Super-Resolution Microscopy Reveals Nanoscale Arrangement of PD-L1 Immune Checkpoint.

PD-L1 is an immune checkpoint protein widely expressed in cancer cells, maintaining the tolerance of antitumor immunity by engaging with its receptor PD-1 on T cells. However, the spatial organization of PD-L1 on the cell membrane is poorly understood. Here, we employ stochastic optical reconstruction microscopy (STORM) integrated with analytical methods to quantitatively reveal the nanoscale arrangement of PD-L1 in breast cancer cells. Results show that PD-L1 is distributed randomly on the cell membrane as monomers. Further, treatments with drugs such as IFN-γ, 2-DG, and simvastatin modulate the density of PD-L1 without affecting its monomeric state. By STORM, combining 2nd antibody-induced cross-linking and fluorescence recovery after photobleaching, we demonstrate that PD-L1 exhibits high lateral mobility and clustering capability. Additional results reveal that paraformaldehyde, a common fixative, is inadequate for fixing PD-L1 for super-resolution imaging. Finally, we observed that PD-L1 forms nanoclusters at cell-cell conjugation sites between breast cancer cells and T cells. Overall, these findings based on STORM extend our understanding of the nanoscale organization of PD-L1 on the membrane.