In this study, we assessed the functional role of SR-BI for the uptake and cross-presentation of HCV by human DCs. We demonstrate that (i) SR-BI is required for the binding and uptake of HCV by human DCs and (ii) SR-BI-mediated uptake results in trafficking into the MHC class I pathway, followed by efficient cross-presentation to HCV-specific CD8+ T-cells. Taken together, our results reveal a novel function for SR-BI for antigen uptake and presentation and identify a novel mechanism whereby DCs can capture and process viral antigens.
SR-BI and its splicing variant SR-BII are physiologically relevant HDL receptors with an identical extracellular loop. SR-BII differs from SR-BI at the C terminus, which is reported to confer an intracellular localization on SR-BII (64
). Using defined antibodies targeting the cytoplasmic tail or extracellular loop of SR-BI, we could show that human immature DCs express SR-BI. These findings are in line with the results of two previous studies demonstrating that SR-BI is expressed on monocyte-derived DCs, as well as on plasmacytoid and myeloid DCs (10
). In contrast to our findings, Yamada et al. (67
) observed a higher level of SR-BI expression on the surface of monocytes using a different anti-SR-BI antibody. These differences could be due to different epitopes recognized by the antibodies or different protocols of monocyte isolation used in their study and ours. In our study, as well as in the study of Buechler et al. (10
), SR-BI expression was induced during the differentiation of monocytes into DCs, indicating that SR-BI may play a specific role for DC function. Since SR-BI has been shown to represent a host cell entry factor for HCV infection of human hepatoma cells (25
), we explored its role in viral antigen capture and presentation by DCs. Using an HCV-LP-based model system (5
), we demonstrate that SR-BI is required for the binding and uptake of HCV-LP into DCs. Since previous results have shown that C-type lectins, such as mannose receptor or DC-SIGN, were not sufficient to mediate HCV-LP binding to DCs (5
), SR-BI may represent one of the key DC surface proteins binding HCV particles on DCs. This novel SR-BI function is further supported by the observation that HDL enhanced the binding of HCV-LP to DCs, whereas oxidized LDL and polyanionic ligands reduced HCV-LP binding. Since the presence of HDL did not inhibit but rather enhanced HCV-LP binding, it is unlikely that HCV and HDL compete for the SR-BI HDL binding domain. The highly reproducible enhancement of HCV-LP binding by HDL may rather point to a more-efficient interaction of SR-BI with HCV, e.g., as a result of a conformational change induced by HDL. These findings are in line with findings observed for the infection of human hepatoma cells with recombinant HCVpp and HCVcc (6
). The significant modulation of HCV-LP binding by HDL and LDL provides a link between lipid metabolism and antigen recognition and may suggest that lipoproteins may interfere with the DC-antigen interaction.
Antigen cross-presentation offers a solution by permitting DCs to crossover exogenous antigens for access to the class I MHC peptide-loading machinery. This mechanism enables DCs to raise immune responses against pathogens, like viruses, that do not infect them (1
). Since robust HCV infection of DCs has not been documented either in vivo (49
) or in vitro (17
), it is likely that the cross-presentation of HCV antigens represents an important mechanism for the induction of antiviral CD8+
T-cell responses. This hypothesis is further supported by our data clearly demonstrating that productive infection of DCs is not required for efficient HCV antigen presentation. This observation extends previous findings for human immunodeficiency virus (HIV). DCs efficiently cross-present HIV antigens captured from both live and apoptotic infected CD4+
T cells, whereas HIV presentation after direct infection of DC was not detectable even with a high amount of replicative virus (42
). Since HCV does not replicate efficiently in DCs (49
), it is likely that the acquisition of HCV antigens for cross-presentation by SR-BI might be a critical point for the development of an early immune response at the early stages of HCV infection. However, the development of a strong T-cell immunity is restricted to antigen-capturing DCs which have been exposed to a stimulus that leads to their maturation. We have previously demonstrated that HCV-LPs induce a small but significant upregulation of the costimulatory molecules CD80 and CD83 (5
). In this study, HCV-LP-pulsed DCs were stimulated with CD40L overnight to ensure sufficient DC maturation. In vivo studies suggest that CD40 is provided by NK lymphoctes in an early DC-NK lymphocyte interaction (21
). Since this interaction likely takes place at the site of infection and in secondary lymphoid organs, the maturation of HCV-LP-pulsed DCs by CD40L could reflect the scenario for antigen presentation in an acute HCV infection.
HCV-LP cross-presentation was markedly inhibited in the presence of anti-SR-BI, suggesting that SR-BI is involved in the trafficking of viral antigens toward the MHC class I pathway. This finding suggests that SR-BI may act as an immunoreceptor facilitating the intracellular accumulation of viral antigens and triggering processing and cross-presentation. This hypothesis is further supported by recent data demonstrating that SR-BI mediates bacterial adhesion and cytosolic accumulation (60
). Moreover, other members of the growing SR family, SR-A and LOX-1, have been shown to be involved in the uptake and trafficking of exogenous antigens toward the MHC class I pathway (18
). Since the anti-SR-BI antibody used in this study may also target the large extracellular loop of SR-BII, we cannot exclude a role for SR-BII in viral antigen uptake and cross-presentation.
Interestingly, HCV-LP cross-presentation could not be completely inhibited by anti-SR-BI, suggesting that additional receptors are involved in targeting HCV-LPs into the MHC class I pathway. Recent studies have shown that the initiation of HCV infection is dependent on a cooperativity between SR-BI and CD81 (31
). In contrast to the findings for HCVcc infection, CD81 did not appear to play a major role in HCV-LP binding and cross-presentation in DCs. These data suggest that SR-BI is the main HCV capture receptor on DCs, while a cooperative action of SR-BI and CD81 is required for efficient HCV infection of hepatocytes. Furthermore, these data illustrate the difference in HCV entry pathways in hepatocytes and DCs. In hepatocytes, HCV enters by clathrin-mediated endocytosis, followed by an HCV envelope membrane fusion process for the delivery of the HCV genome into the cytosol (3
). In contrast, classical MHC class I presentation requires the transfer of the exogenous antigens from the endosome or phagosome into the cytosol, where the antigens are degraded by proteasomes into oligopeptides. The peptides are then transported by the transporter associated with antigen processing into the endoplasmic reticulum and are bound to MHC class I molecules. In an alternative pathway, peptides may be generated within the endocytotic compartment and the resulting peptides are then bound to recycling MHC class I molecules (1
). Further studies analyzing the molecular mechanisms of HCV-LP processing and presentation are in progress. Preliminary studies demonstrated that lactacystin, a highly specific inhibitor of proteasomal antigen processing, did not inhibit HCV-LP cross-presentation (Fig. ). These results may indicate that alternative MHC class I processing and presentation pathways could be involved in HCV-LP cross-presentation or that additional, as-yet-unidentified cytosolic proteases downstream of the proteasome could participate in HCV-LP processing and presentation. Interestingly, several viral epitopes have been identified that are produced or presented more efficiently when proteasome activity is impaired or altered, including viral epitopes from influenza virus (39
) and HIV (14
). Studies are under way to analyze these mechanisms in detail.
In this study, we used an HCV-LP-based model system to assess the molecular mechanisms of HCV particle uptake and presentation by human DCs (5
). HCV-LPs are generated by self-assembly of HCV structural proteins in insect cells (7
) and are characterized by morphological, biophysical, and antigenic properties similar to those of infectious virions (22
). Furthermore, the binding and uptake of HCV-LPs to target cells appear to require a set of viral epitopes and cellular host factors similar to that required by infectious HCV (2
). Although we cannot exclude the possibility that the virus-like particle concentration in our in vitro experiments may exceed the concentration of circulating infectious viral particles interacting with DCs in vivo, studies in animal models, including mice and chimpanzees, have shown that HCV-LPs used in amounts as in this study are appropriate for HCV-LP uptake and presentation by DCs in vivo. Indeed, in vivo studies have demonstrated that HCV-LPs induce a strong antiviral humoral and cellular immune response, including HCV-specific T-helper cells and cytotoxic T lymphocytes, in primates, including chimpanzees (30
). The quantity and quality of HCV-LP-induced cellular immune responses against the HCV structural proteins appear to be similar to the immune responses induced by the infectious virus (30
). Moreover, HCV-LP-induced T-cell responses result in control of HCV infection in the chimpanzee in vivo (20
). These findings and the successful use of virus-like particles of other viruses, including HIV (11
), hepatitis B virus (56
), papillomavirus (36
), and parvovirus (44
), for the study of virus uptake and antigen presentation in DCs indicate that the interaction of HCV-LPs with DCs represents an appropriate model system to study the molecular mechanisms of HCV particle uptake and presentation of HCV structural proteins.
To confirm the validity of the HCV-LP model system, as well as the role of SR-BI for HCV uptake into DCs, we produced high-titer, gradient-purified HCVcc and studied HCVcc uptake by using anti-E2-specific immunofluorescence and confocal LSM. Using this method and purified anti-SR-BI IgG, recently shown to inhibit HCVcc infection of hepatoma cells (69
), we demonstrate that anti-SR-BI IgG specifically inhibits the uptake of HCVcc into DCs (Fig. ). These findings demonstrate the relevance of the HCV-LP model system for the study of HCV particle uptake and confirm the specificity of the anti SR-BI serum used for the study of HCV-DC interaction.
In conclusion, we have demonstrated that SR-BI mediates HCV-LP and HCVcc uptake into human DCs, indicating that SR-BI may represent a cell-surface receptor for the recognition of viral antigens. The inhibition of HCV-LP cross-presentation by anti-SR-BI antibody suggests that SR-BI is implicated in trafficking exogenous viral antigens toward the MHC class I presentation pathway. Taken together, these findings support a novel function of SR-Bs for viral antigen uptake and recognition. In addition, the SR-BI-viral antigen interaction may represent a novel target for therapeutic or preventive strategies aiming at the induction of efficient antiviral immune responses.