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，我们确定了三个已定义的序列，它们介导(A)配体的摄取，(B)靶向更深的内体间隔和(C)循环回DC表面。这使得DEC205成为一种高度专门化的吸收内吞作用受体。除树突状细胞外，DEC205仅由脑内皮细胞(ECs)表达。虽然ECs不是经典的抗原提呈细胞，但我们的初步数据显示，DEC205介导配体被ECs摄取，然后递送给T细胞。因此，DEC205可能是连接内皮细胞和免疫系统功能的“连接物”分子。为了详细分析DEC205在ECs中的功能，我们将在其结构域中引入靶向突变，并跟踪DEC205及其配体通过内体隔间的细胞内路线。DEC205的可能的适配分子，即内体分选复合体的成员，将通过与抗DEC205抗体的共标记和免疫沉淀进行分析。为了在体内研究正常和炎症的脑内皮细胞，我们将使用实验性变态反应性脑脊髓炎(EAE)模型。我们将把代理配体偶联到抗DEC205抗体上，并靶向正常和EAE诱导的小鼠的脑内皮细胞。此后，我们将分析在EAE或健康状态下，ECs对抗原的摄取、抗原向T细胞的提呈以及它们的分化(即Treg、Th1、Th2、无能)。综上所述，我们将确定DEC205受体在血管内皮和免疫系统的界面上的功能。

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.