Etudes du ciblage intracellulaire des molecules non classiques du complexe majeur d'histocompatibilite de classe II.

摘要

HLA-DM and HLA-DO are non-classical MHC class II molecules that modulate the loading of antigenic peptides on molecules such as HLA-DR in B-cells. HLA-DM catalyzes the peptide exchange and HLA-DO influences the peptide repertoire by negatively modulating the activity of HLA-DM. Peptide exchange occurs in class II-rich compartments of the endocytic pathway where they both accumulate. These compartments include multivesicular bodies in which HLA-DR and -DM interact at the level of the internal membranes. Although the targeting of HLA-DO to endosomal compartments is attributed to sorting information within DM cytoplasmic tail, we have investigated the presence of consensus sorting signals within the cytoplasmic domains of HLA-DO. HLA-DR/DO chimeric and mutagenic molecules were transfected into HeLa cells. Our results show the presence of a functional sorting motif within the beta chain. Interestingly, these results suggested that DO can modify DM sorting within endosomal compartments. This hypothesis was later demonstrated by others and prompted us to study the regulation of MVB/exosomal sorting of MHC molecules.;Mature peptide-loaded HLA-DR molecules end-up at the cell surface but also accumulate in exosomal vesicles. These small structures are shed presumably through the fusion of multivesicular bodies with the plasma membrane and are obtained by high speed centrifugation of the culture medium. Despite their co-localization with HLA-DR in MHC II-rich compartments, the presence of HLA-DM in exosomes remains to be demonstrated. In the second part of this work, we show by immunoblot that DM is present in exosomal fractions of the 721.45 B lymphoblastoid cell line. A similar conclusion was reached when looking at exosomes from HeLa cells transfected with CIITA. To gain insight into the intracellular mechanisms regulating exosome entry and cargo retention, HLA-DM was transfected into HeLa cells. Interestingly, in these conditions, DM was excluded from exosomes. However, it was found in exosomes when its YXXL sorting motif was inactivated by site-directed mutagenesis or following co-expression of HLA-DR. Altogether, these results demonstrate that sorting signals in the cytoplasmic domains of transmembrane proteins can regulate exosome entry and raise the intriguing possibility that protein-protein interactions can modulate the activity of such signals.