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：
8932879
负责人：
Brian T Fife
金额：
$ 40万
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/8932879
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 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 pediatric patients prevent public health relevance 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（由适用提供）：对于1型糖尿病（T1D）影响的300万美国人无法治愈。即使每日胰岛素治疗延长了预期寿命，T1D仍会降低生活质量，并导致生命威胁并发症。 T1D是由自反应性T细胞介导的胰岛素产生β细胞死亡引起的。为了治愈T1D，我们必须删除或控制自反应性T细胞。直到最近，鉴定自反应性T细胞一直非常困难。但是，肽-MHC四聚体技术和富集方法的最新进展使我们能够在带有T1D的小鼠模型和人类中识别，跟踪和询问单个CD4+ T细胞克隆。我们已经生成并验证了2 HLA DQ8和3 HLA DR4糖尿病相关的四聚体试剂。该建议的目标是利用这些新型肽：MHC II四聚体试剂来跟踪和表型CD4+ T细胞克隆参与人类T1D发病机理。当前有两个局限性，阻止了这些T1D生物标志物在诊所成功使用。第一个问题是，由于有限的试剂颜色组合，不能简单地分析多个T细胞克隆。该分析必须对每个T细胞四聚体进行顺序进行。在PBMC样品中，单个CD4+自反应性克隆极为罕见，必须使用双染色方案使用特定的T细胞四聚体以两种不同的颜色正确识别。其次，由于许多新发作和患有T1D的风险的患者都是儿童，因此我们受样本量的限制。在此提案中，我们建议通过使用Cytof和流式细胞术来克服这两个局限性。这些研究的结果可能会导致对早期糖尿病诊断和新型生物标志物进行综合分析的新方法，以监测临床环境中的免疫功能和治疗效率。拟议的研究的基本原理是，在T1D期间，有了更好的测定，可以识别和跟踪多个自我反应性T细胞群体，我们将能够开发转化方法，以选择性地消除自我毁灭性T细胞以治愈自身免疫性糖尿病。