Multiplex immune analysis of antigen specific CD4+ T cells in autoimmune diabetes

自身免疫性糖尿病中抗原特异性 CD4 T 细胞的多重免疫分析

基本信息

批准号：
9091431
负责人：
Brian T Fife
金额：
$ 68.46万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-17 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9091431
关键词：
Affect Alleles American Antigens Autoantibodies Autoantigens Autoimmune Diabetes Beta Cell Biological Assay Biological Markers CD4 Positive T Lymphocytes Cell surface Cells Cessation of life Child Clinic Clinical Color Cytometry Development Diabetes Mellitus Diagnosis Diagnostic Dyes Flow Cytometry Frequencies Generations Goals Health Heavy Metals Human Immune Individual Insulin Insulin-Dependent Diabetes Mellitus Label Lead Life Life Expectancy Link Mediating Methods Monitor Monitoring Clinical Trials Pancreas Pathogenesis Patient risk Patients Peptide/MHC Complex Peptides Peripheral Blood Mononuclear Cell Phenotype Population Production Proinsulin Protocols documentation Quality of life Reagent Research Risk Sampling Self-control as a personality trait Serum Sorting - Cell Movement Staging Staining method Stains Streptavidin System T-Lymphocyte TNFRSF10A gene Technology Testing Time Treatment Efficacy cell type cytokine disease diagnosis disorder risk fluorophore immune function insulin dependent diabetes mellitus onset monomer mouse model novel novel marker pediatric patients prevent rapid detection screening transcription factor translational approach

项目摘要

DESCRIPTION (provided by applicant): There is no cure for the 3 million Americans affected by Type 1 diabetes (T1D). Even though daily insulin treatments prolong life expectancy, T1D diminishes the quality of life and leads to life threatening complications. T1D is caused by self-reactive T cell mediated death of insulin producing beta cells. In order to cure T1D, we must remove or control the self-reactive T cells. Until recently, identifying self-reactive T cells has been very difficult. However, recent advances in peptide-MHC tetramer technology and enrichment approaches have allowed us to identify, track and interrogate individual CD4+ T cell clones in both mouse models and humans with T1D. We have generated and validated 2 HLA DQ8 and 3 HLA DR4 diabetes relevant tetramer reagents. The goals of this proposal are to utilize these novel peptide:MHC II tetramer reagents to track and phenotype CD4+ T cell clones involved in human T1D pathogenesis. Currently there are two limitations preventing the successful use of these T1D biomarkers in the clinic. The first problem is that multiple T cell clones cannot be simultaneously analyzed due to limited reagent color combinations. This analysis must be done sequentially for each T cell tetramer. This is further complicated by the fact that individual CD4+ self-reactive clones are exceedingly rare in PBMC samples and must be correctly identified using a double staining protocol using the specific T cell tetramer in two distinct colors. Secondly, we are limited by sample volumes due to the fact that many new onset and patients at risk for developing T1D are children. In this proposal we propose to overcome these two limitations though two distinct but complementary multiplexing assays using CyTOF and flow cytometry. Results from these studies could lead to novel methods for a comprehensive analysis for earlier diabetes diagnosis in humans and novel biomarkers to monitor immune function and therapeutic efficacy in the clinical setting. The rationale for the proposed research is that with a better assay to identify and track multiple self-reactive T cell populations during T1D we will be able to develop translational approaches to selectively eliminate self-destructive T cells to cure autoimmune diabetes.

描述（由申请人提供）： 300 万患有 1 型糖尿病 (T1D) 的美国人无法治愈，尽管每日胰岛素治疗可以延长预期寿命，但 T1D 会降低生活质量并导致危及生命的并发症。自身反应性 T 细胞介导产生胰岛素的 β 细胞死亡为了治愈 T1D，我们必须去除或控制自身反应性 T 细胞。然而，肽-MHC 四聚体技术和富集方法的最新进展使我们能够识别、跟踪和询问患有 T1D 的小鼠模型和人类中的个体 CD4+ T 细胞克隆，我们已经生成并验证了 2 个 HLA DQ8 和 3 个 HLA DR4。该提案的目标是利用这些新型肽：MHC II 四聚体试剂来追踪与人类 T1D 相关的 CD4+ T 细胞克隆并对其进行表型分析。目前，有两个因素阻碍了这些 T1D 生物标志物在临床上的成功使用。第一个问题是，由于试剂颜色组合有限，无法同时分析多个 T 细胞克隆。这种分析必须对每个 T 细胞四聚体依次进行。由于单个 CD4+ 自身反应性克隆在 PBMC 样品中极其罕见，必须使用两种不同颜色的特定 T 细胞四聚体的双染色方案来正确识别，这一事实使情况变得更加复杂。其次，我们受到样品量的限制。由于许多新发 T1D 患者和有患 T1D 风险的患者都是儿童，因此我们建议通过使用 CyTOF 和流式细胞术的两种不同但互补的多重检测来克服这两个限制。对人类早期糖尿病诊断进行全面分析，并使用新的生物标志物来监测临床环境中的免疫功能和治疗效果。拟议研究的基本原理是，通过更好的检测方法来识别和跟踪 T1D 期间的多个自身反应性 T 细胞群。我们将能够开发转化方法选择性消除自毁性 T 细胞来治疗自身免疫性糖尿病。