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. 2021 Jan 7;9(1):e001466. doi: 10.1136/jitc-2020-001466

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© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Activation of the AIM2 inflammasome pathway is triggered by CAR-T-induced tumor cell DNA release. (A) The level of double-stranded DNA (dsDNA) in the supernatant of Raji cells cocultured with CD19 CAR-T cells or mock T cells was evaluated by using the dsDNA assay kit (***p<0.001). (B) The cellular DNAs were prelabeled by 5-ethynyl-2’-deoxyuridine (EdU) in live Raji cells and the macrophages labeled with CellTracker deep red (CTDR) before the coculture. The proportion of CTDR+/EdU+ cells, which represent the macrophages phagocytozing the tumor cell DNAs, was analyzed by flow cytometry (***p<0.001). (C, D) The co-localization of the tumor cell DNAs with the cytosolic DNA sensor AIM2 in the microphages was examined by confocal microscopy and quantified (D, ***p<0.001). (E) The binding of AIM2 to ASC in the macrophages derived from THP-1 cells (M-THP-1) cocultured with CAR-T and Raji cells was evaluated by co-immunoprecipitation. (F) The expression of mature IL-1β and cleaved caspase-1 in the coculture supernatants and pro-caspase-1, pro-IL-1β, and AIM2 in the M-THP-1 cocultured with CAR-T/Raji cells in the presence of the caspase-1-specific inhibitors VX765 or ac-YVAD-cmk was evaluated by western blot. (G) The concentration of IL-1β in supernatants of the coculture of CAR-T/Raji/M-THP-1 in the presence of VX765 or ac-YVAD-cmk was examined by ELISA assay (**p<0.01, ***p<0.001). Data are presented as mean±SEM and analyzed by by two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test or by one-way ANOVA followed by Dunnett’s test. CAR-T, chimericantigen receptor T; CM, culture medium; WCL, whole cell lysate.