Ligand-dependent preTCR function

配体依赖性 preTCR 功能

• 批准号: 10655336
• 负责人: ELLIS L REINHERZ
• 金额: $ 77.24万
• 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/10655336
• 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; Genes; Genetic Transcription; Human; Hydrophobicity; Immune; Immune response; In Vitro; Individual; Ligands; Ligation; Lipids; MHC Interaction; Mediating; Molecular; Mus; Mutation; NMR Spectroscopy; Peptide/MHC Complex; Peptides; Population; Process; Proliferating; Role; Signal Transduction; Specific qualifier value; Specificity; Stromal Cells; Sulfoglycosphingolipids; Surface; System; T-Cell Receptor; 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; optic tweezer; 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.

摘要 CD 4 + T细胞介导的适应性免疫识别是自我与非自我识别所必需的， 胸腺中通过α和β基因重排产生的分布性T细胞受体（TCR）。雕塑 通过阳性和阴性选择的T细胞库发生在CD 4 + CD 8+双阳性（DP） 胸腺TCR首次出现的胸腺发育阶段。在DP阶段之前，各个供应链关联 与不变的前T细胞受体（pT β）结合，在早期胸腺细胞表面形成前TCR。pT β由一个类似于C β的 缺乏V结构域的IG结构域。通过前TCR的信号传导被认为是配体独立的、自主的， 过程但我们最近的数据显示，情况并非如此。使用NMR光谱，两种不同的preTCR之间的相互作用 观察到包括典型的V β CDR环和疏水性的V β链和肽/MHC（pMHC）配体。 在未配对的preTCR V β结构域的分子表面上可接近的补丁。这些观察结果被 概括和扩展使用生物膜力探针（BFP）和光镊（OT）的分析， 完整的早期胸腺细胞上的前TCR。前TCR V → CDR和补丁残基突变影响早期胸腺细胞 在胎儿胸腺器官培养物（FTOC）和OP 9- DL 4基质细胞依赖性分化系统。前TCR-pMHC相互作用触发胸腺细胞钙流， 揭示了配体依赖性信号传导。在这里，我们追求三个目标来定义preTCR机械生物学。在目标1中， 我们将使用胸腺细胞亚群的高通量下一代测序（NGS）来确定胸腺细胞的亚群， 在体外使用表达单链pMHC的前TCR-配体相互作用的结果导致的库变化 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 小鼠我们将鉴定在胸腺发育过程中选择缺失或存活的β链，并进行RNAseq 在用每种类型转导的B6 Rag 2-/-胸腺细胞上，确定preTCR连接的关系， 生物力学和转录组在群体和单细胞水平。在目标2中，结构特征和 前TCR的机械生物学将用单分子（SM）OT实验来询问，以评估前TCR的机械生物学。 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，通过NMR的结构研究与 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 和未连接的preTCR构象异构体。在目标3中，我们将确定与EGFR和preTCR不同的EGFR TCR是否在 缺乏延长的C β链FG环元件，在力下表现出键延长。人力和 具有确定的CD 1c或CD 1d配体特异性以及与T22相互作用的G8受体的小鼠TCR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 <$V <$模是完整的，但C <$C <$取代了C <$C <$常数模。B6 Rag 2-/-胸腺细胞转导 在配体存在或不存在的情况下，评估野生型TCR与嵌合TCR的进展。