Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

Endophilin A3-mediated clathrin-independent endocytosis (EndoA3-mediated CIE) mediates the internalization of immunoglobulin-like proteins, including key immune synapse components. Here, we identify ICAM1 as a novel EndoA3-dependent cargo, alongside ALCAM. We demonstrate that both proteins subsequently undergo retromer-dependent retrograde transport to thetrans-Golgi network (TGN) in cancer cells. From there, they undergo polarized redistribution to the plasma membrane, where they contribute to immune synapse formation between cancer cells and cytotoxic CD8 T cells. Disruption of EndoA3 or retromer components significantly impairs the activation of autologous cytotoxic CD8 T cells, as demonstrated by decreased cytokine production. Concomitantly, we observed a reduced localization of ICAM1 at the immune synapse, indicating impaired immune synapse integrity. Indeed, cancer cells lacking EndoA3-mediated CIE or retromer form enlarged immune synapses that fail to restore full T cell activation, suggesting a compensatory attempt by T cells to overcome the defective synapse. Together, these findings reveal that EndoA3-mediated CIE and retrograde transport act in concert in cancer cells to relocate immune synapse components via the Golgi, thereby promoting the activation of cytotoxic CD8 T cells. Our study paves the way for the design of future therapeutic strategies targeting these pathways to enhance T cell-mediated anti-tumor immunity.