Endolysosomal processing of exogenous antigen into major histocompatibility complex class I-binding peptides.
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-I-processing 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 BFA-treated cells after intracellular MHC-I-binding. Peptide exchange in Kb was demonstrated as the gradual appearance of SIINFEKL/Kb complexes on BFA-treated 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.
Chen
L
L
Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden.
Jondal
M
M
D000818:Animals; D017951:Antigen Presentation / Q000276:immunology*; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D004527:Egg Proteins / Q000276:immunology; D011992:Endosomes / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D008247:Lysosomes / Q000276:immunology*; D010047:Ovalbumin / Q000276:immunology; D010446:Peptide Fragments
D004527:Egg Proteins; D015395:Histocompatibility Antigens Class I; C110366:OVA-8; D010446:Peptide Fragments; D010047:Ovalbumin