Figure 4.

CAR-T therapy induces macrophage phenotype switch by upregulating PD-L1 and IDO. (A, B) The macrophages derived from human PBMCs were preincubated with CAR-T and Raji cells for 24 hours and then co-incubated with CAR-T or mock T cells for 36 hours. The CAR-T cells (A) or mock T cells (B) were then isolated and incubated with Raji/Luc cells and then luciferase activity was measured to exhibit the cytotoxicity of CAR-T cells. (C) The expression of PD-L1 and IDO in the M-PBMCs after coculturing with CAR-T and Raji cells was examined by western blot. (D–G) M-PBMCs (D) or macrophages derived from THP-1 cells (M-THP-1) (F) were preincubated with CAR-T and Raji cells for 24 hours and then cocultured with the activated autologous T cells labeled with CFSE. Flow cytometry analysis of cell division by dilution of CFSE was used to assess the effect of the macrophages on T cell proliferation. The divided T cells were quantified (*p<0.05, ***p<0.001) (E, G). (H) Flow cytometry was employed to evaluate the level of PD-L1 and IDO in M-THP-1 after coculturing with CAR-T and Raji cells (***p<0.001). (I) M-THP-1 cells were labeled with CellTracker Deep Red (CTDR) and then cocultured with CAR-T and Raji cells. The expressions of PD-L1 and IDO were analyzed by confocal microscopy. (J) The effects of the macrophages on T cell proliferation in the presence of PD-L1 antibody or IDO inhibitor (1-MT) were examined by flow cytometry analysis of cell division by dilution of CFSE. (K) The divided T cells were quantified (***p<0.001). Data are presented as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s test. ANOVA, analysis of variance; 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.