High-resolution microscopy reveals distinct lytic granule organization and release in CTLs and anti-Her2 CAR-T Cells at the immune synapse
Abstract
2Bioinformatics, Jerusalem College of Technology
3Shraga Segal Department of Microbiology and Immunology, Ben-Gurion University of the Negev
4National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev
5Immunology and Advanced CAR-T Cell Therapy Laboratory, Research & Development Department, Tel-Aviv Sourasky Medical Center
6Dotan Center for Advanced Therapies, Tel-Aviv Sourasky Medical Center and Tel Aviv University
Cytotoxic T cells (CTLs) and Chimeric Antigen Receptor T cells (CAR-Ts) mediate target cell killing by releasing lytic granules that contain granzymes and perforin. This release process remains poorly understood in both T cells and CAR-Ts due to spatiotemporal limitations in imaging the immune synapse (IS). Here, we employed high and superresolution microscopy to study the organization and dynamic release of lytic granules across the IS formed between CTLs or anti-Her2 CAR-Ts with their target cells. Lytic granules were enriched at the center of the IS formed by CTLs, but not by CAR-Ts. The CAR-Ts formed poor synapses as they did not spread well on HER2+ breast cancer cells (JimT). Still, they could properly mount Ca++ influx and demonstrate segregation of CARs from CD45. We could further follow the CAR-T dynamic release of the granules toward individual target cells and their eventual killing. Our results shed light on the processes required for efficient clearing of solid tumors by the anti-Her2 CAR-T cells.