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

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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.