Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

Abstract Endophilin A3-mediated clathrin-independent endocytosis (EndoA3-mediated CIE) contributes to 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 follow retromer-dependent retrograde transport to the trans-Golgi network (TGN) in cancer cells. From there, we propose that 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 affects the response of autologous cytotoxic CD8 T cells, as evidenced by reduced cytokine production and secretion, but increased lytic activity, while proliferation and later activation marker expression remain intact. This is accompanied by diminished ICAM1 density at the immune synapse, where we observe it arriving via polarized vesicular transport, indicating altered synapse organization. Indeed, cancer cells lacking EndoA3-mediated CIE or retromer form enlarged immune synapses that fail to sustain full T cell cytokine secretion, suggesting a compensatory attempt by T cells to overcome the defective synapse, while likely promoting more transient contacts that potentially favor serial killing. 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 fine-tuning the balance between cytotoxic T cell cytokine secretion and lytic activity. These insights contribute to a better understanding of the mechanisms governing immune synapse formation and organization, providing a necessary foundation for the long-term identification of new strategies to enhance T cell–mediated anti-tumor immunity. Significance Statement This study uncovers a novel mechanism by which clathrin-independent endocytosis (CIE) and retrograde transport collaborate to regulate immune synapse dynamics in cancer cells. We identify ICAM1 as a new cargo of Endophilin A3-mediated CIE, highlighting its role in the polarized redistribution of immune synapse components critical for cytotoxic CD8 T cell activation. By linking a specific CIE mechanism and retrograde transport to immune synapse function, our findings provide new insights into cancer-immunity interactions and suggest potential therapeutic strategies to enhance immune responses by targeting protein trafficking pathways. Competing Interest Statement The authors have declared no competing interest. Footnotes Competing Interest Statement: The authors declare no competing interest. New experiments have been performed and new datasets are now added. This led to a substantial revision of the manuscript (title, authors, abstract, results and figures, discussion, supporting information). The following keypoints were particularly improved: 1. Improved characterization of EndoA3 expression and knockdown effects on cancer cell morphology, as well as ICAM1 and ALCAM surface levels; 2. Enhanced characterization of the ICAM1 polarization process, including new spinning-disk live-cell imaging data tracking ICAM1-positive carriers in cancer cells and demonstrating polarized transport toward the contact zone with CD8 T cells (new Figure 4); 3. Expanded analysis of CD8 T cell response, with new data showing the impact of modulating EndoA3-mediated endocytosis in cancer cells on CD8 T cell activation markers (PD-1, CD137, and Tim-3), proliferation, degranulation, and cytotoxic activity; 4. Inclusion of additional relevant literature and expanded discussion elements.