NOD小鼠共刺激分子ICOS与CD28缺失导致CD4+ T细胞向炎性Th2 分化的机制

Both TCR stimulation and co-stimulatory signals such as CD28 and ICOS are required for T cell activation and differentiation. The depletion of CD28 or ICOS in T cell block Th2 differentiation. Abnormal T cell activation and differentiation in NOD mouse leads to autoimmune disease such as type 1 diabetes, knocking out both CD28 and ICOS in NOD mice with CRISPR/Cas9, which also cues its autoimmune diabetes. Surprisingly, the CD28-/-ICOS-/- mouse suffers from autoimmune ocular conjunctivitis, which is caused by Th2 inflammation. We also found CD28-/- CD4+ T cells is incapable expressing Th2 regulatory transcription factor GATA3 under TCR stimulation, however this situlation can be turned over with additional ICOS deletion. However, the mechanism behind the resulted Th2 inflammation after ICOS and CD28 deletion in NOD mouse is unknown. On the aim of deciphering the understanding this mechanism, we will focus on several key signaling molecules downstream of TCR, and estimate STAT6 phosphorylation among ICOS-/-CD28-/- and NOD WT and CD28-/- CD4 Tcell. We will also study the CD28-/-ICOS-/- T cell development with deploying the Marily H-Y Ag transgenic mouse strain, as center tolerance deficiency may also be an important cause for certain autoimmune disease. It is worth noting that the ICOS/CD28 dual blocking antibody for autoimmune diseases treatment is under clinic trials. However, we are getting opposite result from our system. We believe this project will provide valuable information for clinical treatment and risk assessment.

共刺激分子CD28或ICOS的缺失都可以阻断CD4+T细胞Th2分化。NOD小鼠T细胞活化分化异常导致1型糖尿病等自身免疫病，利用CRISPR/Cas9敲除其CD28与ICOS，NOD小鼠1型糖尿病消失。不解的是，CD28与ICOS缺失的CD4+ T细胞高表达Th2细胞转录因子GATA3及其细胞因子IL4、导致严重自身免疫性眼结膜炎。而NOD小鼠T细胞缺失ICOS与CD28导致Th2炎症的机制未明，本项目将研究双基因敲除后TCR下游关键信号分子的活性，分析ICOS缺失逆转CD28-/-CD4+T细胞Th2分化过程中关键分子如STAT6的活化水平；探索双基因敲除小鼠胸腺发育，明确其Th2自身免疫炎症是否受中枢耐受影响。值得指出的是，ICOS与CD28阻断抗体正在成为治疗自身免疫病的药物，本课题将研究人CD28-/- ICOS-/- T细胞Th2分化，为临床治疗及风险评估提供全新证据。

共刺激分子CD28或ICOS的对T细胞分化和激活起着重要作用。目前，有临床研究正尝试通过同时拮抗的CD28、ICOS双靶向抗体抑制自身免疫性疾病。我们的研究结果显示，同时去除NOD小鼠T细胞的CD28与ICOS分子将促进炎性Th2细胞分化，导致NOD小鼠患自身免疫结膜炎。具体的，通过引入与野生型NOD小鼠（H2g7）异源的MHCii单倍型（H2b）小鼠为受体进行T细胞过继转移实验，我们发现H2g7受体小鼠患病，而异源的H2b小鼠不患病；此结果证明CD28-/-ICOS-/-小鼠T细胞胸腺阴性选择过程中没有将对结膜组织中自身抗原敏感T细胞去除；炎性CD28-/-ICOS-/- Th2细胞源于其胸腺T细胞发育过程中的阴性选择异常。我们还证明了HVEM分子对炎性CD28-/- ICOS-/- Th2细胞分化的抑制作用。通过CRISPR-Cas9进一步去除HVEM分子后，CD28-/- ICOS-/- HVEM-/-小鼠自身免疫结膜炎发病时间提前至出生后第3周。同时，我们还发现TCR-NFkB信号通路在CD28-/- ICOS-/-细胞中异常增高；TCR-NFkB通路可能促进CD28-/- ICOS-/- Th2细胞分化。总而言之，我们的结果显示同时去除CD28和ICOS将导致NOD小鼠患炎性Th2介导的自身免疫疾病；同时拮抗CD28/ICOS的临床尝试存在一定风险。