Cell surface interactome analysis identifies TSPAN4 as a negative regulator of PD-L1 in melanoma

PD-L1 functions as a co-inhibitory checkpoint ligand constraining anti-tumor immunity by binding to PD-1 on immune cells. Although antibody blockade therapy displays clear therapeutic responses, it is not effective in all cancer patients, indicating modulation by other factors. We applied cell surface proximity biotinylation combined with mass spectrometry in melanoma cells to identify novel proteins regulating PD-L1 function. Our findings reveal that membrane organizing protein Tetraspanin-4 (TSPAN4) interacts with PD-L1, where both proteins colocalize on migrasomes and retraction fibers. We demonstrate that TSPAN4 negatively affects PD-L1 protein levels and inhibits PD-L1 lateral mobility on the plasma membrane. TSPAN4 knockdown results in a reduced PD-L1 degradation rate, and enhanced PD-L1 interaction with CMTM6 as the molecular mechanism. Consequently, increased PD-L1 levels in TSPAN4 knockdown melanoma cells leads to enhanced PD-1 binding, and more efficient immune checkpoint blockade through inhibition of T cell responses. This study identifies TSPAN4 as a negative regulator of PD-L1 at the cell surface of melanoma cells, suggesting that TSPAN4 may represent a novel target for improving immune checkpoint therapy in cancer patients.