Cell surface interactome analysis identifies <scp>TSPAN4</scp> as a negative regulator of <scp>PD</scp> ‐ <scp>L1</scp> in melanoma

PD-L1 is a key immune checkpoint ligand that suppresses antitumor immunity by engaging PD-1 on T cells. While therapeutic blockade of PD-L1/PD-1 interactions has shown clinical benefit, many patients fail to respond, indicating modulation by other factors. Here, we identified a novel regulatory axis in which the membrane-organizing protein tetraspanin-4 (TSPAN4) modulates PD-L1 in melanoma cells. Using cell surface proximity biotinylation coupled with mass spectrometry, we discovered that TSPAN4 physically associates with PD-L1, with both proteins colocalizing on migrasomes and retraction fibers. Mechanistically, we show that TSPAN4 negatively regulates PD-L1 protein levels by enhancing its degradation and restricting its lateral mobility at the plasma membrane. Loss of TSPAN4 stabilized PD-L1, promoted its interaction with CMTM6, and increased PD-L1 surface availability for PD-1 binding. Functionally, TSPAN4 knockdown in melanoma cells led to more efficient immune checkpoint blockade through PD-1 on T cells. This study identifies TSPAN4 as a negative regulator of PD-L1 at the cell surface of melanoma cells suggesting that targeting TSPAN4 may offer a new therapeutic strategy to enhance immune checkpoint blockade in melanoma and other cancers.