Investigating the role of Grb2, Dok-3, Cbl and dynein in antigen internalization to provide the second signal for B cell activation

B细胞的激活需要两个时间分离的信号：(I)B细胞受体(BCR)与抗原的连接，以及(Ii)一种共刺激信号，能够最大限度地激活B细胞，包括增殖、分化和抗体产生。BCR的抗原结合导致一系列细胞内效应器和适配器的募集，这些效应器和适配器最终介导细胞内信号传递，导致抗原内化到内小体。内体内的抗原被处理，装载到MHC II类分子上，并呈现在B细胞表面。MHC-II类抗原复合体招募同源的CD4T辅助细胞，为B细胞的激活提供第二信号。最近发现，不变的自然杀伤T细胞(iNKT细胞)和B细胞固有的Toll样受体9(TLR9)也可以提供第二种信号。从这些实验中，似乎内体间隔在协调控制B细胞激活结果的组合信号方面发挥了关键作用。然而，将抗原递送到内体的分子事件尚不清楚。在这个项目中，我们将研究BCR介导的抗原内化的机制以进行内体比较，特别是接头蛋白Grb2和Dok-3，泛素连接酶Cb1和微管运动蛋白dynein在这一过程中的作用。我们将揭示这些分子如何通过CD4T细胞、iNKT细胞或TLR9连接参与第二个激活信号的传递，以及这如何影响体内B细胞的命运。这项研究的结果将有助于理解在保护性抗体反应和自身免疫性疾病期间B细胞激活的组合信号。此外，这一信息很可能代表了许多细胞表面受体的运动和内化的一般机制。

Activation of B cells requires two temporally separated signals: (I.) the ligation of the B cell receptor (BCR) with antigen, and (II.) a costimulatory signal, enabling the maximal activation of B cells, including proliferation, differentiation and antibody production. Antigen engagement of the BCR leads to the recruitment of an array of intracellular effectors and adaptors which mediate intracellular signalling ultimately resulting in antigen internalization into endosomes. Antigen within endosomes is processed, loaded onto MHC class II-molecules and presented on the B cell surface. MHC class II-antigen complexes recruit cognate CD4 T helper cells, which provide the second signal for B cell activation. Recently it became evident that invariant natural killer T cells (iNKT cells) and B cell intrinsic Toll-like receptor 9 (TLR9) engagement in endosomes can also provide the second signal. From these experiments it appears that the endosomal compartment plays a critical role in co-ordinating the combinatorial signals that govern the outcome of B cell activation. However, the molecular events underlying antigen delivery to endosomes is unclear. In this project, we will investigate the mechanisms for BCR-mediated antigen internalization to endosomal comparments, and in particular the role of the adaptor proteins Grb2 and Dok-3, the ubiquitin ligase Cbl and the microtubule motor protein dynein in this process. We will uncover how these molecules participate in the delivery of the second signal for activation either through CD4 T cells, iNKT cells or TLR9 ligation, and how this influences B cell fate in vivo. Results from this study will contribute to the understanding of the combinatorial signalling that underlies the activation of B cells during both, protective antibody responses and autoimmune diseases. Furthermore, this information is likely to represent a general mechanism to couple movement and internalization of numerous cell surface receptors.