Fig. 7. CASTAT5 potentiates the efficacy of CD19CAR T cell therapy in a murine B-cell lymphoma model.

The schema depicts the timeline of the experimental procedures. A20WT tumor cells were subcutaneously inoculated to mice. When tumor sizes reached 170 mm², mice were randomly grouped and received either no treatment (No Tx), CTX only, CTX + control CD19CAR T cells, or CTX + CASTAT5 CD19CAR T cells. (A) Expression of CAR and CASTAT5 in transduced T cells. Total T cells purified from CD45.1 mice were transduced to express CD19CAR alone or co-express CD19CAR and CASTAT5. Representative dot plots show the levels of CD19CAR and CASTAT5 in transduced T cells. CD19CAR was measured by anti-Fab stain and CASTAT5 was evaluated by Thy1.1 expression. (B) Tumor growth curves of each mouse under each condition are shown. The numbers indicate the number of tumor-free mice among treated mice at the end point. (C) Relapsed tumors did not lose CD19 expression. Mice treated with CD19CAR T cells had initial tumor regression followed by relapse. Relapsed tumors were resected and processed into single cell suspension for evaluation of CD19 expression by flow-cytometry. Tumors from untreated mice were used for comparison. Representative dot plots shown represent the co-stain of CD19 and B220. (D) Frequencies of host B cells in blood. At the indicated time points, tail blood was collected and the presence of host B cells in blood was evaluated by CD19 and B220 co-stain. (E) Detection of CD19CAR viral vector in bone marrow by PCR. 30 days after T cell transfer, a cohort of mice were killed and bone marrow aspirates were collected. The presence of CD19CAR T cells was evaluated by PCR detection of the CD19CAR viral vector. (F) Mice cured by CTX+CASTAT5 CD19CAR T cells were resistant to A20WT re-challenge. Live A20WT tumor cells were subcutaneously injected to cured mice and a group of naive mice used as controls. Tumor sizes in mice 3 weeks after tumor inoculation are shown.