Classical MHC class I peptide presentation of a bacterial fusion protein by bone marrow-derived dendritic cells

Dendritic cells (DC) expanded in the presence of GM-CSF from the bone marrow of C57BL/6 mice process Gram-negative bacteria expressing the model antigen Crl-OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257 – 264) processed by DC co-incubated with E. coli expressing Crl-OVA, which contains the K^b-binding OVA(257 – 264) epitope, occurs by a cytosolic MHC-I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1^−/− mice co-incubated with E. coli expressing Crl-OVA did not result in K^b presentation of OVA(257 – 264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257 – 264) on K^b when C57BL/6 DC phagocytosed and processed E. coli expressing Crl-OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257 – 264) processed from bacteria expressing Crl-OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257 – 264) on neighboring bystander K^b-expressing antigen-presenting cells after BALB/c (H-2^d) DC phagocytosed E. coli expressing Crl-OVA could not be detected. Together, these data support a cytosolic MHC-I presentation pathway for OVA(257 – 264) processed from E. coli expressing Crl-OVA by bone marrow-derived DC.