Intermolecular Interactions in the Immunological Synapse

描述(由申请人提供)：内源性非刺激性多肽通过限制抗原的数量在帮助T细胞激活方面起着重要作用。该项目将确定非刺激性MHC-多肽复合体作用的分子基础。CD8或TCR与非刺激性或正选择性MHCp的相互作用相对于其与抗原性MHCp的结合的重要性将使用单链MHC类I-肽分子来确定，其中肽是固定的，并且CD8或TCR结合位点可以突变。在抗原加非刺激性多肽的识别过程中，TCR和CD8之间的相互作用的诱导将使用FRET显微镜进行测量。FRET还将用于确定非刺激性和抗原性MHCp在抗原识别过程中是否紧密结合在一起。我们将测量在存在或不存在少量抗原的情况下，内源性、非刺激性或阳性选择多肽的早期信号反应，以了解T细胞如何被这些配体预刺激。将测量CD8与非刺激性MHCp本身结合的相对重要性(即CD8的黏附功能)与其在浓缩激酶Lck中的重要性。除与抗原性MHCp结合的TCR外，是否发生其他TCR的磷酸化将使用FRET生物传感器进行测试。T细胞的双光子显微镜将被用来了解T细胞行为如何随着TCR配体的亲和力以及是否存在非刺激性pMHC而改变。表达CD8荧光分子的转基因小鼠将被用来研究CD8在迁移过程中的细胞表面动力学和原位抗原识别。

DESCRIPTION (provided by applicant): Endogenous non-stimulatory peptides are important in aiding T cell activation by limiting quantities of antigen. This project will determine the molecular basis for the role of non- stimulatory MHC-peptide complexes. The importance of CD8 or TCR interaction with a non-stimulatory or positive selecting MHCp, compared to its binding to the antigenic MHCp will be determined using single-chain MHC class I-peptide molecules, where the peptide is fixed, and the CD8 or TCR binding site can be mutated. Induction of interaction between TCR and CD8 during recognition by antigen plus non-stimulatory peptides will be measured using FRET microscopy. FRET will also be used to determine whether the non-stimulatory and antigenic MHCp are brought close together during antigen recognition. We will measure early signaling responses to the endogenous, non- stimulatory or positive selecting peptides in the presence or absence of small amounts of antigen, to understand how the T cells can be pre-stimulated by these ligands. The relative importance of CD8 binding to non-stimulatory MHCp itself (i.e. the adhesion function of CD8) versus its importance in concentrating the kinase Lck, will be measured. Whether phosphorylation of other TCRs than those that bind to the antigenic MHCp occurs will be tested using a FRET biosensor. Two photon microscopy of T cells will be used to understand how T cell behavior alters with the affinity of the ligand for the TCR and with the presence or absence of non-stimulatory pMHC. Transgenic mice that express fluorescent CD8 molecules will be used to investigate cell surface dynamics of CD8 during migration and antigen recognition in situ.