Analytical microtools for discovering autoreactive lymphocytes

描述（由申请人提供）：该申请涉及广泛的挑战领域（04）临床研究和特定挑战主题，04- ar -101：皮肤，关节，肌肉和其他组织疾病的自身免疫-开发试剂和分析方法，以识别，表征，跟踪和抑制人类B细胞和T细胞特异性定义的自身抗原和抗原呈递细胞。自身免疫性疾病，如多发性硬化症（MS）、1型糖尿病（T1D）和类风湿性关节炎（RA）是由活化的自身反应性T细胞和B细胞介导的复杂遗传疾病。长期以来，患者炎症组织中T细胞和B细胞克隆扩增群体的存在表明，自身抗原驱动了疾病的进程。阻碍人类自身免疫性疾病研究的一个主要挑战是自身反应性B细胞和T细胞的可靠鉴定和表征。这些罕见的自反应细胞存在于外周，尽管频率很低（1万分之一）。用于研究这些细胞的技术包括流式细胞术和免疫吸附剂测定，但这些方法要么对检测低频细胞灵敏度不足，要么无法恢复细胞。如果能够直接在体外评估这些疾病中自身反应性B细胞和T细胞的频率、克隆变异和功能反应，那么对自身免疫性疾病病因的理解将有可能取得重大进展。本研究的中心目标是建立一套新的方法，使用微制造系统来分离和检测自反应性B细胞和T细胞。该项目涉及以下方面的合作：wucherpfenen实验室（Dana Farber）拥有MS重组抗原鉴定B细胞和T细胞的专业知识，Hafler实验室（Brigham and Women’s）拥有MS自身反应性T细胞克隆和表征的专业知识，Love实验室（MIT）拥有同时平行分析bbb105单个原代细胞分泌产物的专业知识。具体来说，我们将开发单细胞多参数分析，使用纳米孔阵列从儿童和成人多发性硬化症患者中恢复抗原特异性B细胞。在一个平行的目标，我们也将开发一种互补的方法来识别多发性硬化症患者的自身反应性T细胞。这两个目标共同的微观结构——纳米孔阵列——将使一个集成的技术平台能够全面表征自反应性淋巴细胞的两个区室。我们将使用这些技术来分析儿童多发性硬化症患者的B细胞反应，并评估多发性硬化症患者自身反应性T细胞的频率和克隆多样性。这些研究的结果将是对MS中B和T细胞群广度的前所未有的具体知识，这是以前无法获得的，更广泛地说，是评估许多人类自身免疫性疾病中这些类型的自反应细胞的一般过程。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (04) Clinical Research and specific Challenge Topic, 04-AR-101: Autoimmunity For Diseases Of The Skin, Joints, Muscle And Other Tissues-Develop reagents and analytic methods to identify, characterize, track, and inhibit human B and T cells specific for defined selfantigens, and antigen-presenting cells. Autoimmune diseases such as multiple sclerosis (MS), type 1 diabetes (T1D) and rheumatoid arthritis (RA) are complex genetic diseases mediated by activated, autoreactive T cells and B cells. The presence of clonally expanded populations of T cells and B cells in the inflamed tissue of patients has long suggested that self-antigens drive the disease process. A major challenge hindering the study of human autoimmune diseases is the reliable identification and characterization of autoreactive B and T cells. These rare, self-reactive cells are present in the periphery, albeit at very low frequencies (>1 in 10,000). The technologies used for studying these cells include flow cytometry and immunosorbant assays, but these methods either have insufficient sensitivity for detecting low-frequency cells or the recovery of the cells is not possible. Significant advances in the understanding of the etiologies of autoimmune diseases would be possible if it were feasible to assess the frequency, clonal variations, and functional responses of autoreactive B and T cells in these diseases directly ex vivo. The central goal of this research is to establish a novel set of methods that use microfabricated systems to isolate and detect both self-reactive B and T cells. This project involves a collaboration amongst: the Wucherpfennig lab (Dana Farber) with expertise in recombinant antigens for MS to identify B and T cells, the Hafler lab (Brigham and Women's) with expertise in cloning and characterizing autoreactive T cells in MS, and the Love lab (MIT) with expertise in simultaneous parallel analyses of secreted products from >105 single primary cells. Specifically, we will develop single-cell multiparametric assays using arrays of nanowells to recover antigen-specific B cells from pediatric and adult MS patients. In a parallel aim, we will also develop a complementary method to identify autoreactive T cells in MS patients. The microstructure common to both of these aims-the arrays of nanowells-will enable an integrated technology platform capable of comprehensive characterization of both compartments of selfreactive lymphocytes. We will use these technologies to profile B cell responses in pediatric MS patients and evaluate the frequencies and clonal diversity in self-reactive T cells from MS patients. The outcome of these studies will be specific unprecedented knowledge on the breadth of B and T cell populations in MS that has not been accessible previously, and more broadly, a general process for evaluating these types of self-reactive cells in many human autoimmune diseases. PUBLIC HEALTH RELEVANCE: Autoimmune diseases are challenging to study because the cells involved in the disease-self-reactive B and T cells-are rare. Understanding the nature and diversity of these cells involved in the progression of diseases such as multiple sclerosis and type 1diabetes would aid in the development of new therapies, but the cells are difficult to detect and isolate with existing technologies. The aim of this project is to develop new technologies based on microfabricated systems that will allow the isolation and characterization of both B and T cells relevant for autoimmune diseases, but also for other human diseases.