Expression And Regulation Of Negative Costimulatory Molecule Tim3in Tumor Infiltrating T Lymphocytes

Objective：Based on the different expression of Tim3in peripheral blood T cells and tissues(cancer tissues and adjacent cancer tissues) infiltrating lymphocytes (TILs), toinvestigate whether T-bet/Eomes is involved in regulating the expression of Tim3, andfurther analyze the influence factors of Tim3expression in tumor microenvironment,and to provide experimental basis for the feasibility study of Tim3as a new target forimmunotherapy of tumors.Methods:(1) Cancer tissues, adjacent cancer tissues and peripheral blood was obtainedfrom50patients with non-small cell lung cancer (NSCLC) patients. Peripheral Tlymphocytes and TILs were purified to perform flow cytometric analysis to investgatethe expression of Tim3in TILs.(2)Spleen T lymphocytes from C57BL/6WT and T-bet-/-/Eomes-/-DKO micewere cultured in the condition of Th1differentiation and tested the expression ofTIM-3in peripheral T lymphocytes and TILs by flow cytometry. At the same time,Lewis lung cancer models were established. Spleen T lymphocytes and TILs werepurified to detected the expression of Tim3.(3)Real-Time PCR was performed with SYBR green kit to investigate theexpression of mRNA level of IL-1β、IL-6、IL-10、IL-17、TGF-β、TNF、PD-L1、galectin-9、FoxP3、GATA3、T-bet、Eomes in tumor microenvironment.Result:1. Compared with adjacent normal tissue infiltrating lymphocyte, tumorinfiltrating lymphocytes of patients with non small cell lung cancer significantlyup-regulated the surface expression of Tim3; T cells in peripheral blood is almost noexpression of Tim3. 2. Negative costimulatory molecule Tim3in tumor infiltrating lymphocytes ofmost patients with non small cell lung cancer showed moderate expression; lowexpression was shown in a small percentage of the patients.3. In vitro, the expression of Tim3in T lymphocytes cultured under the conditionof Th1differentiation, especially in CD8+T lymphocytes, was inhibited in T-bet/Eomesdouble gene knockout (DKO) mice.4. In Lewis lung carcinoma model in mouse, Tim3expression in spleen Tlymphocyte was regulated by T-bet/Eomes, while it was not regulated in TILs.5. Compared TimMIDgroup to Tim3LOWgroup, the expression of Eomes mRNAwas significantly increased, the expression of TNF-, IL-1β, IL-10mRNAsignificantly decreased (p<0.01). And no significant difference was shown between thetwo groups in the expression of TGF-β、IL-6、T-bet、Gata3mRNA et al.Conclusion:Compared with adjacent normal tissue infiltrating lymphocytes, the expression ofTim3in TILs was significantly up-regulated；In peripheral blood，there is almost noexpression of Tim3in T lymphocytes. A small percentage of patients showed lowexpression of Tim3in TILs. T-bet/Eomes is involved in regulating the expression ofTim3in peripheral immune organs, while the expression of Tim3in TILs might beinduced by the tumor microenvironment.The expression of Foxp3, IL-17and Galectin-9mRNA in tumor tissues was significantly higher than adjacent normal tissues. TILsshowed a state of high exhaustion in Tim3MIDgroup, and the expression ofinflammatory factor was lower.