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: 10170255
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10170255
• 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.

组织驻留的先天样T细胞亚群（即，NKT、MAIT和先天性类NKT）构成了主要的 淋巴细胞群体在屏障组织，如肺，肠道和皮肤，在那里他们提供了一个关键的第一线， 防御细菌，真菌和病毒，以及它们在抗肿瘤免疫中的积极参与者。天生的- 像T细胞亚群一样，都具有快速产生大量细胞因子和趋化因子的能力， 从而具有塑造先天性和适应性免疫的数量和质量的能力， 应答尽管NKT和粘膜相关不变T（MAIT）先天性类巨噬细胞T细胞亚群是 尽管研究得很好，但对先天性类CD 4 + T细胞群体的了解却少得多，而且它们的身份仍然不明确。我们 最近完成了一项关于SLAM/SAP信号通路在人T细胞中的作用的全面研究， 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， 在产生IL-17和IFN-γ的胸腺T细胞的胸腺发育中。因为一个几乎 所有先天性T细胞亚群都依赖于SLAM/SAP信号通路，这些 结果提出了SAP调节尚未定义的先天样T细胞发育的可能性 子集因此，本提案的目的是明确定义SAP依赖性巨噬细胞T细胞， 子集因为迄今为止鉴定的所有先天性样T细胞的一个特征是表达一种 签名不变的半不变TCR，我们提出：确定SAP依赖和SAP无关的TCR 使用单细胞下一代测序的TCR克隆型。我们将从C57 BL/6 J和C57 BL/6 J中分选出单个巨噬细胞T细胞。 B6.SAP-/-小鼠，并通过测序条形码化扩增子文库来确定配对的TCR克隆型 使用V β-和V β-特异性引物产生。我们将在不同的阶段检查胸腺T细胞。 发育（胚胎第17天、出生、新生儿第6天和成人）以捕获不同的巨噬细胞T细胞波 我们将检查肺和脾以鉴定外周中的SAP依赖性亚群。 在完成这些研究后，我们将明确地鉴定SAP依赖性TCR。 克隆型，这将是至关重要的，我们了解如何SLAM/SAP信号调节发育的 编程的T细胞。此外，我们还将提供一个非常宝贵的高分辨率数据集， 胸腺和外周中的TCR克隆型有助于研究人员更好地了解胸腺细胞的作用。 免疫反应中的T细胞