Analysis of the transport pathway of exogenous antigens in cross-presentation by dendritic cells

抗原交叉呈递在感染和肿瘤免疫中至关重要，其中细胞毒性T淋巴细胞（CTL）由特异性装备有细胞机器的树突状细胞（DC）诱导，以将外源性抗原与主要组织相容性复合物（MHC）I类分子呈递。为了研究抗原交叉呈递的分子机制，我们采用小鼠树突状细胞系DC 2.4和骨髓来源的树突状细胞作为模型系统，所述树突状细胞能够呈递具有MHC I类的可溶性抗原如卵清蛋白（OVA）。我们证明，外源性添加的OVA与内质网（ER）的居民分子，如BiP和钙网蛋白，随后通过Sec 61转运蛋白复合物逆行转运到细胞质和泛素-蛋白酶体途径降解。这一机制与分泌蛋白和膜蛋白的质量控制中的ER相关降解（ERAD）基本相同。为了证实这一点，我们试图从暴露于生物素化OVA（bOVA）的DC 2.4细胞的匀浆中重建体外抗原处理系统。结合的bOVA保留在微粒体部分中，并在存在ATP的情况下与网织红细胞裂解物孵育后排出。当与蛋白酶体抑制剂MG 132孵育时，在微粒体外检测到未降解的bOVA。这表明来自微粒体的逆转录转移的bOVA被蛋白酶体降解。使用链霉亲和素磁珠从细胞匀浆中纯化含有bOVA的囊泡，发现其含有ER分子伴侣和布拉德组分以及TAP 1。这些结果有力地表明，DC加工和降解外源性抗原通过布拉德交叉呈递。

Antigen cross-presentation is critical in infectious and tumor immunity where cytotoxic T lymphocytes (CTLs) are induced by dendritic cells (DC) specifically equipped with cellular machineries to present exogenous antigens with major histocompatibility complex (MHC) class I molecules. To examine molecular mechanisms of antigen cross-presentation, we employed as a model system a murine dendritic cell line DC2.4 and bone marrow-derived dendritic cells capable of presenting soluble antigens such as ovalbumin (OVA) with MHC class I. We demonstrate that exogenously added OVA is associated with the endoplasmic reticulum (ER) resident molecules, such as BiP and Calreticulin, followed by retrograde transport to the cytoplasm through the Sec61 transporter complexes and degradated by ubiqutin-proteasome pathway. This mechanism is essentially the same as that known as the ER-associated degradation (ERAD) in the quality control of secretary and membrane proteins. To confirm this, we attempted to reconstitute an in vitro antigen processing system from homogenates of DC2.4 cells ' exposed to biotinylated OVA (bOVA). Incorporated bOVA was retained in microsomal fractions and discharged upon incubation with reticulocyte lysates in the presence of ATP. Undegraded bOVA was detected outside the microsomes when incubated with MG132, a proteasome inhibitor. This indicates that retrotranslocated bOVA from microsomes was degraded by proteasomes. bOVA-containing vesicles were purified using streptavidin magnetic beads from cell homogenates, and were found to contain ER chaperones and BRAD components together with TAP1. These results strongly suggest that DCs process and degrade exogenous antigens through BRAD for cross presentation.