Induction of immune responses after targeting antigens to DC via Fc receptors
We have obtained data from an analysis of the uptake, conservation and cross-presentation of the model antigen OVA after Fc receptor-mediated uptake by DC and from an analysis of the immunological consequences of the antigen depot. We found that MHC class I presentation is relatively short-lived in contrast to MHC class II. However, CD8 cross-priming capacity of OVA-loaded DC was functionally retained for many days, while peptide-pulsed DC had lost their priming capacity after 24 hours. Strikingly, OVA protein antigen was conserved intracellularly in DC for many days. Antigen-derived peptide presentation was efficiently restored after peptide elution from MHC class I at the cell surface, providing a strong association between intracellular antigen depot and the prolonged cross-priming potency of DC. Characterization of the storage compartments shows that these resemble lysosome-like structures, distinct from MHC class II loading compartments. We speculate that DC have adapted an endogenous compartment facilitating prolonged MHC class I presentation from exogenous antigen for effective CD8 T cell cross-priming. We have further analyzed the immunological consequences of the antigen depot. T cells that were induced by dendritic cells several days after a short antigen pulse had full proliferative capacity and could kill tumors in vivo. Furthermore, we studies the mechanism of cross-presentation of antigen from the antigen depot. By using inhibitors for the proteasome, we observed that the proteasome is crucial in the presentation of the antigen in MHC class I. By using TAP deficient cells, we found that TAP is also necessary for this process. We further characterized the antigen depot by confocal microscopy and found that the antigen co-localized with the lysosomal marker LAMP1, but not with the endosomal marker EEA1, TAP, MHC class I or MHC class II. We conclude that the antigen depot is a storage compartment and not a loading compartment. We have started to examine depot formation by targeting antigen to other receptors. For example, we have found that presentation of TLR ligand- long peptide conjugates was also long-lived, while presentation of OVA, targeted via the mannose receptor, was not long-lived. We will address the requirement of antigen depot formation in dendritic cells for effective tumor elimination, by pulse-loading experiments of short and long peptides, whole protein immune complexes and TLRL-conjugated peptides. Our goal is to better understand the molecular requirements for antigen depot formation in dendritic cells. To accomplish this, we will further characterize the antigen storage compartment by proteomics. In addition, we want to address the mechanism of processing and presentation in MHC class I and MHC class II from the antigen depot in more detail. Furthermore, we plan to address the question why antigen is not degraded as would be expected in compartments with lysosomal characteristics.
Dendritic cells in cancer immunotherapy.
Major histocompatibility complex class II
Major histocompatibility complex class I
Prophylactic immunization with DCs loaded with Ag-IgG immune complexes leads to efficient induction of tumor protection in mice