Comparing CAR and TCR ligation events in the same T cell, Davenport et al. found that CAR stimulation initiated an immune synapse (IS) characterized by unclustered Lck, a smaller actin ring, and diffuse LFA-1, rather than the “bullseye” IS characteristic of TCR stimulation. CARs induced signaling pathways more rapidly and strongly than TCRs, and induced faster lytic granule recruitment and delivery to the synapse. CAR-stimulated T cells also detached from their dying targets earlier, allowing for rapid serial killing of tumor cells.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Author Info: (1) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000

Author Info: (1) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (2) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. Department of Medical Biology, The University of Melbourne, Parkville, VIC 3052, Australia. (3) Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. Department of Medical Biology, The University of Melbourne, Parkville, VIC 3052, Australia. (4) Department of Medical Biology, The University of Melbourne, Parkville, VIC 3052, Australia. Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. (5) Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. Department of Computing and Information Systems, The University of Melbourne, Parkville, VIC 3010, Australia. (6) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (7) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (8) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (9) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (10) Australian Cancer Research Foundation Translational Research Laboratory, Melbourne Health, Parkville, VIC 3050, Australia. Department of Medicine, The University of Melbourne, Parkville, VIC 3010, Australia. (11) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia; phil.darcy@petermac.org paul.neeson@petermac.org jenkins.m@wehi.edu.au. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (12) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia; phil.darcy@petermac.org paul.neeson@petermac.org jenkins.m@wehi.edu.au. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. (13) Cancer Immunology Research, Peter MacCallum Cancer Center, Melbourne, VIC 3000, Australia; phil.darcy@petermac.org paul.neeson@petermac.org jenkins.m@wehi.edu.au. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC 3000, Australia. Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. Department of Medical Biology, The University of Melbourne, Parkville, VIC 3052, Australia. Institute for Molecular Science, LaTrobe University, Bundoora, VIC 3086, Australia.

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