Biology and structure of pMHC receptors functioning as mechanosensors in the [alpha][beta] T-cell lineage

总体总结 T 淋巴细胞利用  T 细胞受体 (TCR) 通过识别来区分自身与非自身 与排列在抗原呈递细胞 (APC) 上的 MHC 分子 (pMHC) 结合的稀疏抗原肽。 通过卓越的特异性和数字敏感性， T 淋巴细胞可以破坏被病毒改变的宿主细胞， 其他传染性病原体或癌变，同时保持正常细胞对应物完好无损。直到 最近，鉴于 TCR 缺乏体细胞突变，目前尚不清楚 TCR 歧视是如何实现的 与 B 细胞受体不同，增强受体-配体亲和力的基因。与传统的配体缔合相反 然而，以抗原抗体相互作用为例，现在很明显，物理力量在 基于 TCR 的非平衡 T 细胞激活。在这里，我们研究了一个总体假设，即  谱系 识别 pMHC 配体的受体，即 TCR 和 preTCR，充当机械传感器，传导 生物力学影响胸腺细胞发育以及 T 细胞抗原识别和激活。两者都 TCR 和 preTCR 利用力诱导与通电和非通电相关的不同受体构象异构体。 充满活力的国家。项目 1 应阐明使用配对驱动 αTCR 机械传感的生物物理特征 通过光镊 (OT) 进行单分子和单细胞测量以确定非平衡动力学 以及受力下能源景观的参数化。反过来，CD8 T 细胞反应，例如抗原特异性 体外触发敏感性和体内细胞增殖、效应和记忆 T 细胞发育将 使用 TCR 逆转录小鼠进行评估。不同群体和单细胞的 RNAseq 分析应定义 力依赖性转录组与 TCR-pMHC 键上的物理负荷之间的联系。项目2应 接下来使用高通量对 preTCR 和 pMHC 相互作用进行可比较的 OT 生物物理研究 对 DN3、DN4、DP 大和 DP 小子集进行世代测序 (NGS)，以确定 TCR 库 MHC 充足和 MHC 缺乏的动物体内外的变化。通过确定  链克隆型 在胸腺细胞发育过程中与特定的单-相互作用时被选择或不允许 链 pMHC 配体，表达这些 preTCR 的胸腺细胞的耦合 RNAseq 分析，OT 分析， 分子动力学 (MD) 以及 NMR 和 X 射线晶体学结构研究，早期的规则 应定义 pMHC 的胸腺选择。  和 TCR 谱系之间的区别 机械力也应类似地进行分析和比较。项目3将开发尖端核磁共振方法 揭示 pMHC 连接后 preTCR 和 αTCR 受体的变构机制，表征主要和 MD Core 辅助的次要状态结构和相互转换动力学可增强原子细节。安 行政核心 (A)、蛋白质生产核心 (B) 和 MD 核心 (C) 将协助所有项目了解如何 Force 赋予 pMHC 的  T 谱系识别以基础和转化的重要性。

OVERALL SUMMARY T lymphocytes utilized  T cell receptors (TCRs) to distinguish self versus non-self through recognition of sparse antigenic peptides bound to MHC molecules (pMHC) arrayed on antigen presenting cells (APC). Through remarkable specificity and digital sensitivity,  T lymphocytes can destroy host cells altered by viruses, other infectious pathogens or cancerous transformations while leaving normal cellular counterparts intact. Until recently, it was unclear how TCR discrimination was achieved, given a lack of somatic mutations of TCR genes to boost receptor-ligand affinity unlike with B cell receptors. Contrary to conventional ligand associations exemplified by antigen-antibody interactions, however, it is now evident that physical force plays a crucial role in non-equilibrium TCR-based T cell activation. Here we investigate the overarching hypothesis that  lineage receptors that recognize pMHC ligands, namely TCRs and preTCRs, function as mechanosensors, transducing biomechanical forces to impact thymocyte development as well as T cell antigen recognition and activation. Both TCRs and preTCRs utilize force to induce different receptor conformers associated with energized and non- energized states. Project 1 shall elucidate biophysical features driving TCR mechanosensing using paired single molecule and single cell measurements via optical tweezers (OT) to determine non-equilibrium dynamics and parameterization of energy landscapes under force. In turn, CD8 T cell responses such as antigen-specific in vitro triggering sensitivity and in vivo cellular proliferation, effector and memory T cell development will be assessed using TCR retrogenic mice. RNAseq analysis of various populations and single cells shall define the connection between force-dependent transcriptomes and physical load on TCR-pMHC bonds. Project 2 shall perform comparable OT biophysical studies on preTCRs and pMHC interactions using high throughput next generation sequencing (NGS) of DN3, DN4, DP large and DP small subsets to determine TCR repertoire changes in MHC-sufficient and MHC-deficient animals in vitro and in vivo. By determining  chain clonotypes that are selected or disallowed during thymocyte developmental progression upon interaction with specific single- chain pMHC ligands, coupled RNAseq analysis of thymocytes expressing those preTCRs, OT profiling, Molecular Dynamics (MD) and NMR and X-ray crystallography structural studies, the rules governing early thymic selection by pMHC shall be defined. Distinctions among  and TCR lineages with respect to mechanical force shall be similarly analyzed and compared. Project 3 shall develop cutting-edge NMR methods to reveal allosteric mechanisms of preTCR and TCR receptors upon pMHC ligation, characterizing major and minor state structures and kinetics of interconversion aided by the MD Core to enhance atomistic detailing. An Administrative Core (A), a Protein Production Core (B) and a MD Core (C) will assist all Projects to discern how force empowers  T lineage recognition of pMHC with basic and translational importance.