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197 Cross-Presentation of HIV-1 Vaccinal Lipopeptides by Human Dendritic Cells
M. Andrieu*1, J. F. Desoutter 1, E. Loing2, D. Hanau3, J. G. Guillet1, and A. Hosmalin1
1Inst. Cochin de Génétique Moléculaire, Paris, France; 2SEDAC-Therapeutics, Lille, France; and 3ETS, Strasbourg, France
Background: Lipopeptides are currently used in vaccine trials. An efficient vaccine against HIV must induce good primary T-cell responses and, for this, must be presented by dendritic cells (DC) as antigen presenting cells. We have shown that a model lipopeptide containing an HLA-A*0201-restricted short epitopic peptide from HIV-1 (Pol476-484) was endocytosed and presented in association with MHC class I molecules by human DC. To determine more precisely this cross-presentation pathway, we have used a longer lipopeptide (Pol461-484), which requires processing to be presented.
Methods: DC were differentiated from peripheral blood monocytes using GM-CSF and IL-4. Pol 461-484 lipopeptide was synthesized by terminal addition of a N-epsilon-palmytoyl-lysine to the HIV-1 Pol461-484 sequence. For confocal microscopy studies, histidine 482 was substituted of by an N-epsilon-rhodamine-lysine. For functional studies, DC were incubated overnight with lipopeptide alone or in the presence of inhibitors before being added to anti-Pol476-484 CD8+ T-cell lines. Surface CD8 expression and intracellular IFN-gamma production were measured by flow cytometry analysis.
Results: Colocalization of the rhodamine-labeled lipopeptide and FITC-Dextran by confocal microscopy showed that the Pol461-484 lipopeptide was rapidly endocytosed into immature DC. Intracellular cytokines assays showed that lipopeptide-pulsed DC stimulated Pol476-484-specific CD8+ T cells. Lipopeptide presentation was not inhibited by monensin, which impairs lysosomal enzymatic degradation by inhibiting endosomal acidification. Conversely, it was inhibited by brefeldin A and by a proteasome-specific inhibitor.
Conclusions: Therefore, the cross-presentation pathway followed by this long lipopeptide in human DC was endocytosis, then egress to the cytoplasm and proteasomal degradation before association with MHC class I molecules.
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