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We characterized the relationships of PD-L1 with lipid rafts in the plasma membrane by adopting a toolbox of fluorescence microscopy techniques which span different spatial resolutions and ultimately leverage single molecule detection. On account of our interest in novel therapeutic approaches to Non-Small Cell Lung Cancer (NSCLC), we selected the HCC827 as model cell line, because we demonstrated it possesses an abundant plasma membrane expression of PD-L1 while being representative of common mutation of EGFR gene in NSCLC. Our findings visually revealed for the first time that PD-L1 is engaged in the raft regions of the plasma membrane where it arranges as polydisperse nanostructured clusters with <100 nm size. Consistently, raft disruption by cholesterol depletion leads to significant alteration of this morphological pattern. Engagement in rafts may confer PD-L1 specific functions in the context of PD-L1/PD-1 mediated immuno checkpoint and the immuno-oncology strategies to modulate it.

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