Analyzing the function of the endocytic receptor DEC205 in vitro and in vivo during delivery and presentation of antigens in brain endothelial cells.

抗原摄取受体DEC 205最初在树突状细胞（DC）中描述。在细胞内结构域内，我们表征了三个确定的序列，介导（a）配体的摄取，（B）靶向更深的内体区室和（c）再循环回到DC表面。 这使得DEC 205成为吸收性内吞作用的高度特化受体。除了DC之外，DEC 205仅由脑的内皮细胞（EC）表达。虽然EC不是经典的抗原呈递细胞，但我们的初步数据显示DEC 205介导配体摄取到EC中，然后呈递给T细胞。因此，DEC 205可以作为连接EC功能与免疫系统功能的“接头”分子。为了详细分析DEC 205在EC中的功能，我们将在其结构域中引入靶向突变，并跟踪DEC 205及其配体通过内体区室的细胞内路由。DEC 205的可能衔接分子，即内体分选复合物的成员，将通过与抗DEC 205抗体的共标记和免疫沉淀进行分析。对于正常和炎症脑内皮细胞的体内研究，我们将利用实验性过敏性脑脊髓炎（EAE）模型。我们将在正常和EAE诱导的小鼠中将替代配体偶联至抗DEC 205抗体并靶向脑EC。此后，我们将分析EAE或健康条件下EC的抗原摄取，抗原向T细胞的呈递，以及它们的分化（即Treg，Th1，Th2，无反应性）。总之，我们将定义DEC 205受体在血管内皮和免疫系统界面的功能。

The antigen uptake receptor DEC205 was originally described in dendritic cells (DCs). Within the intracellular domain we characterized three defined sequences, mediating (a) the uptake of ligands, (b) the targeting to deeper endosomal compartments and (c) the recycling back to the surface of DCs. This makes DEC205 a highly specialized receptor for absorptive endocytosis. Beyond DCs, DEC205 is exclusively expressed by endothelial cells (ECs) of the brain. While ECs are not classical antigen presenting cells, our preliminary data show that DEC205 mediates uptake of ligands into ECs, followed by presentation to T cells. Thus, DEC205 may serve as “linker” molecule connecting the function of ECs with those of the immune system. To analyze the functions of DEC205 in ECs in detail, we will introduce targeted mutations into its domains and follow the intracellular routing of DEC205 and its ligands through endosomal compartments. Possible adapter molecules of DEC205, i.e. members of the endosomal sorting complexes will be analyzed by co-labeling and immunoprecipitation with anti-DEC205 antibodies. For in vivo investigations of normal and inflamed brain endothelia, we will make use of the experimental allergic encephalomyelitis (EAE) model. We will couple surrogate ligands to anti-DEC205 antibodies and target brain ECs in normal and EAE induced mice. Thereafter, we will analyze the antigen uptake by ECs, the presentation of antigens to T cells, as well as their differentiation (i.e. Treg, Th1, Th2, anergy) during EAE or healthy conditions. In summary, we will define the functions of the DEC205 receptor at the interface of the vascular endothelium and the immune system.