Analytical microtools for discovering autoreactive lymphocytes

描述（由申请人提供）：本申请涉及广泛的挑战领域（04）临床研究和特定的挑战主题，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、跟踪和抑制对定义的自身抗原特异的人B和T细胞以及抗原呈递细胞。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性疾病。长期以来，在患者发炎组织中存在克隆扩增的T细胞和B细胞群体表明，自身抗原驱动了疾病过程。阻碍人类自身免疫性疾病研究的主要挑战是自身反应性B和T细胞的可靠鉴定和表征。这些罕见的自我反应细胞存在于外周，尽管频率很低（> 1/10，000）。用于研究这些细胞的技术包括流式细胞术和免疫吸附测定，但这些方法要么检测低频细胞的灵敏度不足，要么无法回收细胞。如果能够直接离体评估自身免疫性疾病中自身反应性B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学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，使用微加工系统分离和检测自身反应性B和T细胞。该项目涉及以下实验室之间的合作：Wucherpfennig实验室（Dana Farber），其在MS重组抗原方面具有专业知识，以识别B和T细胞; Hafler实验室（Brigham and Women's），其在MS中克隆和表征自身反应性T细胞方面具有专业知识;以及Love实验室（MIT），其在同时平行分析来自> 105个单个原代细胞的分泌产物方面具有专业知识。具体来说，我们将开发单细胞多参数检测使用阵列的细胞，以恢复抗原特异性B细胞从儿童和成人MS患者。在一个平行的目标，我们还将开发一种互补的方法来识别MS患者的自身反应性T细胞。这两个目标共同的微结构-微阵列-将使一个综合的技术平台能够全面表征两个隔室的自我反应淋巴细胞。我们将使用这些技术来分析儿科MS患者的B细胞反应，并评估MS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特定的前所未有的知识的广度B和T细胞群体在MS中，还没有访问以前，更广泛地说，一个一般的过程，用于评估这些类型的自我反应细胞在许多人类自身免疫性疾病。 公共卫生相关性：自身免疫性疾病的研究具有挑战性，因为参与疾病的细胞--自身反应性B和T细胞--非常罕见。了解这些细胞的性质和多样性与多发性硬化症和1型糖尿病等疾病的进展有关，将有助于开发新的治疗方法，但这些细胞很难用现有技术检测和分离。该项目的目的是开发基于微制造系统的新技术，该系统将允许分离和表征与自身免疫性疾病相关的B和T细胞，以及其他人类疾病。

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.