Long-Term Inhibition of TIM-3 Expression Potentiates Antitumor Immune Responses of ROR1-Specific Chimeric Antigen Receptor T Cells
XIA Yulong1, JIANG Jihong2, JIN Xin1, SHI Chunqing1, ZHANG Xiaodong1, JIANG Jinzhou1, SONG Qibin3*, CHEN Rui4*, WU Jiong1*
This study aimed to investigate the effects of TIM-3 (T cell immunoglobulin and mucin domaincontaining protein 3) gene silencing on proliferation and antitumor immune responses of ROR1-CAR T cells and its mechanism. shRNA (short hairpin RNA) targeting TIM-3 was employed to knockdown TIM-3, and then the expression of TIM-3 was detected at the mRNA and protein levels by qRT-PCR and Western blot, respectively. ELISA experiments verified the effector cytokines production and cell killing effects of TIM-3-targeted ROR1-CAR T cells on target cells. Finally, antigen-dependent cell proliferation was validated by flow cytometry. The results showed that TIM-3-targeting shRNA could significantly inhibit TIM-3 expression in ROR1-CAR T cells, while TIM-3 knockdown did not affect the percentage and proliferation of ROR1-CAR T cells. ROR1-expressing SW620 cell, a human colon cancer cell line, secretes high levels of Galectin-9 which could directly inhibit the immune response of ROR1-CAR T cells. In addition, long-term inhibition of TIM-3 promoted ROR1-CAR T cell antigen-dependent cytokine production and proliferation. In conclusion, eliminating the interaction of tumor cell-derived Galectin-9 with TIM-3 on the surface of ROR1-CAR T cells can enhance the antitumor activities of ROR1-CAR T cells.
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