TSHR-CAR/Foxp3 mRNA诱导的Treg细胞靶向治疗小鼠Graves病的机制研究

Treatments for Graves’ disease (GD) include antithyroid drugs, radioactive iodine and surgery, but none are ideal for their respective defects. Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. We have previously discovered that there was a significant decrease in the proportion of Treg cell and their suppression function in GD animal. Based on the theory that thyroid stimulating hormone receptor (TSHR) is the autoantigen in GD, we suppose that TSHR-specific Treg cell which designed to selectively target the TSHR involved in the autoimmunity is an effective biological immunotherapy for GD. We propose gene engineering to achieve TSHR-CAR Tregs from CD4+T cells and demonstrate their efficacy in GD model. CD4+ T cells were modified utilizing a mRNA vector system to express a chimeric antigen receptor(CAR) targeting TSHR in trans with the murine Foxp3 gene. The CAR Treg cells were evaluated from mixed lymphocyte reaction in vitro for targeted suppressive capacity, and in vivo to treat GD mice for their location and therapeutic effect. T-cell receptor diversity, an important feature of immune homeostasis, is detected by high-throughput sequencing pretherapy and post-treatment. This project is designed to explore the treatment effect and mechanism of immune target therapy for GD. This research finding could expand to the treatment of other autoimmune diseases in the future.

Graves病（GD）的治疗主要有抗甲状腺药物、放射性碘和外科手术，但这些方式均存在一定缺陷。Treg细胞在维持机体免疫平衡中至关重要, 我们前期发现GD动物Treg细胞比例及免疫抑制功能显著降低。结合TSHR是导致GD的自身抗原，推测TSHR抗原特异性Treg细胞可以有效治疗GD，其机制可能是靶向抑制效应T细胞，重建机体免疫稳态。本研究拟构建TSHR-CAR/Foxp3 mRNA载体，体外诱导CD4+T细胞分化为TSHR特异性CAR Treg细胞，并将该细胞应用于GD小鼠的治疗。通过体外混合淋巴细胞培养，证实CAR Treg细胞靶向结合和免疫抑制能力；通过动物实验观察该细胞对效应T细胞的抑制及对GD的治疗效果；通过免疫组库测序观察治疗前后小鼠TCR组库多样性改变，评估免疫状态。本项目拟探索CAR Treg细胞对GD免疫靶向治疗的效果及机制，该研究未来也可应用于其他自身免疫性疾病的治疗。

Graves甲亢（Graves’disease，GD）在人群中的患病率高达0.5%-2%，其中女性是男性的5-10倍，且发病率有逐年上升的趋势。目前临床上的治疗以抗甲状腺药物治疗为主，但是治疗周期长，复发率高。Graves眼病（Gravesophthalmopathy, GO）是甲亢患者最常见的甲状腺外表现，严重者可发生角膜溃疡、压迫性视神经病变，甚至失明，严重影响患者的生活质量，给患者造成巨大的身心压力。Graves眼病是不可逆性的免疫机制介导的炎症反应，目前临床上的治疗都只能勉强改善其临床表现。Treg细胞在维持机体免疫平衡中至关重要, 我们前期发现GD动物Treg细胞比例及免疫抑制功能显著降低。本项目中，我们完成了小鼠Graves病及GO动物模型的诱导，分别应用磁珠法和流式细胞术成功分选CD4+CD25+ Treg细胞，观察其对动物模型的治疗效果。另外，我们还尝试使用硒及一些新型免疫抑制剂干预Graves病及GO动物模型，观察其对Treg细胞的影响。.该项目按时、按研究计划完成，培养2名博士研究生，相关成果包括：已发表SCI论文2篇（影响因子共8.9分），待发表SCI论文2篇，国内外会议口头发言3次，专利申请1项（受理中），软件著作权1项（已获批），西安交通大学校级新医疗新技术1项。