Analytical microtools for discovering autoreactive lymphocytes

用于发现自身反应性淋巴细胞的分析微型工具

• 批准号: 7936882
• 负责人: John Christopher Love
• 金额: $ 49.99万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-23 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936882
• 关键词: Address; Adult; Affect; Animal Model; Antibodies; Antigen-Presenting Cells; Antigens; Area; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmunity; Autologous; B-Lymphocytes; Behavior; Biological Assay; Biological Factors; Blood Circulation; Cell Separation; Cell Therapy; Cells; Cerebrospinal Fluid; Characteristics; Childhood; Chronic; Clinical; Clinical Research; Cloning; Collaborations; Complex; Data; Dendritic Cells; Deposition; Development; Disease; Disease Progression; Elements; Etiology; Flow Cytometry; Frequencies; Goals; Hereditary Disease; Human; Image; Immunology; Immunophenotyping; Immunotherapy; In Vitro; Individual; Insulin-Dependent Diabetes Mellitus; Interleukin-17; Joints; Knowledge; Light; Love; Lymphocyte; Measures; Mediating; Mediator of activation protein; Methods; Micromanipulation; Monitor; Multiple Sclerosis; Muscle; Nature; Outcome Study; Outcomes Research; Pathogenicity; Patients; Peptides; Population; Printing; Process; Protein Microchips; Publishing; Reagent; Recombinant Antibody; Recombinants; Recovery; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Rheumatoid Arthritis; Sampling; Serum; System; T-Lymphocyte; Technology; Testing; Tissues; To autoantigen; Variant; Woman; autoreactive B cell; autoreactive T cell; base; cytokine; high throughput screening; human disease; interest; lymph nodes; model development; monocyte; monomer; new technology; novel; peripheral blood; public health relevance; response; skin disorder

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.

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.自身免疫性疾病，例如多发性硬化症（MS），1型糖尿病（T1D）和类风湿关节炎（RA）是由活化的自动反应性T细胞和B细胞介导的复杂遗传疾病。长期以来，在患者发炎的组织中，克隆扩展的T细胞和B细胞的存在表明，自我抗原驱动了疾病过程。阻碍人类自身免疫性疾病的研究的重大挑战是自动反应性B和T细胞的可靠鉴定和表征。这些罕见的自反应性细胞存在于外围，尽管频率非常低（> 10,000分之一）。用于研究这些细胞的技术包括流式细胞仪和免疫吸附测定法，但是这些方法无法为检测低频细胞的灵敏度不足，或者无法恢复细胞。如果可以在这些疾病中直接在体内评估自身反应性B和T细胞的频率，克隆变化和功能反应，那么对自身免疫性疾病的病因的理解将有可能取得重大进展。这项研究的核心目标是建立一组新型的方法，该方法使用微生物系统来隔离和检测自反应性B和T细胞。该项目涉及：Wucherpfennig Lab（Dana Farber）之间的合作，具有重组抗原的专业知识，用于识别B和T细胞，Hafler Lab（Brigham and妇女）在克隆和表征MS的自动反应性T细胞方面具有专业知识，并在MS中具有专业知识，并具有同时分泌分析的MS（MIT）的专业知识（MIT）。具体而言，我们将使用纳米维尔阵列开发单细胞多参数测定，以从小儿和成人MS患者中恢复抗原特异性B细胞。在平行目的中，我们还将开发一种补充方法来鉴定MS患者的自动反应性T细胞。这两个目的共有的微观结构 - 纳米维尔阵列将实现一个综合技术平台，能够全面表征自我反应性淋巴细胞的两个隔室。我们将使用这些技术来介绍小儿MS患者的B细胞反应，并评估来自MS患者的自我反应性T细胞中的频率和克隆多样性。这些研究的结果将是关于MS中B和T细胞种群广度的特定前所未有的知识，而MS的广度尚未获得，并且更广泛地是评估许多人类自身免疫性疾病中这些类型的自我反应性细胞的一般过程。 公共卫生相关性：自身免疫性疾病在研究中具有挑战性，因为与疾病自我反应性B和T细胞有关的细胞很少见。了解这些细胞的性质和多样性，涉及多发性硬化症和1糖尿病类型的疾病进展将有助于开发新疗法，但是这些细胞很难通过现有技术检测和分离。该项目的目的是开发基于微生物系统的新技术，该技术将允许与自身免疫性疾病以及其他人类疾病相关的B和T细胞的隔离和表征。