T-bet和Eomes在WNT信号介导的CD8+干细胞样记忆T细胞形成中的作用机制研究

The recently identified T memory stem cells(TSCM) are capable of reconstituting the full diversity of memory and effector T lymphocytes on serial transplantation in mice, indicating that these cells are endowed with the stem cell-like attributes of self-renewal capacity and multi-potency. These qualities make TSCM a particularly attractive subset to employ in adoptive immunotherapies for cancers. The instructive signals guiding the formation of TSCM have just begun to be investigated; WNT signaling promoted the generation of TSCM, but the underlying mechanism still remains unclear. In our previous work, we showed that combined T-bet and Eomes deficiency resulted in severe reduction in the number of effector/central memory T cells but an increase in the percentage of CD62Lhigh CD44low Sca-1+ T cells which were similar to the phenotype of memory stem T cells. These data indicated that T-bet and Eomes may play a role in regulating the balance between the “stemness” and differentiation of memory T cells. In this proposed research project, we intend to explore the role and the underlying mechanism of T-bet and Eomes in the formation of TSCM promoted by WNT signaling. Our project intends to reveal a new role for T-bet and Eomes in the formation of TSCM and establish a newly improved strategy of enhancing the anti-tumor effect of adoptive transfer therapy with TSCM.

干细胞样记忆T细胞（TSCM）做为新发现的记忆细胞亚群，具有良好的增殖、自我更新和多向分化能力，在过继性免疫细胞移植治疗肿瘤方面具有良好的应用前景。激活WNT信号通路可以诱导Naïve T细胞更多地分化成为TSCM细胞，但调控的信号分子及其作用机制尚未阐明。本项目组前期研究发现敲除转录因子T-bet和Eomes可以促进TSCM表型特征的CD8+记忆T细胞产生，提示T-bet和Eomes可能参与调控Naïve T细胞向TSCM细胞分化，但机制尚不清楚。本项目拟通过体内外实验研究，旨在阐明T-bet和Eomes在WNT信号通路介导的Naïve T细胞向TSCM细胞分化过程中的生物学作用及相关分子机制。藉此，为Naïve T细胞向TSCM细胞的分化提供新的理论基础，为过继性细胞免疫治疗肿瘤提供新的策略。

干细胞样记忆T细胞(TSCM)做为新发现的记忆细胞亚群，具有良好的增殖、自我更新和多向分化能力，在过继性免疫细胞移植治疗肿瘤方面具有良好的应用前景，但调控Naïve T细胞分化成为TSCM细胞的信号分子及其作用机制尚未阐明。本项目研究采用体外TWS119激活WNT信号通路的方式，诱导活化的Naïve CD8+ T细胞向TSCM细胞分化，分析其对转录因子T-bet和Eomes的表达水平的影响及可能的分子调控机制。研究结果表明高浓度（7uM）的TWS119处理可以明显下调活化后的CD8+ T细胞上转录因子T-bet和Eomes的mRNA表达水平。敲除T-bet和Eomes基因并未影响TWS119诱导活化后Naïve CD8+ T细胞更多的向TSCM细胞分化。这也与我们前期研究发现同时敲除T-bet和Eomes会导致肿瘤抗原特异性的具有干细胞样记忆T细胞表型特征的T细胞比例显著上调的结果相符合。而使用Wnt/β-catenin/TCF特异性的阻断剂阻断后，T-bet和Eomes的mRNA表达水平不受影响。进一步研究发现，高浓度的TWS119处理可以明显抑制CD8+ T细胞内转录因子STAT1上701位酪氨酸的磷酸化水平。提示高浓度TWS119处理可能通过下调细胞内STAT1的磷酸化水平从而下调T-bet和Eomes的表达水平，其通过抑制细胞内T-bet和Eomes表达，将这两个转录因子维持在适合的水平上从而促进TSCM细胞产生。