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Super-Resolution Microscopy Reveals Nanoscale Arrangement of PD-L1 Immune Checkpoint
Fulin Xing1, Jianyu Yang1, Fen Hu1
1The Key Laboratory of Weak-Light Nonlinear Photonics of Education Ministry, School of Physics and TEDA Institute of Applied Physics, Nankai University, Tianjin 300071, China.
Abstract:
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.
Insights
Programmed death-ligand 1 (PD-L1) is randomly distributed as monomers on cancer cell membranes. Super-resolution microscopy reveals PD-L1
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Programmed death-ligand 1 (PD-L1) is a key immune checkpoint protein expressed on cancer cells.
- PD-L1 engages with PD-1 on T cells, suppressing antitumor immunity.
- The nanoscale organization of PD-L1 on the cell membrane is not well understood.
Purpose of the Study:
- To quantitatively reveal the nanoscale arrangement of PD-L1 on breast cancer cell membranes.
- To investigate the effects of drug treatments on PD-L1 organization.
- To assess the mobility and clustering capabilities of PD-L1.
Main Methods:
- Stochastic optical reconstruction microscopy (STORM) was employed for super-resolution imaging.
- Analytical methods were integrated to quantify PD-L1 distribution.
- Techniques included fluorescence recovery after photobleaching and antibody-induced cross-linking.
Main Results:
- PD-L1 was found to be randomly distributed as monomers on the cell membrane.
- Drug treatments (IFN-γ, 2-DG, simvastatin) altered PD-L1 density but not its monomeric state.
- PD-L1 demonstrated high lateral mobility and clustering potential, forming nanoclusters at cell-cell junctions.
Conclusions:
- PD-L1 exists as monomers on the cell membrane, with its density modulated by certain drugs.
- PD-L1 exhibits dynamic behavior, including lateral mobility and clustering.
- Paraformaldehyde fixation is unsuitable for preserving PD-L1 structure in super-resolution imaging.
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