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DEC205最初是在树突状细胞（dc）中发现的。在细胞内结构域中，我们描述了三个确定的序列，介导(a)配体的摄取，(b)靶向更深的内体室和(c) dc表面的再循环。这使得DEC205成为吸收性内吞作用的高度特化受体。除dc外，DEC205仅由脑内皮细胞（ECs）表达。虽然ECs不是典型的抗原呈递细胞，但我们的初步数据显示DEC205介导配体进入ECs，然后呈递到T细胞。因此，DEC205可能作为连接ECs功能与免疫系统功能的“连接物”分子。为了详细分析DEC205在ECs中的功能，我们将在其结构域引入靶向突变，并通过内体室跟踪DEC205及其配体的细胞内路径。DEC205可能的适配器分子，即内体分选复合物的成员将通过与抗DEC205抗体共标记和免疫沉淀来分析。对于正常和炎症脑内皮的体内研究，我们将使用实验性过敏性脑脊髓炎（EAE）模型。我们将替代配体偶联抗dec205抗体，靶向正常和EAE诱导小鼠的脑ECs。之后，我们将分析ECs对抗原的摄取，抗原向T细胞的呈递，以及它们在EAE或健康状态下的分化（即Treg， Th1, Th2, energy）。综上所述，我们将明确DEC205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.