Ligand-dependent preTCR function

配体依赖性 preTCR 功能

• 批准号: 10438679
• 负责人: ELLIS L REINHERZ
• 金额: $ 77.91万
• 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/10438679
• 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; 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细胞介导对自我与非自我歧视必不可少的自适应免疫识别 通过和基因重排在胸腺中产生的分布式T细胞受体（TCR）。雕刻 通过正选择和负选择的T细胞库发生在CD4+ CD8+双阳性（DP）处 胸腺发育的阶段首先出现TCR。在DP阶段之前，单个连锁协会 与不变的pre-t（PT）一起，在早期胸腺细胞表面形成了prestrc。 pt由c IG域缺乏V域。通过Pretcr发出信号被认为是与配体无关的，自主的 过程。我们最近的数据表明。使用NMR光谱法，两个不同的pretcr之间的相互作用 观察到涉及规范VCDR环和疏水性的链条和肽/MHC（PMHC）配体 在未配对的PretcrV结构域的分子表面上可访问。这些观察已经 使用生物膜力探针（BFP）和光学镊子（OT）分析进行了概括和扩展 完整的早期胸腺细胞上的pretcr。 PretcrVCDR和斑块残留物的突变会影响早期胸腺细胞 胎儿胸腺器官培养（FTOC）和OP9-的增殖和发育进展到DP阶段 DL4基质依赖性分化系统。 Pretcr-PMHC相互作用触发胸腺细胞钙通量， 揭示了依赖配体的信号传导。在这里，我们追求三个目标来定义Pretcr机械生物学。在AIM 1中， 我们将使用胸腺细胞亚群的高吞吐量下一代测序（NGS）来确定 曲目在体外使用单链PMHC表达在体外发生变化 基质和胸腺细胞祖细胞以及通过在B6 MHC中进行体内分析足够且缺乏 老鼠。我们将在胸腺发育过程中确定选择用于损失或生存的链条并执行RNASEQ 在B6 rag2 - / - 胸腺细胞中用每种类型翻译，确定了Pretcr连接的关系， 人群和单细胞水平的生物力学和转录组。在AIM 2中，结构特征和 pretcrs的机械生物学将通过单分子（SM）OT实验进行询问，以评估 不同的prestcrs对结构过渡，债券寿命，紧凑和扩展之间的影响 国家及其与胸腺发展的关系。此外，NMR与 项目3，Core B，Core C和X射线晶体学应定义连接的单个分子种群 和无罪的prestr构象。在AIM 3中，我们将确定与TCR和Prepcr不同的TCR是否在 缺乏拉长的C链FG环元件，在力下表现出明显的键延长。人类和 具有定义的CD1C或CD1D配体规范以及与T22相互作用的鼠标TCR将是 使用SM，单分子单细胞（SMSC）和单细胞（SC）OT分析与Chimeric版本进行比较 其中vv模块完好无损，但cc取代了cc常数模块。 B6 rag2 - / - 胸腺细胞翻译 在存在或不存在配体的情况下，将评估野生型与嵌合TCR的进展。