Ligand-dependent preTCR function

摘要  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)。雕刻的 通过正向和负向选择的 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  链关联 与不变的前 T (pT) 结合，在早期胸腺细胞表面形成前 TCR。 pT 由 C 样结构组成 Ig 结构域缺少 V 结构域。通过 preTCR 的信号传导被认为是一种不依赖于配体的、自主的信号传导 过程。我们最近的数据表明情况并非如此。使用 NMR 光谱，研究两种不同的 preTCR 之间的相互作用 观察到  链和肽/MHC (pMHC) 配体涉及规范的 V CDR 环和疏水性 在未配对的 preTCR V 结构域的分子表面上可访问的补丁。这些观察结果已 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 preTCR。 preTCR V CDR 和补丁残基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通量， 揭示配体依赖性信号传导。在这里，我们追求定义 preTCR 机械生物学的三个目标。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  使用单链 pMHC 表达在体外由于前 TCR-配体相互作用而导致的谱库变化 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 老鼠。我们将识别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， 群体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 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，与以下机构合作进行 NMR 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 和未连接的 preTCR 构象异构体。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，在力作用下明显键延长。无论是人类还是 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 模块完好无损，但 CC 取代了 CC 常数模块。 B6 Rag2-/- 胸腺细胞转导 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。

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.