Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation

Abstract

Targeting antigens to dendritic cell (DC)-specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8 ؉ T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies. IntroductionDendritic cells (DCs) play a key role in initiating adaptive immune responses by capturing antigens and presenting them to T cells. The discovery of receptors that are mainly expressed by DCs, such as several members of the C-type lectin receptor (CLR) family, allows for vaccination strategies that target antigens directly to DCs in vivo. 1 Targeted delivery of antigens through CLRs stimulates antigen presentation and results in immunity when DC maturation stimuli are coadministered. 2 Although most of the vaccines currently on the market mainly induce humoral responses, many DC-targeted vaccines also induce strong cytotoxic T cell (CTL) responses. 1 This is attributed to the ability of certain DC subsets to present exogenous antigens on MHC class I, a process called cross-presentation.CLRs bind carbohydrate structures in a Ca 2ϩ -dependent manner via their carbohydrate recognition domain (CRD). The CRD binds specific mannose, galactose, or fucose structures present on self or non-self-proteins. 3 Human DC-SIGN represents a member of the CLR family that has been explored for DC vaccination strategies. The extracellular part of DC-SIGN is composed of a C-terminal CRD and a neck region consisting of 7 complete, and 1 incomplete, 23-residue tandem repeat regions. DC-SIGN has been demonstrated to recognize many pathogens, including viruses, bacteria, fungi, and parasites. 4 After binding, these pathogens are internalized and pathogen-derived antigens are presented via MHC class I and II molecules to CD8 ϩ and CD4 ϩ T cells, respectively. 5,6 By analogy, vaccination strategies targeting antigens to the CR...

Enhanced receptor–clathrin interactions induced by N -glycan–mediated membrane micropatterning

“…Cell surface glycan-based interactions are thought to increase signaling and residence time of the EGFR by recruiting the receptor away from caveolae (14). Because DC-SIGN internalizes via CCPs (20,24,25), we investigated whether the glycan-based mesoscale confinement exhibited by DC-SIGN influences its interaction with clathrin. We performed SPT on CHO cells cotransfected with either wtDC-SIGN or N80A, and clathrin light-chain (CLC)-YFP ( Fig.…”

“…We focused on the transmembrane glycoprotein dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) given its importance in supporting primary immune responses such as pathogen recognition and uptake on immature dendritic cells (imDCs), signaling, and cell adhesion (6,(18)(19)(20). Moreover, DC-SIGN contains a single N-glycosylation site, organizes in nanoclusters at the cell membrane (19,(21)(22)(23), and internalizes bound antigens via CPPs for subsequent processing and presentation to T cells (20,(24)(25)(26). Our work provides insights on how surface glycanmediated interactions tune spatiotemporal micropatterning of receptors on the cell membrane, potentially regulating interactions with the endocytic machinery and underscoring the importance and complex role of surface glycans on cell membrane organization and function.…”

Criteria for Dendritic Cell Receptor Selection for Efficient Antibody-Targeted Vaccination

“…For example, DEC205 (CD205), a molecule that traffics to late endosomes (4)(5)(6), is considered a superior receptor for MHC I cross-presentation (7)(8)(9)(10). Alternatively, MHC I cross-presentation is efficiently enhanced by receptors that traffic to early, but not late, endosomes (5,6,11,12). Therefore, several trafficking routes downstream of receptor internalization can promote MHC I cross-presentation.…”

Molecular Mechanisms of TLR2-Mediated Antigen Cross-Presentation in Dendritic Cells

“…In particular, Ags conjugated to Abs are targeted to either the early or the late endosome through CD40, the mannose receptor, or DEC205 (11). Efficient cross-presentation is mediated by either prolonged residence of the Ag in the early endosome or decreased degradation in the late endosome (11)(12)(13). Dendritic cell (DC) cross-priming of the TLRmediated CTL response during microbial infection may not only upregulate costimulatory molecules but also regulate this process of cross-presentation (14)(15)(16)(17).…”