Single-cell transcriptome profiling of disease-related lymphocytes in patients with multiple sclerosis

多发性硬化症(MS)是一种常见的自身免疫性疾病，其病程多变且不可预测。其特点是脱髓鞘、中枢神经系统淋巴细胞浸润和炎性损害。淋巴细胞渗透的原因及其细胞和分子靶点仍然是个谜。我们建议扩展CRC128的项目A5“极早期多发性硬化症适应性免疫反应的天气学分析：B细胞和T细胞的谱系和靶抗原”，其中我们关注与疾病相关的B细胞和T细胞及其在极早期MS发病机制中的参与。在这个项目中，我们通过NGS和生物信息学分析建立了来自脑脊液(CSF)的单个细胞的完整转录组分析。我们完成了第一系列实验，将受试者的单细胞转录与已确诊的多发性硬化症和前驱多发性硬化症以及对照组进行了比较。在所有这些实验中，脑脊液样本都是在MS的静止阶段获得的，初步数据显示，在急性发作期间，脑脊液中可能会检测到转录本的戏剧性变化。令人惊讶的是，我们发现在急性发作期间接受腰椎穿刺术的两名患者的淋巴细胞亚群组成存在显著差异。因此，当在急性发作期间获得细胞时，与MS的静止期相比，不同的CD4+和CD8+T细胞亚群缺失，不同的亚群可能表达特有的激活和归巢标记，因此我们目前假设这些细胞可能已经迁移到大脑中，在那里它们可能导致病变的形成和疾病的恶化。这些研究的继续是有限的，因为这样的实验极其昂贵。在这里，我们申请额外的资金来分析更大的患者队列。我们计划研究10名在疾病急性发作和静止期分别获得细胞的患者，5名进行性多发性硬化症患者，以及5名非炎症性神经疾病患者。我们还将对其中10名患者的外周血细胞进行研究。对这个队列的分析将使我们能够确认初步结果。我们乐观地认为，我们可以证实我们的数据，这样我们就可以更深入地了解在MS急性发作期间离开脑脊液的细胞的特征。我们预计，这些结果可能有助于解释为什么特定的T细胞亚型进入脑组织，以及它们如何成功地越过血脑屏障。这可能会揭示MS的免疫病理机制的重要见解，也可能具有很高的临床益处。我们的观察可能会引导我们寻找新的生物标记物来预测疾病进程，有助于患者分层和治疗选择，并可能为影响T细胞迁移行为和防止脑组织攻击的合理治疗铺平新的道路。

Multiple sclerosis (MS) is a prevalent putative autoimmune disease with a variable and unpredictable disease course. It is characterized by demyelination, lymphocyte infiltrations in the central nervous system, and inflammatory lesions. The reasons for lymphocyte infiltration and their cellular and molecular targets are still enigmatic. We propose to extend project A5 "Synoptic analysis of adaptive immune reactions in very early multiple sclerosis: Repertoires and target antigens of B- and T-cells" of CRC128, where we focus on putatively disease-related B- and T-cells and their involvement in the pathogenesis of very early MS. In this project, we established whole transcriptome analyses of single cells from cerebrospinal fluid (CSF) by NGS and bioinformatics analyses. We completed a first series of experiments where we compared single cell transcriptomes of subjects with established and prodromal MS, and controls. In all these experiments, CSF samples were obtained at quiescent stages of MS. Preliminary data revealed that dramatic changes in the transcriptomes may be detected in CSF during an acute attack. Surprisingly, we found striking differences in the composition of lymphocyte subpopulations in two patients who underwent lumbar puncture during an acute attack. Thus, when cells were obtained during an acute attack, distinct subpopulations of CD4+ and CD8+ T cells were missing as compared to quiet stages of MS. Different subpopulations may express characteristic activation and homing markers, therefore we currently assume that these cells might have migrated into the brain where they probably contributed to lesion formation and deterioration of the disease. Continuation of these studies is limited because such experiments are extremely costly. Here we apply for additional funding to analyze a larger cohort of patients. We are planning to study 10 patients each where cells were obtained during an acute attack and at a quiet stage of the disease, 5 patients with progressive MS, and 5 subjects with non-inflammatory neurological diseases. From 10 of these patients we will additionally investigate cells from peripheral blood. Analysis of this cohort will allow us to confirm the preliminary results. We are optimistic that we may substantiate our data so that we may gain deeper insight in the characteristics of the cells that leave the CSF during an acute MS attack. We expect that these results may significantly contribute to explaining why particular T cell subtypes enter brain tissue and how they succeed in crossing the blood-brain barrier. This may reveal important insights into the immunopathology of MS, and may also have high clinical benefit. Our observations may guide us towards new biomarkers that predict disease courses, contribute to patient stratification and therapy selection, and may moreover pave new ways for rational therapies that influence the migrational behavior of T cells and prevent attack of brain tissue.