自身反应性CD4+ T细胞在缺血性脑损伤中的作用机制研究

Autoimmunity emerges against brain-derived antigens in patients with stroke and in mice with middle cerebral artery occlusion (MCAO). However, mechanisms for the genesis of autoimmunity in the context of the specific microenvironment of the central nervous system (CNS) have not been investigated in depth. Furthermore, the impacts of autoimmunity on clinical outcome of stroke are not clear. Autoimmune responses are largely orchestrated by T cells of the CD4+ subset; therefore, we will focus on CD4+ T cell mediated-autoimmunity and its impact on stroke. For this purpose, we have adopted T cell receptor (TCR) transgenic mice in which the majority of CD4+ T cells recognize a human myelin oligodendrocyte glycoprotein (MOG) epitope (2D2). The availability of preexisting CD4+ auto-(myelin-) reactive T cells as well as the convenience of isolating these cells in large quantities makes this model very suitable for studying the autoimmunity mediated by CD4+ T cells. To establish a foundation for our investigation, we have demonstrated that CD4+ T cells infiltrated into peri-infarcted area in brain sections of patients with ischemic stroke. In MCAO mice, 2D2 CD4+ T cells readily home to the peri-ischemic regions. Further, mice with autoreactive (2D2) CD4+ T cells infiltrating in the brain developed an exacerbated form of stroke after MCAO, as reflected by more severe neurological deficit and larger lesion size revealed by high-field rodent magnetic resonance imaging (7T MRI). The stroke severity in MCAO mice with autoreactive CD4+ T cells appeared to correlate with the magnitude of brain inflammation. Importantly, we found that 2D2 CD4+ T cells can recognize a non-MOG, neuroantigen in stroke. Based on these findings, our central hypothesis is that the activation of CD4+ T cells and abundance of brain antigens released abruptly during brain ischemia facilitate these CD4+ T cells to recognize a larger spectrum of neuroantigens and diversify the immune response; and that the existence of these autoreactive CD4+ T cells accelerates brain infarction. The overall goal of this proposal is to determine the genesis of autoimmunity in ischemic stroke and explore its clinical relevance. Our specific aims are to: (1) Characterize the diversified 2D2 CD4+ T cell responses after brain ischemia; (2) Determine the underlying mechanisms of the diversification of 2D2 CD4+ T cell responses after brain ischemia(3) Determine the impact of diversified 2D2 CD4+ T cell responses on ischemic brain injury. The outcome of this proposal will provide a better understanding of the genesis of autoimmunity after stroke, as well as the impact of autoimmunity on post-ischemic brain injury.

脑缺血后机体产生针对神经抗原的自身免疫反应，但自身免疫反应产生的机制及其对缺血性脑卒中预后的影响还不清楚。自身免疫反应主要由CD4+T细胞介导，因此本课题将关注CD4+T细胞。我们发现缺血性卒中病人尸检脑片有大量CD4+T细胞浸润。为进一步研究脑缺血后T细胞反应的变化，我们使用了2D2 小鼠，其CD4+T细胞转基因表达能识别特定神经髓鞘抗原（MOG35-55）的T细胞受体。脑缺血后2D2小鼠CD4+T细胞能识别新的神经抗原且脑损伤加重。我们推测CD4+T细胞在脑缺血后反应出现多样化，可被多种神经抗原活化，从而加重脑损伤。我们将使用（2D2）CD4+T细胞研究：1 ）脑缺血后不同阶段T细胞抗原反应多样化的时间和空间特征。2）脑缺血造成T细胞反应多样化的机制。3）针对神经抗原的T细胞反应多样化对缺血性脑卒中的影响及其机制。该研究将揭示脑缺血引起自身免疫反应的机制，及其在脑损伤中的作用。

脑卒中后外周的免疫细胞迅速迁移至中枢神经系统接触暴露的脑抗原，通过炎症因子的释放、细胞毒作用及抗原呈递的自身脑组织的免疫反应损坏脑组织。脑缺血后机体产生针对神经抗原的自身免疫反应已有报道，但该自身免疫反应产生机制及其对临床结果的影响尚存在争议。自身免疫反应主要由CD4+ T细胞介导，因此本项目主要关注CD4+ T细胞亚型。我们应用T细胞受体（TCR）转基因小鼠（2D2），这种TCR可以识别髓鞘少突糖蛋白（MOG35-55）的抗原表位。通过短暂性大脑中动脉闭塞模型和光化学皮层缺血性卒中模型，我们发现脑缺血后脑内固有小胶质细胞呈递神经抗原，造成CD4+ T细胞反应多样化；同时识别神经抗原的自身反应性CD4+ T细胞通过改变脑内的炎性环境加重脑内炎症反应，从而进一步加重缺血性脑损伤；进一步我们在缺血性卒中病人的尸检脑片中发现存在T细胞活化以及MOG特异性T细胞存在。以上结果阐明了自身反应性CD4+ T细胞在缺血性脑损伤中的作用。另一步，在该项目执行过程中，我们通过神经影像学手段，探索脑内转输或浸润细胞的变化与炎性病灶的位置关系，使用结合荧光染料的新型铁氧化物纳米微粒（MIRB），追踪探索转输的CD4+T细胞在活体脑缺血动物中的组织分布及动态变化，为该项目的完成提供活体研究可行的技术手段。综上，该研究有助于进一步了解自身免疫反应参与缺血性脑卒中发病过程，进而增加我们对于卒中发病机制的理解，为阻断T细胞的招募和激活来减轻缺血性脑损伤提供了新的靶点。