Defining the SAP-dependent and SAP-independent gamma delta TCR repertoire

定义 SAP 相关和 SAP 独立的 gamma delta TCR 指令集

• 批准号: 10043222
• 负责人: JONATHAN E BOYSON
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-22 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043222
• 关键词: Adaptor Signaling Protein; Adult; Affect; Applications Grants; Area; Bacteria; Bar Codes; Birth; Cells; Characteristics; Data; Data Set; Dependence; Development; Embryo; Exhibits; Family; Frequencies; Hematopoietic; Immune response; Impairment; Interferons; Interleukin-17; Interleukin-4; Investigation; Laboratories; Libraries; Lung; Lymphocyte; Lymphoid Tissue; Microbe; Mucous Membrane; Mus; Organ; Pathway interactions; Peripheral; Play; Population; Receptor Signaling; Research Personnel; Resolution; Role; SH2D1A gene; SLAM family receptor; Shapes; Signal Pathway; Signal Transduction; Skin; Spleen; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; T-cell receptor repertoire; Thymus Gland; Tissues; Tumor Immunity; Virus; adaptive immune response; anti-tumor immune response; antimicrobial; chemokine; cytokine; fungus; neonate; next generation; next generation sequencing; novel; receptor

The tissue-resident innate-like T cell subsets (i.e., NKT, MAIT, and innate-like T) constitute major lymphocyte populations in barrier tissues like the lung, gut, and skin, where they provide a critical first line of defense against bacteria, fungi, and viruses and where they are active players in anti-tumor immunity. Innate- like T cell subsets all share the ability to rapidly producing large amounts of cytokines and chemokines, and thereby possess the ability to shape both the quantity and quality of both the innate and adaptive immune responses. Although the NKT and mucosal-associated invariant T (MAIT) innate-like  T cell subsets are well-studied, far less is known of the innate-like  T cell populations, and their identity remains ill-defined. We recently completed a comprehensive investigation into the role of the SLAM/SAP signaling pathway in  T cell development and function. This study revealed that the SLAM/SAP signaling pathway is a critical determinant in the thymic development of both IL-17- and IFN--producing  T cells. Since one of the hallmarks of nearly all innate-like T cell subsets is a dependence on the SLAM/SAP signaling pathway for their development, these results raise the possibility that SAP regulates the development of, as yet undefined innate-like  T cell subsets. Therefore, the objective of this proposal is to unambiguously define the SAP-dependent  T cell subsets. Because a characteristic feature of all innate-like T cells identified so far is the expression of a signature invariant of semi-invariant TCR, we propose to: identify the SAP-dependent and SAP-independent  TCR clonotypes using single-cell next-generation sequencing. We will sort single  T cells from C57BL/6J and B6.SAP-/- mice, and determine the paired TCR clonotypes by sequencing barcoded amplicon libraries generated using V- and V-specific primers. We will examine thymic  T cells at different stages of development (embryonic day 17, birth, day 6 neonate, and adult) to capture distinct waves of  T cell development, and we will examine lung and spleen to identify the SAP-dependent subsets in the periphery. Upon completion of these studies, we will have unambiguously identified the SAP-dependent  TCR clonotypes, which will be critical in to our understanding how SLAM/SAP signaling regulates the developmental programming of  T cells. In addition, we will have provided an invaluable high-resolution dataset of the paired  TCR clonotypes in both the thymus and periphery to investigators seeking to understand better the role of  T cells in the immune response.

组织驻留的先天T细胞亚群(即NKT、MAIT和先天T)构成了主要的 肺、肠道和皮肤等屏障组织中的淋巴细胞群，在这些组织中，它们提供了关键的第一线 对细菌、真菌和病毒的防御，以及它们在抗肿瘤免疫中的积极作用。与生俱来 就像T细胞亚群一样，所有的亚群都有快速产生大量细胞因子和趋化因子的能力，并且 从而拥有塑造先天免疫和获得性免疫的数量和质量的能力 回应。尽管NKT和粘膜相关不变量T(MAIT)先天类T细胞亚群是 尽管研究很充分，但对先天类T细胞群的了解要少得多，它们的身份也仍然不明确。我们 最近完成了对SLAM/SAP信号通路在T细胞中作用的全面研究 发展和功能。这项研究表明SLAM/SAP信号通路是一个关键的决定因素 在胸腺发育过程中产生IL-17和干扰素-的T细胞。因为这几乎是一个标志 所有先天类T细胞亚群的发育依赖于SLAM/SAP信号通路，这些 结果提高了SAP调节尚未明确的先天T细胞发育的可能性 子集。因此，这项提议的目标是明确地定义SAP依赖的T细胞 子集。因为到目前为止发现的所有先天类T细胞的一个特征是表达一个 基于半不变的特征不变量，我们提出：识别SAP依赖和SAP非依赖的TCR 使用单细胞下一代测序的TCR克隆型。我们将对C57BL/6J中的单个T细胞进行分选 B6.SAP-/-小鼠，通过条码扩增文库测序确定成对的TCR克隆型 用V-和V-特异的引物产生。我们将检测胸腺T细胞在不同阶段的 发育(胚胎第17天、出生、第6天新生儿和成人)以捕获不同波的T细胞 我们将检查肺和脾，以确定外周SAP依赖的亚群。 在完成这些研究后，我们将毫不含糊地确定依赖SAP的TCR 克隆类型，这对于我们理解SLAM/SAP信号如何调节发育至关重要。 T细胞的编程。此外，我们还将提供一个宝贵的高分辨率配对数据集 胸腺和外周的TCR克隆型研究人员试图更好地了解的作用 免疫反应中的T细胞。