Processing and presentation of exogenous antigen by dendritic cells

Dendritic cells (DC) play a key role in the immune system. In this thesis, we have investigated and characterized pathways for the processing and presentation of exogenous OVA on MHC-I ("cross-presentation") and how these were influenced by the purine nucleoside Adenosine (Ado), an immune regulator that accumulates to high concentrations at sites of inflammation and tumor growth. In addition, we established a method to detect the binding of glycolipids containing tumor-associated carbohydrate antigens (TACAs) to conserved CD 1 1d molecules expressed on viable mouse cells, including DC. Immature (iDC) and activated (aDC), but to a lesser degree resting DC (rDC), were found to express a fully functional, highly efficient TAP-independent vesicular MHC-I processing pathway (VP). In this VP, OVA-derived SIINFEKL/Kb complexes were formed through peptide exchange in recycling Kb molecules. Such complexes were also formed at the cell surface by peptide regurgitation. This high efficient VP could be restored in rDC by cellular activation using TLR ligands, such as CpG (binding to TLR- and LPS (binding to TLR-4) and also by the mere mechanical disruption of cells, the latter occurring without the development of an activated phenotype and the secretion of IL-12 in cells. Reactivation of the high activity VP in rDC was not dependent on type 1 interferon or IL-12 but required the adaptor protein MyD88. We also confirmed that the cysteine protease Cathepsin S (Cat S) was required for the formation of the SIINFEKL/Kb complexes in the VP. In addition, we found that Ado had a strong inhibitory effect on the low degree VP seen in rDC and that this effect was mediated by the A, Ado receptor and involved the retention of intracellular SIINFEKL/Kb complexes. Four newly synthesized TACAs containing glycolipids were studied for the binding to CD1d on viable cells by the use of specific anti-TACA mAbs and flow cytometry. By this procedure specific binding could be visualized and quantitated. A method like this might be useful for the controlled loading of glycolipid antigens into DC and the possible use of such cells for immunotherapy