Ligand-dependent preTCR function

摘要 T细胞通过克隆介导对自我与非自我辨别至关重要的适应性免疫识别 分布T细胞受体(TCR)通过和基因重排在胸腺中产生。雕刻品 通过阳性和阴性选择的T细胞谱系发生在CD4CD8双阳性(DP) 胸腺发育的阶段，即TCR首次出现的阶段。在DP阶段之前，各个链关联 在不变的前T细胞受体(Pre-T，PT)的作用下，在早期胸腺细胞表面形成前TCR。PT由类似于C的 IG结构域缺乏V结构域。通过前TCR的信号被认为是一种不依赖配体的、自主的 进程。我们最近的数据显示情况并非如此。用核磁共振波谱研究两个不同的前TCR之间的相互作用 观察到链和肽/MHC(PMHC)配体包括规范的VCDR环和疏水的 在未配对的前TCRV结构域的分子表面可访问的补丁。这些观察结果已经被 使用生物膜力探头(BFP)和光镊子(OT)分析 完整早期胸腺细胞上的前TCRs。前TCRV、、CDR和补丁残留物突变对早期胸腺细胞的影响 胎儿胸腺器官培养(FTOC)和OP9-2的增殖和发育进展至DP期 DL4基质细胞依赖的分化系统。PreTCR-pMHC相互作用触发胸腺细胞钙流量， 揭示了配体依赖的信号。在这里，我们追求三个目标来定义TCR前机械生物学。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来确定 体外表达单链pMHC的前TCR-配体相互作用引起的谱系变化 通过体内分析B6 MHC的充分性和缺陷性 老鼠。我们将确定在胸腺发育过程中选择丢失或存活的链，并进行RNASEQ 在每种类型转导的B6 Rag2/-胸腺细胞上，确定前TCR连接的关系， 种群和单细胞水平的生物力学和转录组。在目标2中，结构特征和 前TCRs的机制生物学将通过单分子(SM)OT实验进行询问，以评估 不同的前TCR对结构转变、键寿命、紧凑型和延伸型之间跳跃的影响 状态及其与胸腺发育的关系。此外，核磁共振的结构研究与 项目3，核心B，核心C和X射线结晶学将定义连接的单个分子布居状态 和未连接的前TCR构象。在目标3中，我们将确定TCR与TCR和前TCR是否不同 缺少拉长的C链FG环元件，在受力下表现出键的延长。无论是人类还是 具有定义的CD1c或CD1d配体特异性的小鼠TCR以及与T22相互作用的G8将是 SM、单分子单细胞(SMSC)和单细胞(SC)OT分析与嵌合版本的比较 其中VV模块保持不变，但CC替换了CC常量模块。转导B6Rag2-/-胸腺细胞 野生型与嵌合型TCR的进展将在存在或不存在配体的情况下进行评估。

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.