靶向小胶质细胞CSF1R的嵌合抗原受体T细胞免疫疗法在阿尔兹海默病中作用机制探索

Microglia are the resident innate immune cells of the central nervous system that mediate brain homeostasis maintenance. Microglia are critical to immune response in the CNS, and microglia-mediated neuroinflammation is a hallmark of Alzheimer's disease (AD). Activated microglia (M1) produce and release several pro-inflammatory mediators, such as nitric oxide, interleukin (IL)-1β, and tumor necrosis factor-alpha (TNF-a), orchestrating neuroinflammation, and neurodegeneration. Neuroinflammation is initially a protective response in the brain, however, uncontrolled inflammatory responses are considered to be a serial pathological interactions in response to β-amyloid (Aβ) accumulation. Thus, the classical M1 activation of microglia is considered the initial pathogenesis in AD. Colony stimulating factor 1 receptor (CSF1R) controls the production, differentiation, and varieties functions of MG in the central nervous system. Our previous findings clearly showed that the increased Aβ clearance through the interactions between microglia and astrocytes (MG-AC) by selective regulations on the tumor necrosis factor α (TNFα)-NALP3 inflammasome pathway, together with CSF1R being involved in the downregulation of inhibitory-checkpoint pathways during M1-M2 polarization of MG in AD. With the supportive preliminary data that there are significant clearance of Aβ by Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) by Targeting Colony stimulating factor 1 receptor of microglia in Alzheimer’s transgenic mice model, the further investigation on the mechanisms the cross-talk of MG-AC by dynamic regulation on CSF1R/CREB1/NALP3 inflammasome pathway with the application of Single cell RNA-seq, RNAi, and live cell imaging, will construct the brand new AD immunotherapy by CSF1R.CAR-T.

小胶质细胞(MG)是中枢神经系统内固有免疫细胞，其中主要起致炎作用的M1表型被认为是导致阿尔兹海默病(AD)脑内β淀粉样蛋白(Aβ)清除障碍，Tau过磷酸化，慢性免疫损害及神经退变进展的主要参与细胞。集落刺激因子-1受体(CSF1R)主要表达于脑内MG，调控MG的活化、吞噬，M1-M2极化等多种功能。前期工作已明确：①选择性干预MG-TNFα/NALP3炎症小体途径可以调节MG-AC交互作用，促进Aβ的清除；②CSF1R在MG的M1-M2极化过程中起“检测点”作用。在初步证实靶向小胶质细胞CSF1R的嵌合抗原受体T细胞免疫疗法(CAR-T)有效促进Aβ清除基础上，拟进一步通过单细胞转录组测序、RNAi、活细胞成像等技术阐明CSF1R/CREB1/NALP3炎症小体途径调控MG-AC协同促进Aβ清除的机制；建立CAR-T靶向MG治疗AD的新型免疫新策略。

本研究在成功制备第四代可诱导CSF1R.CAR-T细胞基础上，开展系列研究：① 可诱导CSF1R.CAR-T细胞有效性及安全性：CSF1R.CAR-T细胞能调控性杀伤靶细胞(CSF1R阳性小胶质细胞)，提高了对AD治疗的靶向性；同时通过可诱导表达系统，实现了对CAR基因表达水平的调控，有效控制细胞因子分泌水平，从而避免了CAR- T细胞发挥治疗作用可能触发的“细胞因子风暴”副作用，及对正常细胞的安全性。② 靶向抑制CSF1R对AD表型的改善及对AD病理的影响：低、中、高剂量的CAR-T细胞治疗都可以明显改善APP/PS1小鼠的自发活动和精神行为，进而阐明了靶向CSF1R对AD特征性病理改变β淀粉样蛋白、tau蛋白以及神经元的调节作用及机制。③ 靶向抑制CSF1R 对小胶质细胞极化的调节作用：首次证实了靶向CSF1R的CAR-T疗法在小胶质细胞M1-M2极化中的作用，其中高剂量组效果更显著。④ 靶向抑制CSF1R对小胶质细胞焦亡的影响及可能机制的探索：与AD组相比，高剂量治疗组和抑制剂组GSDMD蛋白水平明显降低，提示通过影响小胶质细胞焦亡，进一步对上游NLRP3炎症小体、ASC、AIM2及caspase1等因子下调表达。综上，本课题首次证明了靶向CSF1R在有效改善AD模型鼠认知障碍及精神行为症状同时，可以有效减轻AD特征性病理改变，进而提出靶向CSF1R的CAR-T疗法在小胶质细胞M1-M2极化中的可能作用机制，及其对NLRP3-caspase1-GSDMD焦亡的抑制作用，为临床转化提供理论和实验依据。