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Background Blockade of the programmed cell death protein 1 (PD-1) immune checkpoint (ICB) is revolutionizing cancer therapy, but little is known about the mechanisms governing its expression on CD8 T cells. Because PD-1 is induced during activation of T cells, we set out to uncover regulators whose inhibition suppresses PD-1 abundance without adversely impacting on T cell activation.Methods To identify PD-1 regulators in an unbiased fashion, we performed a whole-genome, fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in primary murine CD8 T cells. A dual-readout design using the activation marker CD137 allowed us to uncouple genes involved in PD-1 regulation from those governing general T cell activation.Results We found that the inactivation of one of several members of the TMED/EMP24/GP25L/p24 family of transport proteins, most prominently TMED10, reduced PD-1 cell surface abundance, thereby augmenting T cell activity. Another client protein was cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which was also suppressed by TMED inactivation. Treatment with TMED inhibitor AGN192403 led to lysosomal degradation of the TMED-PD-1 complex and reduced PD-1 abundance in tumor-infiltrating CD8 T cells (TIL) in mice, thus reversing T cell dysfunction. Clinically corroborating these findings, single-cell RNA analyses revealed a positive correlation between TMED expression in CD8 TIL, and both a T cell dysfunction signature and lack of ICB response. Similarly, patients receiving a TIL product with high TMED expression had a shorter overall survival.Conclusion Our results uncover a novel mechanism of PD-1 regulation, and identify a pharmacologically tractable target whose inhibition suppresses PD-1 abundance and T cell dysfunction.