Relapse-free patients with rectal cancer of working age are at risk of disability pension.

An alternative endolysosomal pathway has recently been suggested for the processing of MHC-I-binding peptides, and peptide/MHC-I complexes have been demonstrated in this compartment. However, it remains unclear where in the antigen-presenting cells such peptides are processed, in the endolysosomes themselves or in the proteasomal complex. Here, we have investigated this using monoclonal antibodies specific for the immunodominant SIINFEKL/Kb complex (25-D1) or for the carbohydrate part of Db-or Kb-binding glycopeptides in combination with inhibitors for classical and endolysosomal MHC-Iprocessing pathways. Alternative processing was detected in both wt and TAP1 -/-immature DC (iDC) as the expression of SIINFEKL/Kb complexes on the surface of OVA-treated cells in the presence of Brefeldin A (BFA) or lactacystin and their absence in the presence of the lysosomotropic amines ammonium chloride, chloroquine and methylamine. Internalized Db-and Kb-binding glycopeptides, detected with high specificity using an anti-galabiose (Gal2) monoclonal antibody, were found to appear on the cell surface of BFAtreated cells after intracellular MHC-I-binding. Peptide exchange in Kb was demonstrated as the gradual appearance of SIINFEKL/Kb complexes on BFAtreated cells which earlier had been saturated with another Kb-binding peptide. Our data support the presence of a fully functional endolysosomal processing pathway in iDC guided by the chaperone function of MHC-I molecules.

Cross‐presentation of soluble protein antigens on major histocompatibility complex (MHC) class I by dendritic cells (DC) can occur in vesicular, endolysosomal compartments and be either dependent or independent of TAP peptide transporters. Here we investigate if an immunostimulatory CpG oligodeoxynucleotide can increase the activity in a TAP‐independent endolysosomal vesicular pathway (el‐VP) in vivo as we have earlier found in in vitro cultured DC. We use the in vivo response of CFSE labelled OT‐1 T cells, transgenic for a T‐cell receptor (TCR) that recognizes an ovalbumin (OVA)‐derived peptide (SIINFEKL) presented by H‐2Kb, transferred into TAP1−/− mice, as a functional read‐out for activity in the el‐VP. We have found a poor OT‐1 T‐cell response to soluble OVA which, however, could be strongly enhanced by the simultaneous administration of CpG. This increased responsiveness required both the endolysosomal cathepsin S (CatS) and Toll like receptor (TLR)9, the CpG receptor, both of which are present in the el‐VP. Confocal microscopy demonstrated a co‐localization of H‐2Kb/SIINFEKL and the endolysosomal marker LAMP1 in CD11c positive DC which was markedly increased by CpG administration. No complexes were found in the ER and cis‐Golgi compartments in TAP1−/− mice, indicating the lack of classical MHC‐I processing. In DC isolated from CatS−/− mice the opposite was found, complexes were present in the ER but not in the el‐VP. We conclude that in vivo activation of TLR9 by CpG increases the efficiency of TAP independent el‐VP and that this might contribute to the potent adjuvant activity of this type of compound. The cellular mechanisms remain to be established.