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

 描述（由申请人提供）：目前还没有治愈300万受1型糖尿病（T1 D）影响的美国人。尽管每日胰岛素治疗可以延长预期寿命，但T1 D降低了生活质量并导致危及生命的并发症。T1 D是由自身反应性T细胞介导的胰岛素产生β细胞的死亡引起的。为了治愈T1 D，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在T1 D小鼠模型和人类中识别、跟踪和询问单个CD 4 + T细胞克隆。我们已经产生并验证了2种HLA DQ 8和3种HLA DR 4糖尿病相关四聚体试剂。本提案的目标是利用这些新的肽：MHC II四聚体试剂来跟踪和表型参与人类T1 D发病机制的CD 4 + T细胞克隆。目前，有两个限制因素阻碍了这些T1 D生物标志物在临床中的成功应用。第一个问题是，由于有限的试剂颜色组合，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CD 4+自身反应性克隆在PBMC样品中极其罕见，并且必须使用双染色方案使用两种不同颜色的特异性T细胞四聚体来正确鉴定，这进一步使情况复杂化。其次，我们受到样本量的限制，因为许多新发T1 D患者和有发生T1 D风险的患者都是儿童。在该提案中，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测定来克服这两个限制。这些研究的结果可能导致对人类早期糖尿病诊断进行全面分析的新方法，以及在临床环境中监测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跟踪T1 D期间的多种自身反应性T细胞群，我们将能够开发翻译方法来选择性地消除自我破坏性T细胞，以治愈自身免疫性糖尿病。

 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.