CD28缺失加剧NOD小鼠T细胞活化的机制研究

T cell activation is essential for its role in combating pathogen and tumor cells. CD28 is a critical receptor for T cell activation, through a mechanism called co-stimulation, and blockade of CD28 potently inhibits T cell mediated autoimmunity in C57BL/6 mice. However, CD28 deficiency in the NOD/ShiLtJ (NOD) mice contradictorily resulted in exacerbated autoimmune disorder, such as type 1 diabetes. Regulatory T cells or Tregs are deficient in both strains in the absence of CD28, and crippled immune tolerance mediated by regulatory T cells is not sufficient to explain this paradoxical phenomenon. The exacerbated autoimmunity may be rooted in hyper-activity of effector T cells in NOD mice, but the underlying mechanism is still not known. This proposed project aims at identifying the genetic factors responsible for the discrepancy in the outcome of CD28-/-T cell responsiveness observed in NOD strain and the classic C57BL/6 strain. CD28-/-C57BL/6J mice will be crossed to NOD mice to generate F1 hybrids followed by intercrosses to produce F2 population which includes mice carrying CD28-/-genotype, to be used for quantitative trait locus or QTL mapping. In the CD28-/- F2 population, blood glucose assay as well as immuno-phenotyping of T cell activation and differentiation in the peripheral lymphoid organs will be performed. On the other hand, the CD28-/- F2 animals will be analyzed for chromosomal segregation by means of genome wide scanning using genetic markers that can differentiate C57BL/6J and NOD strains. The candidate genes from such QTL mapping will be verified by the emerging genome editing tool such as the TALENs or CRISPR/Cas9 system to confirm their role in regulating autoimmunity originating from CD28 deficiency in NOD mice. It is very important to note that CD28 deficiency may play a critical role in the autoimmune diseases including diabetes, as clinical findings confirmed that CD28 negative T cells infiltrate affected tissues in autoimmune patients.

T细胞细胞活化是其对抗感染与肿瘤的前提，但其活化失控可导致自身免疫病。CD28是极为重要的T细胞活化受体，阻断CD28可强烈抑制C57BL/6小鼠自身免疫病，相反CD28缺失却严重加剧NOD小鼠自身免疫病。目前已知调节性T细胞差异无法解释上述现象。NOD效应T细胞在CD28缺失条件下仍过度活化是上述现象的根源，且NOD效应T细胞体外TCR刺激增殖水平也有异常，但分子机制皆未明确。为阐明此机制，本课题将CD28-/-C57BL/6与NOD小鼠构建F2代家系，利用高通量流式细胞仪分析F2小鼠T细胞免疫表型并检测血糖水平，全基因组扫描遗传位标定位调控基因，用基因剔除技术验证导致NOD效应T细胞过度活化的基因。本课题将首次明确CD28-/-NOD效应T细胞过度活化的控制基因。大量临床研究表明自身免疫病患者炎症组织有CD28阴性T细胞浸润因此本课题十分重要，研究结果对操控T细胞活性意义重大。

T细胞介导的自身免疫病在人群及动物模型中广泛存在，但CD28缺失加剧NOD小鼠自身免疫性糖尿病机制令人费解。因为CD28是T细胞活化的重要共刺激受体，其缺失可有效阻断T细胞介导的炎症。因此，本项目着重探索CD28缺失加剧NOD小鼠T细胞活化致病的免疫遗传机制、即搜索导致这一表型的调控基因。有趣的是，我们发现至少Ptpn22和ICOS两个分子参与调控NOD小鼠CD28缺失糖尿病更加严重，尤其是ICOS作用显著。我们研究发现ICOS缺失能彻底阻断CD28–/– NOD小鼠T细胞活化导致的糖尿病，但它却促进小鼠患自发性自身免疫性结膜炎。本项目发现， 患病动物眼睑粘膜提取的CD4 T细胞分泌IL-4显著增加，且纯化的CD28–/–ICOS–/– NOD小鼠CD4 T细胞过继转移到T细胞完全缺失的CD3e敲除NOD小鼠，10天内受体小鼠也能患病。此外，CD28–/–ICOS–/– NOD小鼠CD4 T细胞在TCR刺激条件下，能高表达Gata3，患病组织中CD4与Gata3共表达。上述研究证明了ICOS具有抑制CD28–/– NOD小鼠T细胞向Th2细胞分化的功能，ICOS进一步缺失导致严重的自身免疫病。值得指出的是，ICOS 和CD28都是治疗自身免疫病的靶点，本研究表明在自身免疫病易感小鼠中阻断上述共刺激受体可导致自身免疫性疾病更加严重，并阐明了影响辅助T细胞分化的机制，为临床自身免疫病治疗提供了全新的信息。

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