Helios+调节性T细胞负载AchR特异性嵌合抗原受体治疗EAMG的实验研究

Myasthenia gravis (MG) is a common neurological autoimmune disease. Traditional treatments nonspecifically suppress immunoactivation of the whole immune system, resulting in poor curative and large side effect. Treg cells are considered to be the most “intelligent” natural immunosuppressants in the immune system. However, due to its disadvantages of nonspecific and unstability, application of Treg cells in clinical treatment is limited. Chimeric antigen receptor (CAR), invented since the early 21st century, endowed Treg cells the ability to rapidly recognize antigen and be activated. Besides, Helios was newly identified as a key transcription factor for maintaining Treg cell stability. This study is aimed to construct novel nano-vector PCPH NPs loaded with CARαAchR-Helios-Foxp3 coexpression plasmid DNA, and induce Treg cells to be both antigen-specific and functional stability. By observing therapeutic effects and mechanism of these Treg cell treatment in EAMG, this study might provide experimental foundation and theoretical basis for antigen specific Treg cell therapy of MG.

重症肌无力（MG）是神经系统常见的自身免疫性疾病，传统治疗方法均为非特异性的抑制机体免疫反应，疗效欠佳且长期使用副作用大。Treg细胞是机体最“智能”的天然免疫抑制剂，但因其非抗原特异性及在扩增活化过程中的不稳定性等缺陷，在自身免疫性疾病治疗的临床应用受限。本世纪初建立的嵌合抗原受体（CAR）技术能赋予Treg细胞迅速识别抗原并活化的能力，而最新发现Helios是Treg细胞在扩增活化过程中保持稳定性的关键转录因子。本研究拟采用新型纳米载体PCPH NPs负载CARαAchR-Helios-Foxp3共表达质粒诱导生成稳定的抗原特异性Treg细胞，观察其对EAMG的治疗作用，并探讨其作用机制。由于所构建的Treg细胞兼具抗原特异性及稳定性的优点，该方法为探寻临床可行的MG抗原特异性Treg细胞免疫治疗提供实验基础及理论依据。

本研究以寻找最佳抗原特异性方法治疗EAMG为目标。通过构建抗原特异性CAR-FOXP3+Helios+Treg细胞，将其注入EAMG小鼠体内，诱导抗原特异性免疫耐受。研究发现，CAR-FOXP3+Helios+Treg细胞能显著降低EAMG的临床评分，该治疗效果呈部分剂量依赖性。体内实验证实，CAR-FOXP3+Helios+Treg细胞能降低EAMG小鼠血清抗AChR IgG及其亚型IgG1,IgG2b，IgG2c水平，同时脾脏B细胞、浆细胞水平也有所下降，CAR-FOXP3+Helios+Treg细胞能促进EAMG小鼠脾T淋巴细胞由Th17、Th1向Th2、FoxP3+Treg转化。在临床试验中，我们发现全身型MG患者外周血Helios+Treg细胞较眼肌型MG患者和正常人均降低。Helios+ Treg亚群相较于Helios- Treg亚群表达更高水平的FoxP3，而GITR可调控Treg中Helios的表达，这为Helios+Treg细胞应用于MG患者临床治疗奠定了理论基础。