通过细胞重编程及再分化获得“年轻化”的肿瘤特异性杀伤性T细胞用于人原发性肝癌治疗及其机制

We have found that most of cytotoxic T cells (CTLs) in tumor of hepatocellular carcinoma (HCC) patients are at the state of senescence and high expression of inhibitory receptors such as T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3) and programmed death 1(PD-1), when blocked both of them, the function of CTLs can be restored to some extent, while the anti-tumor effect of them is still poor. Recently, we found that T cells can be reprogrammed into induced pluripotent stem cells (iPSCs) and then redifferentiated into CTLs which called “rejuvenated”cells possessed antigen-specific killing activity. We had got the CTL - iPSCs, but whether they owned the same T cell receptor (TCR) gene with CTLs and their capacity of proliferation is yet to be further verified.Therefore, our research will carry out the following works: Firstly, we reprogrammed HCC-CTLs into T-iPSCs and redifferentiated them into CTLs, then detect cell proliferation, activity and whether they carry the same TCR gene with HCC-CTLs. Secondly, we explore the mechanisms of generatin of rejuvenated iPSCs-CTLs as well as the mechanisms of genetic stability of TCR genes. Our research aims to get rejuvenated CTLs for HCC treatment and improve the development of stem cell technology.

我们发现人原发性肝癌(HCC)浸润的特异性杀伤性T细胞(CTL)处于衰老状态并高表达衰老分子Tim-3、PD-1，阻断上述分子后发现CTL功能部分恢复但抗肿瘤效果仍欠佳。最近表明，将外周血T细胞重编程为诱导多能干细胞(iPSC)并再分化为T细胞，可在保留抗原特异性情况下使T细胞彻底“年轻化”。我们预实验将HCC-CTL重编程为T-iPSC，但其是否携带来源T细胞受体(TCR)基因及再分化为年轻化CTL的能力尚待验证。本课题在干细胞分化、抗HCC研究等基础上，拟：1.将HCC-CTL重编程为T-iPSC然后再分化为CTL，并检测其增殖能力提高、免疫活性增强等年轻化特征及高效特异性杀伤HCC情况；2.研究其中通过染色体端粒延长、端粒酶活性增强而使细胞年轻化的机制，通过保持TCR基因遗传稳定而保留其抗原特异性的机制。本课题旨在获得年轻化CTL用于HCC治疗，并促进肿瘤的干细胞治疗机制完善和更新。

肝癌中浸润性CTL细胞免疫无能的肝脏免疫环境状态限制体内免疫系统对肿瘤的杀伤作用，我们将肝癌癌旁组织中衰老的CTLs利用重编程技术使其“年轻化”，改变细胞命运并彻底逆转其免疫无能状态，这较以往仅对HCC组织T细胞进行体外扩增、癌抗原基因修饰TCR等传统细胞免疫治疗，具有质的提升。首先，我们从HCC患者肿瘤组织、癌旁组织、外周血采集分离CD8+T细胞，检测细胞表面免疫功能情况，结果发现原发性肝癌组织CD8+T细胞比例较癌旁组织低且肝癌组织中CD8+T细胞表面抑制性受体表达更多、分泌细胞因子IFN-γ、TNF-α更少，肿瘤组织中心CD8+T细胞免疫功能相比于癌旁组织更衰老；进一步阻断CD8+T细胞表面抑制受体，细胞免疫功能有所恢复。然后，我们将上述获得的癌旁组织中CD8+T细胞运用CD3/28抗体激活增殖，导入仙台病毒四个重编程因子（Oct3/4、Sox2、Klf4和c-Myc），发现获得的细胞具有干细胞特性。细胞免疫荧光证实重编程后的细胞表达干细胞标志蛋白NANOG、SSEA4、TRA1-81等干细胞特征性蛋白；小鼠成瘤实验证实重编程后的细胞能够形成内胚层、中胚层、外胚层样组织，证明其有分化潜能。并且利用CD8+单阳性T细胞培养发育条件（C3H10T1/2滋养细胞、OP9-DL1滋养细胞、VEGF，SCF，FLT-3 和IL-7L等）将iPSCs诱导再分化为CD8+T细胞，结果发现T-iPSC分化的CD8+T细胞保留原有TCR基因型，且细胞表面抑制性受体表达较癌旁组织内CD8+T细胞明显减少。我们成功将肝癌癌旁组织中浸润性CTL重编程为T-iPSC，并将T-iPSC再分化为CD8+T细胞，实现了细胞的年轻化。