Single Cell Transcriptome Analysis of Islet Antigen Reactive Memory CD4+ T Cells in Establshed T1D

项目摘要 胰岛反应性T细胞有助于T1 D的发展和进展。这些细胞可以在较低的 外周血中的频率，并可能提供一个窗口，疾病的进展和β 细胞破坏。在本申请中，我们提出表征胰岛抗原反应性记忆CD 4+（IARM-1）。 使用单细胞RNA测序（RNAseq）检测患有T1 D的个体中的CD 4）T细胞。这个强大的工具将允许 我们来确定是否独特的转录物标签定义IARM-CD 4 T细胞，如果这些标签改变 随着时间的推移，疾病进展。一些初步调查结果表明，对这些问题的扩展分析 细胞将提供T1 D致病过程的独特信息：T1 D供体中的这些细胞具有 在健康对照供体中未观察到扩增的TCR克隆型，它们具有独特的转录物表型，并且 它们表现出免疫调节剂（包括T1 D风险等位基因）的不平衡等位基因表达。我们提出了三 独立但相互关联的具体目标：确定 IARM-CD 4 T细胞中扩增的TCR克隆型。我们将从外周血中分离罕见的IARM-CD 4 T细胞。 T1 D和健康对照（HC）受试者的血液，并评估克隆型扩增随时间的稳定性， 克隆型扩增与C肽损失的程度和/或速率的关系。表征 IARM-CD 4 T细胞的转录表型，并确定其通用性、稳定性和克隆型 这些签名的特殊性。我们将表征IARM-CD 4 T细胞的转录物特征， 随着时间的推移，个体受试者，以确定其稳健性，与扩增的TCR克隆型的相关性，以及 作为C肽损失的程度和/或速率。确定不平衡等位基因的程度和稳定性 免疫基因的表达，包括T1 D风险等位基因，在IARM-CD 4 T细胞中。我们将描述等位基因 免疫基因表达失衡，包括提供信息的T1 D风险等位基因。我们共同预测， 研究将有助于阐明IARM-T细胞的独特特征，提供对免疫学的深入了解， T1 D进展的方面，并确定使用IARM-CD 4 T细胞作为生物标志物的可行性， 治疗目标

PROJECT SUMMARY Islet-reactive T cells contribute to the development and progression of T1D. These cells can be found at low frequencies in the peripheral blood and potentially provide a window into the progression of disease and beta cell destruction. We propose in this application to characterize islet antigen reactive memory CD4+ (IARM- CD4) T cells in individuals with T1D using single cell RNA sequencing (RNAseq). This powerful tool will allow us to determine whether unique transcript signatures define IARM-CD4 T cells and if these signatures change over time with disease progression. Several preliminary findings indicate that an expanded analysis of these cells will provide unique information on the pathogenic processes in T1D: these cells in T1D donors have expanded TCR clonotypes not seen in healthy control donors, they have unique transcript phenotypes, and they exhibit imbalanced allelic expression of immune modulators, including T1D risk alleles. We propose three independent, but interrelated specific aims (SA): Determine the frequency, stability and specificity of expanded TCR clonotypes in IARM-CD4 T cells. We will isolate rare IARM-CD4 T cells from the peripheral blood of T1D and healthy control (HC) subjects and evaluate the stability of clonotype expansion over time and the relationship of clonotype expansion to the extent and/or rate of C-peptide loss. Characterize the transcript phenotypes of IARM-CD4 T cells and determine the generality, stability and clonotype specificity of these signatures. We will characterize the transcript signatures for IARM-CD4 T cells from individual subjects over time to determine their robustness, association with expanded TCR clonotypes, as well as the extent and/or rate of C-peptide loss. Determine the extent and stability of imbalanced allelic expression of immune genes, including T1D risk alleles, in IARM-CD4 T cells. We will characterize allelic imbalance in immune gene expression, including informative T1D risk alleles. Together, we anticipate these studies will help elucidate unique characteristics of IARM-T cells, provide insight into the immunological aspects of T1D progression, and to determine the feasibility of using IARM-CD4 T cells as biomarkers and therapeutic targets.