CD28 Triggering

描述（由申请人提供）：两个信号模型和共刺激的概念在我们对T细胞调控的理解中根深蒂固。现已证实，通过CD28共刺激可对T细胞活化、存活、耐受和分化产生显著影响。然而，尽管有相当大的兴趣和努力，CD28共刺激的调控和机制仍然知之甚少。CD28信号是通过将胞质信号分子募集到CD28胞质尾部（CT）结构域内的特定基序来介导的。为了了解与CD28的管腔结构域结合的配体如何转导细胞质结构域的变化以启动信号传导，我们建立了FRET实验。CFP和YFP融合至CD28的CT末端。CD28是一种二硫链二聚体，当CD28- cfp和CD28- yfp共表达时，高水平的FRET被定向表达，这表明CT结构域的末端在CD28二聚体内非常接近。当CD28被募集到免疫突触时，FRET水平降低，表明CD28二聚体内CT结构域的方向发生了一些结构变化。令人惊讶的是，即使在没有CD28配体结合的情况下，FRET的这种变化也不是由CD28配体结合介导的，而是通过TCR信号介导的。这表明TCR信号可以激活CD28，可能影响配体结合（类似于整合素激活的内向外信号传导）、免疫突触募集或信号转导。本R21应用的总体目标是确定TCR介导的CD28 CT结构域方向变化的机制和功能后果。我们将通过两个特定的目标来做到这一点：1，确定与TCR诱导CD28 CT结构域方向变化相关的分子事件。2、确定TCR信号是否调控CD28配体结合。

DESCRIPTION (provided by applicant): The two signal model and the concept of costimulation are well engrained in our understanding of T cell regulation. It is well established that costimulation through CD28 can have a dramatic effect on T cell activation, survival, tolerance, and differentiation. However, in spite of considerable interest and effort, the regulation and mechanism of CD28 costimulation are still poorly understood. CD28 signaling is mediated through the recruitment of cytosolic signaling molecules to specific motifs within the cytosolic tail (CT) domain of CD28. In an effort to understand how ligand binding to the lumenal domain of CD28 could transduce changes to the cytosolic domains to initiate signaling, we established a FRET assay. CFP and YFP were fused to the end of the CT of CD28. CD28 is a disulfide-linked dimer and when CD28-CFP and CD28-YFP were co-expressed, high level of FRET was directed, indicating that the ends of the CT domains are in close proximity within the CD28 dimer. When CD28 was recruited to the immunological synapse, the level of FRET was reduced, indicating that there was some structural change in the orientation of the CT domains within the CD28 dimer. Surprisingly, this change in FRET was not mediated by CD28 ligand binding, but instead was mediated through TCR signaling, even in the absence of CD28 ligand binding. This suggests that TCR signaling can activate CD28, possibly impacting on ligand binding (in analogy to inside-out signaling for integrin activation), immunological synapse recruitment, or signal transduction. The overall goal of this R21 application is to identify the mechanism and functional consequence of this TCR- mediated change in the orientation of the CD28 CT domains. We will do this through two Specific Aims. 1, Determine the molecular events associated with TCR induced changes in the orientation of the CD28 CT domains. 2, Determine whether TCR signaling regulates CD28 ligand binding. PUBLIC HEALTH RELEVANCE: T cell activation requires the specific recognition of pathogen-specific proteins by the TCR and co-signaling through costimulatory molecules, most notably CD28. Although the function of CD28 is well described, the molecular events associated with CD28 function as not well understood. We have made the surprising finding that the TCR may modify the conformation of CD28 and so regulate the function of CD28.