Characterization of the MHC Class II Peptide Loading Complex

描述(申请人提供)：B细胞和CD4T细胞之间的相互作用是由多肽-MHC II类复合体介导的，并支持体液免疫反应的充分发展。B细胞是抗原特异性的抗原提呈细胞，在B细胞受体(BCR)介导的同源抗原的结合和内化之后，免疫相关的抗原处理发生。本实验室已经证实，BCR介导的抗原加工发生在抗原(Ag)-BCR泛素化之后，并导致表达具有独特功能和生化特性的衍生多肽-II类复合体(称为I型复合体)。推动这个项目的基本假设是，Ag-BCR复合体的加工发生在MHC II类多肽负载复合体(PLC)中，该复合体位于MHC II类富含抗原的处理舱中。为了验证这一假设，我们将扩展我们的新的初步数据，并采用生化方法进一步定义B细胞中处理通过BCR介导的或液体相内吞作用(目标1)内化的抗原的II类PLC的分子组成。我们还将利用“FRET显微镜”方法来研究完整B细胞中第二类PLC形成的动力学(目标2)。这个项目的总体目标是 建议更好地了解BCR介导的抗原处理后第二类多肽负载的分子机制，并确定第二类多肽负载是否发生在含有特定抗原肽来源的PLC中(即，Ag-BCR复合体)。

DESCRIPTION (provided by applicant): Interactions between B cells and CD4 T cells are mediated by peptide-MHC class II complexes and support full development of a humoral immune response. B cells are antigen-specific antigen presenting cells, where immunologically relevant antigen processing occurs subsequent to B cell receptor (BCR)-mediated binding and internalization of cognate antigen. This laboratory has established that BCR-mediated antigen processing occurs subsequent to antigen (Ag)-BCR ubiquitination and results in expression of derivative peptide-class II complexes (termed "Type I" complexes) with unique functional and biochemical properties. The underlying hypothesis driving this project is that processing of Ag-BCR complexes occurs within an MHC class II peptide-loading complex (PLC) located in MHC class II enriched antigen processing compartments. To test this hypothesis, we will extend our new preliminary data and take a biochemical approach to further define the molecular composition of the class II PLC in B cells processing antigen internalized either via BCR-mediated or fluid-phase endocytosis (Aim 1). We will also utilize a "FRET microscopy" approach to study the dynamics of class II PLC formation in intact B cells (Aim 2). The overall goal of this proposal is to gain a better understanding of the molecular mechanism of class II peptide loading subsequent to BCR-mediated antigen processing, and to determine if class II peptide loading occurs within a PLC containing a dedicated source of antigenic peptide (i.e., Ag-BCR complexes).