Hypocretin特异性CD4+T细胞介导1型发作性睡病发病的机制研究

Narcolepsy type 1 (NT1), affects about 0.05% of the general population, is a chronic sleep disorder caused by the loss of neurons that produce hypocretin (HCRT). It is well documented that this disorder has an autoimmune origin and HCRT is the candidate NT1 autoantigen. A contribution of CD8+ T cells in T1N is suggested by a murine model. Prevalence of T1N increased severalfold in China following the 2009 “Swine Flu” H1N1 influenza pandemic, and influenza peptides are likely to be critically involved. We suspected that H1N1 influenza peptides mimic hypocretin binding DQ6, then some CD4+ T cells can be activated that leads to the destruction of hypocretin-producing neurons and provoke the onset of NT1. We have identified DQ6-restricted T epitopes in prepro-HCRT and screened HCRT tetramer-reactive cells and found that some CD4+ T cells were cross-reactive. We will explore the pathogenesis mechanism that how influenza peptides mimic HCRT to trigger NT1 using an approach that combines single cell index sorting, deep sequencing of single T cell mRNA transcripts and T cell activation assay. The study is expected to propose a new influenza peptide-mediated NT1 pathogenesis mechanism and to provide theoretical basis for the development of NT1 drugs and influenza vaccines.

1型发作性睡病（NT1）是由下丘脑食欲素(HCRT)神经元缺失引起的一种慢性睡眠障碍，影响0.05%的总人口。文献报道NT1是一种自身免疫病，HCRT是潜在的NT1自身抗原。小鼠模型中发现HCRT通过CD8+T介导的细胞毒作用引起NT1。2009年甲型H1N1大流行之后，NT1患病率增加数倍，推测流感肽在NT1发病中发挥关键作用。我们提出科学假说：流感肽通过模拟HCRT结合DQ6，活化HCRT反应性CD4+T细胞，攻击HCRT神经元引起NT1。前期我们鉴定得到DQ6高亲和力HCRT抗原肽，筛选到HCRT-DQ6特异性CD4+T细胞，发现HCRT反应性CD4+T细胞存在交叉反应。后期我们将利用单细胞分选、TCR mRNA深度测序、T细胞活化实验等探究流感肽模拟HCRT引起NT1发病的机制。该研究有望提出一种新的流感抗原肽介导NT1发病的机制，为NT1药物及流感疫苗的开发提供理论依据。

1型发作性睡病(NT1)是由产生下丘脑食欲素(HCRT)的神经元选择性缺失引起的睡眠障碍。大量研究表明NT1与HLA-DQB1*0602等免疫相关基因存在较强疾病关联。2009年甲型H1N1流感大流行期间，H1N1感染或疫苗接种后，NT1发病率显著增加，上述遗传学和流行病学证据表明NT1是T细胞介导的自身免疫病，但下丘脑神经元损伤的确切机制尚不清楚。鉴于HCRT前体是NT1潜在的自身抗原，本项目主要研究内容包括HCRT前体中DQ6限制性表位鉴定、HCRT特异性CD4+T细胞筛选及其诱导NT1发病的机制研究。课题组在HCRT前体中鉴定得到四个较高亲和力的DQ6结合抗原肽，包括HCRT1-13、HCRT25-37、HCRT56-69和HCRT87-100。通过HCRT-DQ6 tetramer染色，在NT1患者中筛选到体内克隆增殖的HCRT特异性CD4+T细胞，通过单细胞分选和TCR测序，课题组在不同NT1个体中鉴定到相同的T细胞克隆，并发现交叉反应性TCR27可交叉识别HCRT87-97以及具有同源序列的抗原肽，进一步通过T细胞活化实验证实DQ6限制性提呈HCRT87-100给TCR27时，可以触发TCR27介导的T细胞活化。该研究进一步阐明了HCRT特异性CD4+T细胞介导NT1发病的机制，为NT1药物及H1N1流感疫苗的开发提供理论依据。