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)通过识别来区分自体和异体 与MHC分子结合的稀疏抗原肽(PMHC)排列在抗原提呈细胞(APC)上。 通过显著的特异性和数字敏感性，T淋巴细胞可以摧毁被病毒改变的宿主细胞， 其他感染性病原体或癌变，而保持正常细胞对应的完好无损。直到 最近，由于缺乏TcR的体细胞突变，尚不清楚tcr歧视是如何实现的。 与B细胞受体不同的是，基因可以提高受体-配体的亲和力。与传统的配基缔合相反 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T细胞受体的非平衡T细胞活化。在这里，我们调查了血统的最重要的假设 识别pMHC配体的受体，即TCRs和preTCRs，作为机械传感器发挥作用，转导 影响胸腺细胞发育以及T细胞抗原识别和激活的生物力学力量。两者都有 TCR和Pre TCR使用力诱导与通电和非通电相关的不同受体构象 充满能量的状态。项目1将阐明使用配对技术驱动TCR机械传感的生物物理特征 通过光钳(OT)测量单分子和单细胞以确定非平衡动力学 以及在武力作用下的能源景观的参数化。反过来，CD8 T细胞反应，如抗原特异性 在体外触发敏感性和体内细胞增殖、效应和记忆性的T细胞发育 使用TCR逆转录基因小鼠进行评估。不同群体和单个细胞的RNAseq分析应定义 力依赖转录本与TCR-pMHC键上的物理负荷之间的联系。项目2将 使用高通量NEXT对前TCR和pMHC相互作用执行类似的OT生物物理研究 DN3、DN4、DP大小亚集的代序列测定以确定TCR谱系 MHC充足和MHC缺陷动物在体外和体内的变化。通过确定链克隆类型 在胸腺细胞发育过程中与特定的单细胞相互作用时被选择或不被允许的基因 链pMHC配体，表达这些前TCR的胸腺细胞的耦合RNAseq分析，OT图谱， 分子动力学(MD)、核磁共振和X射线晶体结构研究，早期的规则 应定义pMHC对胸腺的选择。和TCR谱系之间的差异 机械力应进行类似的分析和比较。项目3将开发尖端的核磁共振方法 为了揭示前TCR和受体在pMHC连接时的变构机制，主要和 微态结构和动力学的相互转化协助MD核心，以加强原子细节。一个 行政核心(A)、蛋白质生产核心(B)和MD核心(C)将协助所有项目识别如何 FORCE使T血统对pMHC的识别具有基本和翻译重要性。

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.