Probing the mechanistic basis for T cell fate decisions (R01)

标题：探索T细胞命运决定的机制基础。 CD 4 + T细胞使用高度敏感和特异性的模块化生物传感器来调查身体的微生物病原体 或肿瘤。TCR是这种行为的主要媒介。检测抗原上MHC的含量 提呈细胞的肽（pMHC）来源于微生物病原体或肿瘤，并中继pMHC特异性 信息通过细胞膜传递到免疫受体酪氨酸激活基序（ITAM）。 相关的CD 3 γε、CD 3 δε和CD 3 γ ε信号传导模块。CD 4将Lck募集至TCR-CD 3复合物， MHC的同时结合。通过Lck的ITAM磷酸化的数量和质量提供了ITAM磷酸化的基础。 指导CD 4 + T细胞命运决定。然而，TCR-CD 3-pMHC-CD 4的单个亚基如何与TCR-CD 3-pMHC-CD 4的亚基结合， 宏复合体的配合和共同作用，以推动CD 4 + T细胞的命运决定仍然是完全确定的。我们 工作假设是这些分子以与较不复杂的受体类似的机械原理起作用 系统，如细胞因子受体，由此受体相关的细胞内信号传导酶及其 底物保持在代表“关闭”的空间关系中; pMHC-接合和相互作用 然后，TCR-CD 3复合物和CD 4之间的细胞外相互作用定位细胞内信号传导 结构域以适当的空间关系持续足够的持续时间以启动和增强信号传导。我们 首要目标是了解这种复杂分子机制的内部运作， 或模仿其形态和功能，设计具有独特治疗功能的新型模块化生物传感器。期间 在上一个资助期内，我们建立了多个实验平台，研究 TCR-CD 3亚基的质膜（JM）区。这使我们能够报告一个 一种机械开关，将pMHC特异性信息从TCR-pMHC界面传递到T细胞 细胞膜与CD 3受体信号传导模块的胞质质膜区域的连接。此外，我们执行了 TCR-CD 3-pMHC-CD 4大复合物结构的第一次实验分析，发现 CD 4 JM区邻近CD 3异二聚体，而CD 3 γ-D位于TCR的相对侧。 我们还鉴定了TMD和CD 4细胞外结构域中高度保守的残基， 对于CD 4的Lck独立和Lck依赖功能。最后，我们获得了TCR的功能证据- 我们将其解释为MHC扫描的证据。此次更新的目标 应用是解构TCR-CD 3-pMHC-CD 4大复合物的分子机制， 操作和表征这些机制在体内的后果。我们的工作将产生基本的 深入了解CD 4 + T细胞命运决定的关键决定因素，并为开发 具有平移潜力的新型模块化生物传感器。

TITLE: Probing the mechanistic basis for T cell fate decisions. CD4+ T cells use highly sensitive and specific modular biosensors to survey the body for microbial pathogens or tumors. The TCR is the chief mediator of this behavior. It surveys the contents of MHC on antigen presenting cells for peptides (pMHC) derived from microbial pathogens or tumors, and relays pMHC-specific information across the cell membrane to the ten immunoreceptor tyrosine-based activation motifs (ITAMs) of the associated CD3γε , CD3δε, and CD3ζζ signaling modules. CD4 recruits Lck to the TCR-CD3 complex upon concurrent binding of MHC. The quantity and quality of ITAM phosphorylation by Lck provides the base set of instructions that inform CD4+ T cell fate decision. Yet, how the individual subunits of the TCR-CD3-pMHC-CD4 macro-complex fit and work together to drive CD4+ T cell fate decisions remains to be fully defined. Our working hypothesis is that these molecules operate on a similar mechanistic principle to less complex receptor systems, such as cytokine receptors, whereby receptor-associated intracellular signaling enzymes and their substrates are held in a spatial relationship that represents “off”; pMHC-engagement and reciprocal extracellular interactions between the TCR-CD3 complex and CD4 then positions the intracellular signaling domains in the appropriate spatial relationship for a sufficient duration to initiate and potentiate signaling. Our overarching goal is understand the inner workings of this complex molecular machinery so that we can modify or imitate its form and function to design novel modular biosensors with unique therapeutic functions. During the previous funding period we built multiple experimental platforms to study the spatial relationship between the juxtamembrane (JM) regions of the TCR-CD3 subunits. These allowed us to report the identification of a mechanical switch that relays pMHC-specific information from the TCR-pMHC interface across the T cell membrane to the cytosolic juxtamembrane regions of the CD3ζζ signaling module. In addition, we performed the first experimental analysis of the architecture of the TCR-CD3-pMHC-CD4 macrocomplex and found that the CD4 JM region is proximal to the CD3 heterodimers, while CD3ζζ resides on the opposite side of the TCR. We also identified highly conserved residues in the TMD and extracellular domains of CD4 that are important for CD4's Lck-independent and Lck-dependent functions. Finally, we obtained functional evidence for TCR- intrinsic specificity for MHC that we interpret as evidence for MHC scanning. The goals for this renewal application are to deconstruct the molecular mechanisms by which the TCR-CD3-pMHC-CD4 macrocomplex operates and characterize the consequences of these mechanisms in vivo. Our work will yield fundamental insights into the key determinants of CD4+ T cell fate decisions and provide a blueprint for the development of novel modular biosensors with translational potential.