Ligand-dependent preTCR function

配体依赖性 preTCR 功能

• 批准号: 10020601
• 负责人: ELLIS L REINHERZ
• 金额: $ 79.84万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-29 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10020601
• 关键词: Address; Autoimmunity; Behavior; Biology; Biomechanics; CD3 Antigens; CD8B1 gene; Cell Lineage; Cells; Collaborations; Core Protein; Data; Development; Discrimination; Elements; Event; Exhibits; Fetal Thymic Organ Culture; Gene Rearrangement; Genetic Transcription; Human; Hydrophobicity; Immune; Immune response; In Vitro; Individual; Ligands; Ligation; Lipids; MHC Interaction; Mature T-Lymphocyte; Mediating; Molecular; Mus; Mutation; NMR Spectroscopy; Peptide/MHC Complex; Population; Process; Role; Signal Transduction; Specific qualifier value; Specificity; Stromal Cells; Structure; Sulfoglycosphingolipids; Surface; System; T-Cell Development; T-Cell Receptor; T-Cell Receptor Genes; T-Lymphocyte; TCR Activation; Thymocyte Development; Thymocyte Selection; Thymus Gland; X-Ray Crystallography; adaptive immunity; biophysical analysis; biophysical properties; combat; conformer; experimental study; in vivo; laser tweezer; mechanotransduction; next generation sequencing; notch protein; pathogen; peptide structure; progenitor; receptor; receptor function; release of sequestered calcium ion into cytoplasm; single molecule; thymocyte; transcriptome; transcriptome sequencing

ABSTRACT  T cells mediate adaptive immune recognition essential for self vs. non-self discrimination via clonally distributed T cell receptors (TCRs) generated in the thymus through  and  gene rearrangements. Sculpting of the T cell repertoire through both positive and negative selection occurs at the CD4+CD8+ double positive (DP) stage of thymic development where  TCRs first appear. Prior to the DP stage, individual  chains associate with the invariant pre-T (pT), forming preTCRs on the surface of early thymocytes. pT consists of a C-like Ig domain lacking a V domain. Signaling through preTCR was considered a ligand-independent, autonomous process. Our recent data show otherwise. Using NMR spectroscopy, interactions between two distinct preTCR  chains and peptide/MHC (pMHC) ligands are observed involving canonical V CDR loops and a hydrophobic patch accessible on the molecular surface of the unpaired preTCR V domain. These observations have been recapitulated and extended using biomembrane force probe (BFP) and optical tweezers (OT) analyses of preTCRs on intact early thymocytes. Mutation of preTCR V CDR and patch residues impacts early thymocyte proliferation and developmental progression to the DP stage in both fetal thymic organ culture (FTOC) and OP9- DL4 stromal cell-dependent differentiation systems. PreTCR-pMHC interaction triggers thymocyte calcium flux, revealing ligand-dependent signaling. Here we pursue three aims to define preTCR mechanobiology. In Aim 1, we will use high throughput next generation sequencing (NGS) of thymocyte subpopulations to determine  repertoire changes as a consequence of preTCR-ligand interaction in vitro using single chain pMHC expressing stroma and thymocyte progenitors as well as by performing in vivo analysis in B6 MHC sufficient and deficient mice. We shall identify  chains selected for loss or survival during thymic development and perform RNAseq on B6 Rag2-/- thymocytes transduced with each type, determining the relationship of preTCR ligation, biomechanics and transcriptome at population and single cell level. In Aim 2, structural features and mechanobiology of the preTCRs will be interrogated with single molecule (SM) OT experiments to assess the impact of diverse preTCRs on structural transitions, bond lifetimes, hopping between compact and extended states and their relationship to thymic development. In addition, structural studies by NMR in collaboration with Project 3, Core B, Core C and X-ray crystallography shall define individual molecular population states of ligated and unligated preTCR conformers. In Aim 3, we will determine if TCRs, distinct from TCRs and preTCRs in lacking the elongated C chain FG loop element, manifest bond prolongation under force. Both human and mouse TCRs with defined CD1c or CD1d ligand specificities as well as G8  that interacts with T22 will be compared using SM, single molecule single cell (SMSC) and single cell (SC) OT analyses with chimeric versions in which VV module is intact but CC replaces the CC constant module. B6 Rag2-/- thymocyte transduced wild type  versus chimeric TCRs will be assessed for progression in the presence or absence of ligands.

抽象的  T 细胞通过克隆介导适应性免疫识别，这对自我与非自我歧视至关重要 通过  和  基因重排在胸腺中产生分布式 T 细胞受体 (TCR)。 通过阳性和阴性选择的 T 细胞库发生在 CD4+CD8+ 双阳性 (DP) 处 胸腺发育阶段，其中  TCR 首次出现，在 DP 阶段之前，各个  链会结合。 与不变的前 T (pT) 一起，在早期胸腺细胞表面形成前 TCR，由 C 样结构组成。 缺乏 V 结构域的 Ig 结构域被认为是配体独立的、自主的。 我们最近的数据显示，两种不同的 preTCR 之间的相互作用并非如此。 观察到  链和肽/MHC (pMHC) 配体涉及规范的 V CDR 环和疏水性 未配对的 preTCR V 结构域的分子表面上存在补丁，这些观察结果已得到证实。 使用生物膜力探针（BFP）和光镊（OT）分析进行概括和扩展 完整早期胸腺细胞上的前TCR 前TCR V CDR 和补丁残基的突变影响早期胸腺细胞。 胎儿胸腺器官培养物 (FTOC) 和 OP9- 中增殖和发育进展至 DP 阶段 DL4 基质细胞依赖性分化系统。PreTCR-pMHC 相互作用触发胸腺细胞钙流， 在这里，我们追求定义 preTCR 机械生物学的三个目标。 我们将使用胸腺细胞亚群的高通量下一代测序 (NGS) 来确定  使用单链 pMHC 表达在体外观察前 TCR-配体相互作用导致的谱库变化 基质和胸腺细胞祖细胞以及通过在 B6 MHC 充足和缺乏中进行体内分析 我们将鉴定在胸腺发育过程中选择丢失或存活的  链并进行 RNAseq。 对用每种类型转导的 B6 Rag2-/- 胸腺细胞进行分析，确定 preTCR 连接的关系， 目标 2、结构特征和群体和单细胞水平的生物力学和转录组。 preTCR 的机械生物学将通过单分子 (SM) OT 实验来评估 不同 preTCR 对结构转变、键寿命、紧凑型和扩展型之间跳跃的影响 状态及其与胸腺发育的关系此外，与 NMR 合作进行结构研究。 项目 3、核心 B、核心 C 和 X 射线晶体学应定义连接的单个分子群体状态 在目标 3 中，我们将确定 TCR 是否与 TCR 和 preTCR 不同。 缺乏伸长的 C 链 FG 环元件，在力作用下，键会延长。 具有确定的 CD1c 或 CD1d 配体特异性的小鼠 TCR 以及与 T22 相互作用的 G8  将被 使用 SM、单分子单细胞 (SMSC) 和单细胞 (SC) OT 分析与嵌合版本进行比较 其中 VV 模块完整，但 CC 取代了 CC 恒定模块转导的 B6 Rag2-/- 胸腺细胞。 将评估野生型TCR与嵌合TCR在存在或不存在配体的情况下的进展情况。