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

在 αβ T 细胞谱系中充当机械传感器的 pMHC 受体的生物学和结构

• 批准号: 10020596
• 负责人: MATTHEW J LANG
• 金额: $ 244.43万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-29 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10020596
• 关键词: Acute; Affinity; Animals; Antibodies; Antigen-Presenting Cells; Antigens; Appearance; Autoimmune Diseases; Autoimmunity; Automobile Driving; Binding; Biological; Biological Assay; Biology; Biomechanics; Biophysics; Blood; CD8-Positive T-Lymphocytes; CD8B1 gene; Cancerous; Cell Compartmentation; Cell Lineage; Cell Proliferation; Cells; Cellular biology; Chemicals; Communicable Diseases; Coupled; Development; Discrimination; Engineering; Epithelial; Epithelium; Epitopes; Fostering; Hematopoietic stem cells; Immunity; Immunologic Deficiency Syndromes; In Vitro; Individual; Infection; Infectious Agent; Influenza A virus; Kinetics; Ligands; Ligation; Link; Lymphocyte; Malignant Neoplasms; Mature T-Lymphocyte; Measurement; Mediating; Memory; Methods; Minor; Molecular; Motion; Mus; Patients; Peptides; Performance; Peripheral; Phase; Play; Population; Positioning Attribute; Process; Production; Proteins; Receptor Cell; Receptors, Antigen, B-Cell; Recombinant Proteins; Regulation; Relaxation; Role; Sensitivity and Specificity; Somatic Mutation; Source; Specificity; Structure; Structure of thymic medulla; Surface; System; T cell response; T memory cell; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Cell Receptor Genes; T-Lymphocyte; T-Lymphocyte Subsets; T-cell receptor repertoire; TCR Activation; Techniques; Testing; Thymocyte Development; Thymus Gland; Tissue-Specific Gene Expression; Tissues; Variant; Virus; X-Ray Crystallography; adaptive immunity; base; biophysical analysis; biophysical properties; cellular pathology; cellular transduction; comparative; conformer; design; digital; effector T cell; experimental study; in silico; in vivo; laser tweezer; mechanical force; mechanotransduction; molecular dynamics; next generation sequencing; pathogen; progenitor; receptor; receptor binding; receptor function; secondary lymphoid organ; simulation; single cell analysis; single molecule; stem cells; thymocyte; transcriptome; transcriptome sequencing; vector; Acute; Affinity; Animals; Antibodies; Antigen-Presenting Cells; Antigens; Appearance; Autoimmune Diseases; Autoimmunity; Automobile Driving; Binding; Biological; Biological Assay; Biology; Biomechanics; Biophysics; Blood; CD8-Positive T-Lymphocytes; CD8B1 gene; Cancerous; Cell Compartmentation; Cell Lineage; Cell Proliferation; Cells; Cellular biology; Chemicals; Communicable Diseases; Coupled; Development; Discrimination; Engineering; Epithelial; Epithelium; Epitopes; Fostering; Hematopoietic stem cells; Immunity; Immunologic Deficiency Syndromes; In Vitro; Individual; Infection; Infectious Agent; Influenza A virus; Kinetics; Ligands; Ligation; Link; Lymphocyte; Malignant Neoplasms; Mature T-Lymphocyte; Measurement; Mediating; Memory; Methods; Minor; Molecular; Motion; Mus; Patients; Peptides; Performance; Peripheral; Phase; Play; Population; Positioning Attribute; Process; Production; Proteins; Receptor Cell; Receptors, Antigen, B-Cell; Recombinant Proteins; Regulation; Relaxation; Role; Sensitivity and Specificity; Somatic Mutation; Source; Specificity; Structure; Structure of thymic medulla; Surface; System; T cell response; T memory cell; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Cell Receptor Genes; T-Lymphocyte; T-Lymphocyte Subsets; T-cell receptor repertoire; TCR Activation; Techniques; Testing; Thymocyte Development; Thymus Gland; Tissue-Specific Gene Expression; Tissues; Variant; Virus; X-Ray Crystallography; adaptive immunity; base; biophysical analysis; biophysical properties; cellular pathology; cellular transduction; comparative; conformer; design; digital; effector T cell; experimental study; in silico; in vivo; laser tweezer; mechanical force; mechanotransduction; molecular dynamics; next generation sequencing; pathogen; progenitor; receptor; receptor binding; receptor function; secondary lymphoid organ; simulation; single cell analysis; single molecule; stem cells; thymocyte; transcriptome; transcriptome sequencing; vector

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.

总结 T淋巴细胞利用T细胞受体（TCR）通过识别来区分自我与非自我 与抗原呈递细胞（APC）结合的稀疏抗原性胡椒体（PMHC）。 通过显着的特异性和数字敏感性，淋巴细胞可以破坏因病毒而改变的宿主细胞， 其他感染性病原体或取消转化，同时使正常的细胞对应物完好无损。直到 最近，鉴于缺乏TCR的体细胞突变，尚不清楚如何实现TCR歧视 与B细胞受体不同，基因可以增强受体配体亲和力。与常规配体关联相反 然而，以抗原抗体相互作用为例，现在有证据表明，物理力在 基于非平衡的基于TCR的T细胞激活。在这里，我们研究了血统的总体假设 识别PMHC配体的接收器，即TCR和Pretcrs，充当机理，转换 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。两个都 TCR和Pretcrs利用力诱导与能量和非通电和非受体构象体 充满活力的状态。项目1应阐明使用配对的生物物理特征驱动TCR机制求解 通过光学镊（OT）确定非平衡动力学的单分子和单细胞测量 和力景观的参数化。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，效应子和记忆T细胞的发育将是 使用TCR后源性小鼠评估。 RNASEQ对各种人群和单细胞的分析应定义 TCR-PMHC键上的力依赖性转录组与物理负载之间的联系。项目2应 使用高吞吐量对prestcrs和PMHC相互作用进行可比的OT生物物理研究 DN3，DN4，DP和DP小子集的生成测序（NGS），以确定TCR曲目 体外和体内MHC富裕和缺乏MHC的动物的变化。通过确定链插型 在胸腺细胞发育过程中选择或不允许的，与特定的单次相互作用 链PMHC配体，表达这些假定的胸腺细胞的RNASEQ分析，OT分析， 分子动力学（MD）和NMR和X射线晶体学结构研究，规则提前 应定义PMHC的胸腺选择。相对于tr的区别 机械力应类似地分析和比较。项目3应开发最先进的NMR方法 在PMHC连接后揭示了PMHC受体的变构机制，以大专业和 MD核心的次要状态结构和互连的动力学可增强原子化细节。一个 行政核心（a），蛋白质生产核心（b）和MD核心（C）将帮助所有项目，以辨别如何 武力赋予对PMHC的谱系识别，具有基本和翻译的重要性。