Figure 5.

Activation of the AIM2 inflammasome triggered by CAR-T treatment induces macrophage phenotype switch. (A) Macrophages derived from THP-1 cells (M-THP-1) or M-PBMCs were treated with recombinant human IL-1β. The expression of PD-L1 and IDO in the macrophages was detected by western blot. (B, C) M-THP-1 infected with AIM2-shRNA expressing lentivirals were preincubated with CAR-T and Raji cells for 24 hours and then cocultured with the activated autologous T cells labeled with CFSE. T cell division was evaluated by flow cytometry (***p<0.001). (D, E) The expression of PD-L1 and IDO in M-THP-1 after coculturing with CAR-T and Raji cells in response to AIM2 knocking down or caspase-1 inhibitor (VX765 or ac-YVAD-cmk) treatment was detected by western blot. (F, G) M-THP-1 were preincubated with CAR-T and Raji cells for 24 hours in the presence of the AIM2 inhibitor thalidomide and then cocultured with the activated autologous T cells labeled with CFSE. The effects of the macrophages on T cell proliferation were examined by flow cytometry. The divided T cells were quantified (***p<0.001). (H) In response to thalidomide and IL-1β treatment, the expression of PD-L1 and IDO in M-THP-1 cocultured with CAR-T and Raji cells detected by western blot. Data are presented as mean±SEM and analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. CAR-T, chimericantigen receptor T; CFSE, carboxyfluorescein succinimidyl ester; IDO, indoleamine 2,3-dioxygenase; M-PBMCs, macrophages derived from PBMCs; PBMCs, peripheral blood mononuclear cells; PD-L1, programmed cell death-ligand 1.