Structural basis of CD28 and CTLA-4 interactions with CD80, CD86, and the CD80-PD-L1 heterodimer on artificial and cellular membranes

SUMMARY The opposing actions of the co-stimulatory receptor CD28 and the co-inhibitory receptor CTLA-4, mediated by their interactions with B7-family ligands, govern the balance between T-cell activation and immune tolerance. We determined the cryo-EM structures of CD28 and CTLA-4 bound to CD80, CD86, and the CD80-PD-L1 heterodimer under two-dimensional membrane confinement. We show that CD28-CD80, CD28-CD86, and CTLA-4-CD86 form discrete complexes with closed-leg, cross-leg, and open-leg configurations, respectively, whereas CTLA-4 assembles into extended linear clusters and two-dimensional lattices with CD80. PD-L1 binding to CD80 disrupts CTLA-4-CD80 clustering by preventing CD80 homodimerization, while preserving CD28-CD80 binding through a pronounced architectural rearrangement. We further confirm the formation of linear and two-dimensional CTLA-4-CD80 clusters in cell-derived membrane fragments using cryo-EM. Together, these findings demonstrate that immune checkpoint signaling is governed not only by receptor-ligand affinity but also by membrane-imposed geometry and the competitive reorganization of higher-order assemblies at the immunological synapse.