The activation and function of T cells is guided by immune checkpoint and co-stimulatory signals encountered on the surface of their interaction partners. Despite the importance of these molecules in T cell immunoregulation, little is known about the spatial organization and surface architecture of these proteins. To address this knowledge gap, we employed multiplexed, super-resolution DNA-PAINT imaging on the surface of individual mouse cDC1s and melanoma cells to simultaneously map the single protein distributions of 6 different proteins: the co-stimulatory molecules CD80 and CD86, the immune checkpoints PD-L1 and PD-L2, and MHC-I and MHC-II. These data reveal unappreciated and complex spatial organization associated with immune activation vs repression, and identify CD80 as a key “remodelling” factor that disrupts formation of immunosuppressive surface complexes.