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

• 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
• 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

 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 万美国人患有 1 型糖尿病 (T1D)，目前无法治愈。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。 T1D 是由自身反应性 T 细胞介导的产生胰岛素的 β 细胞死亡引起的。为了治愈T1D，我们必须去除或控制自身反应性T细胞。直到最近，识别自身反应性 T 细胞一直非常困难。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 糖尿病相关四聚体试剂。该提案的目标是利用这些新型肽：MHC II 四聚体试剂来追踪参与人类 T1D 发病机制的 CD4+ T 细胞克隆并对其进行表型分析。目前，这些 T1D 生物标志物在临床上的成功使用存在两个限制。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。必须对每个 T 细胞四聚体依次进行该分析。由于单个 CD4+ 自身反应克隆在 PBMC 样品中极为罕见，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，我们受到样本量的限制，因为许多新发 T1D 患者和有患 T1D 风险的患者都是儿童。在本提案中，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。这些研究的结果可能会带来对人类早期糖尿病诊断进行综合分析的新方法，以及监测临床环境中免疫功能和治疗效果的新生物标志物。这项研究的基本原理是，通过更好的检测方法来识别和追踪 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来选择性消除自我破坏性 T 细胞，从而治愈自身免疫性糖尿病。