TMED10 inhibition suppresses cell-surface PD-1 expression and overcomes T cell dysfunction
PD-1 immune checkpoint blockade (ICB) is revolutionizing cancer therapy, but little is known about the mechanisms governing its expression. In a whole-genome, dual- marker FACS-based CRISPR-Cas9 screen in primary murine CD8 T cells, we found that inactivation of the EMP24/GP25L/p24 protein family, most prominently TMED10, reduced PD-1 cell-surface stability, thereby augmenting T cell activity. Treatment with TMED inhibitor AGN192403 led to lysosomal degradation of the TMED-PD-1 complex and reduced PD-1 expression in intratumoral CD8 T cells in mice, thus reversing T cell dysfunction. Clinically corroborating these findings, single-cell RNA analyses  revealed a positive correlation between TMED expression in tumor-infiltrating CD8 T cells (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. Our results uncover a novel mechanism of PD-1 regulation, and provide a possible opportunity for PD-1 small molecule inhibition Overall design: C57BL/6 Rag2-/- mice were inoculated with B16F10-OVA cells. On day 4, ACT was performed, administering CD8+ OT-I sgCtrl or sgTmed10. On day11, spleen and tumor were isolated and digested. Tumor samples were analyzed using flow cytometry and RNA sequencing. Moreover, CD8+ bead isolation was performed to enrich for intratumoral CD8+ T cell population ahead of RNA sequencing. Overall, RNAseq data was derived from T cells before inoculation, from treated tumors and ultimately, from CD8+ enriched intratumoral fractions.