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

总体汇总 T淋巴细胞利用自身T细胞受体（TCR）通过识别来区分自我与非自我 稀疏的抗原肽结合到排列在抗原呈递细胞（APC）上的MHC分子（pMHC）。 通过显着的特异性和数字敏感性，抗病毒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性转化，同时保持正常细胞对应物完整。直到 最近，由于缺乏TCR的体细胞突变，人们还不清楚是如何实现TCR的识别的 与B细胞受体不同，与常规配体缔合相反 然而，以抗原-抗体相互作用为例，现在很明显，物理力在其中发挥着至关重要的作用 基于非平衡介导TCR的T细胞活化。在这里，我们调查的首要假设， 识别pMHC配体的受体，即TCR和preTCR，起机械传感器的作用， 生物力学力影响胸腺细胞发育以及T细胞抗原识别和活化。两 受体TCR和前TCR利用力诱导与能量化和非能量化受体相关的不同受体构象。 能量状态。项目1应阐明生物物理学特征驱动的双列TCR机械感应使用配对 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 以及在外力作用下能量景观的参数化。反过来，CD 8 T细胞应答，如抗原特异性 体外触发敏感性和体内细胞增殖、效应T细胞和记忆T细胞发育将被 使用TCR逆转录小鼠评估。各种群体和单细胞的RNAseq分析将定义细胞的RNA序列。 力依赖性转录组和TCR-pMHC键上的物理负荷之间的连接。项目2将 使用高通量对preTCR和pMHC相互作用进行可比较的OT生物物理学研究 DN 3、DN 4、DP大亚群和DP小亚群的世代测序（NGS），以确定TCR T细胞库 在体外和体内的MHC-充足和MHC-缺乏的动物中的变化。通过确定β链克隆型 在胸腺细胞发育过程中，当与特定的单- 链pMHC配体，表达那些前TCR的胸腺细胞的偶联RNAseq分析，OT谱， 分子动力学（MD）和核磁共振和X射线晶体学结构研究， 应定义pMHC的胸腺选择。在以下方面， 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pMHC连接后preTCR和pMHC TCR受体的变构机制，表征了主要和 次要的状态结构和相互转换的动力学的MD核心的帮助下，以提高原子的细节。一个 管理核心（A）、蛋白质生产核心（B）和医学博士核心（C）将帮助所有项目辨别如何 力赋予pMHC的BMPT谱系识别以基本和翻译重要性。

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.