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Background Junction Adhesion Molecule Like (JAML), is a newly identified co-stimulatory molecule for γδ T cells, tumor-infiltrating CD8 + T cells (TILs), and tissue-resident memory (TRM) cells. JAML contains a PI3K binding motif in its intracellular domain (YMTM), similar to CD28. The JAML ligand, coxsackie and adenovirus receptor (CXADR), is expressed on epithelial cells, and JAML-CXADR engagement is necessary for leukocyte transmigration.1 2 JAML is enriched in tumor-infiltrating CD8+ TRM cells in multiple cancer types, and targeting JAML with monoclonal agonist antibodies promotes CD8+ and γδ T cell anti-tumor immunity, alone or combined with immune checkpoint blockade against PD-1 and CTLA-4.3 4 Therefore, we hypothesized that JAML could be used as an alternative co-stimulatory molecule in the chimeric antigen receptor (CAR) construct to promote CD8+ T cell survival and function.Methods We generated two types of JAML CARs by replacing the CD28 co-stimulatory or transmembrane domains in a CD19-targeting CAR. Healthy donor T cells were activated and transduced to manufacture JAML CAR-T cells. In vitro functional assays were performed by co-culturing the CAR-T cells with antigen-positive and antigen-negative cell lines. Transcriptomic analysis and mTOR activity measurements were also conducted.Results JAML CAR-T cells had similar manufacturability to CD28 CAR-T cells in terms of viability, cell size, and expansion. They maintained CAR expression but with lower surface levels compared to CD28 CAR-T cells. JAML CAR-T cells had a higher proportion of juvenile phenotype cells (CD45RA +CCR7+, CD27+CD28+, CD127+CD25-) and lower tonic signaling compared to CD28 CAR-T cells. In functional assays, JAML CAR-T cells exhibited similar cytotoxicity to CD28 CAR-T cells, with higher CD8+ T cell proliferation and skewing while lower in cytokine production. JAML CAR-T cells showed comparable mTOR activation, as measured by S6 phosphorylation, to CD28 CAR-T cells upon antigen stimulation. Transcriptomic analysis revealed enrichment of cytotoxic genes associated with NK and CD8+ cytotoxic T cells and lower exhaustion signatures in JAML CAR-T cells.Conclusions In conclusion, we propose JAML as a novel co-stimulatory molecule for CAR-T cell therapy that can promote the functional activity and survival of CD8 + CAR-T cells.References Witherden DA, Verdino P, Rieder SE, Garijo O, Mills RE, Teyton L, Fischer WH, Wilson IA, Havran WL. The junctional adhesion molecule JAML is a costimulatory receptor for epithelial γδ T cell activation. Science. 2010 3;329(5996):1205-10.Verdino P, Witherden DA, Havran WL, Wilson IA. The molecular interaction of CAR and JAML recruits the central cell signal transducer PI3K. Science. 2010 3;329(5996):1210-4.Eschweiler S, Wang A, Ramírez-Suástegui C, von Witzleben A, Li Y, Chee SJ, Simon H, Mondal M, Ellis M, Thomas GJ, Chandra V. JAML immunotherapy targets recently activated tumor-infiltrating CD8+ T cells. Cell reports. 2023;42(2).McGraw JM, Thelen F, Hampton EN, Bruno NE, Young TS, Havran WL, Witherden DA. JAML promotes CD8 and γδ T cell antitumor immunity and is a novel target for cancer immunotherapy. Journal of Experimental Medicine. 2021 4;218(10).