Site-specific tumor immunoediting following CAR T cell therapy. (A–E) B cell lymphomas were established by i.v. injection of 0.5 × 106 Eµ-myc-DEVD cells in C57BL/6 mice after sublethal irradiation (4 Gy). 7 d later, mice were injected i.v. with CAR T cells or control activated T cells (untransduced) or left untreated. (A) CAR T cell therapy prolongs mice survival. Log-rank test was used for statistical analysis. ***, P < 0.001. Data are compiled from three independent experiments (n = 8 mice in the control group and n = 13 mice in the CAR T cell group). (B) Representative images of the bone marrow on day 21 after CAR T cell therapy. Images show massive tumor infiltration (live tumor cells in gray), a few occasional CAR T cells (green), and rare apoptotic tumors (blue). Representative of 16 images from two independent experiments. Scale bars represent 20 µm. (C) Disappearance of CAR T cells at late time points. Bar charts showing the percentage of CAR T cells among CD8+ T cells at the indicated time points in the bone marrow (BM) and in lymph nodes (LN). Data are compiled from several time points from three independent experiments (n = 3 mice per group). (D and E) Emergence of CD19-negative tumor cells is detected in the bone marrow but not in lymph nodes. Representative FACS plots (D) and bar charts (E) showing CD19 expression in tumors cells (identified based on their fluorescence) in the bone marrow and lymph nodes. Analyses were performed between days 28 and 32. Data are compiled from three independent experiments. FSC, forward scatter. Mann–Whitney test was for statistical analysis. **, P < 0.01. (F–H) CAR T cell exhibit a reduced cytotoxic activity in lymph nodes. (F) Experimental setup. (G) The in vivo killing of transferred CD19+ splenocytes was compared in the bone marrow and lymph nodes. (H) Frequency of CAR T cells among total cells as analyzed by flow cytometry. (I) Strong PD-L1 expression in myeloid cells is detected in the lymph nodes, but not the bone marrow, following CAR T cell therapy. Representative histograms (upper panel) and compiled quantification (lower graph). Each dot represents one mouse. Data are compiled from three independent experiments. Wilcoxon matched-pairs signed rank test was used for statistical analysis. **, P < 0.01; *, P < 0.05.
