCWaters EOrozco CTRobinson MAGiovacchini DHinze CfalseRigid, bivalent CTLA-4 binding to CD80 is required to disrupt the cis CD80/PD-L1 interaction.The CTLA-4 and PD-1 checkpoints control immune responses and are key targets in immunotherapy. Both pathways are connected via a cis interaction between CD80 and PD-L1, the ligands for CTLA-4 and PD-1, respectively. This cis interaction prevents PD-1-PD-L1 binding but is reversed by CTLA-4 trans-endocytosis of CD80. However, how CTLA-4 selectively removes CD80, but not PD-L1, is unclear. Here, we show CTLA-4-CD80 interactions are unimpeded by PD-L1 and that CTLA-4 binding with CD80 does not displace PD-L1 per se. Rather, both rigidity and bivalency of CTLA-4 molecules are required to orientate CD80 such that PD-L1 interactions are no longer permissible. Moreover, soluble CTLA-4 released PD-L1 only at specific expression levels of CD80 and PD-L1, whereas CTLA-4 trans-endocytosis released PD2024-01-01T00:00:00Z2024 Sep2026-07-16T06:56:18.668Z2026-07-09T13:10:41.553ZS-EPMC76187553927786010.1016/j.celrep.2024.114768