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

 描述(申请人提供)：300万美国人患有1型糖尿病(T1D)，目前尚无治愈方法。尽管每天接受胰岛素治疗可以延长预期寿命，但T1D会降低生活质量，并导致威胁生命的并发症。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，我们必须清除或控制自身反应性T细胞。直到最近，识别自身反应性T细胞一直是非常困难的。然而，多肽-MHC四聚体技术和浓缩方法的最新进展使我们能够在患有T1D的小鼠模型和人类模型中识别、跟踪和询问单个CD4+T细胞克隆。我们已经合成并验证了2个HLADQ8和3个HLADR4糖尿病相关四聚体试剂。这项建议的目标是利用这些新的多肽：MHC II四聚体试剂来追踪与人类T1D发病相关的CD4+T细胞克隆及其表型。目前，有两个限制阻碍了这些T1D生物标记物在临床上的成功应用。第一个问题是，由于试剂颜色组合的限制，无法同时分析多个T细胞克隆。这种分析必须对每个T细胞四聚体按顺序进行。更复杂的是，单个的CD4+自反应克隆在PBMC样本中非常罕见，必须使用两种不同颜色的特定T细胞四聚体的双重染色方案来正确识别。其次，我们受到样本量的限制，因为许多新发病和有患T1D风险的患者都是儿童。在这项建议中，我们建议通过两种截然不同但互为补充的多路分析方法来克服这两个限制，使用细胞周期图和流式细胞术。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记物。提出这项研究的基本原理是，有了更好的方法来识别和跟踪T1D期间的多个自我反应性T细胞群体，我们将能够开发出翻译方法，选择性地消除自我破坏性T细胞，以治疗自身免疫性糖尿病。

 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.