Multivesicular late endosomes – MHC class II-enriched compartments in intestinal epithelial cells and origin of MHC class II-loaded exosomes

Background and aims: Intestinal epithelial cells (IEC) are suggested to present antigens to CD4+ T cells and were shown to release A33-positive, MHC class II/peptide-loaded exosomes with immunogenic properties. Dependent on inflammatory stimuli antigen presentation by IEC might be tolerogenic or pro-inflammatory. Here we investigated the subcellular compartments within IEC involved in antigen processing and class II peptide loading. Methods: Ovalbumin (OVA) was luminally injected into jejunal or ileal loops of TnfΔ(ARE) mice which spontaneously develop a Crohn's like ileitis, and C57/129SvEv controls. Specimens were taken from OVA-incubated loops at different times up to two hours. MHC class II, invariant chain (Ii), A33 and OVA were subcellularly visualised using fluorescence and cryo electron microscopy. Tissue levels of IL-12 and IFN-gamma were assessed using quantitative real-time RT-PCR. Results: MHC class II and Ii expression was found in the ileal epithelium of Tnf and control mice, but not detected in the jejunal epithelium. In ileal IEC of Tnf and control mice, the majority of MHC class II and Ii was localised in multivesicular late endosomes (MVLE). OVA accumulated in these MVLE one hour after injection. OVA targeting to MVLE was also seen in jejunal IEC, lacking MHC class II and Ii. Internal vesicles of MVLE were labeled for A33, and A33-positive exosomes were identified in intercellular spaces of the epithelium. Compared to control jejunum and ileum as well as Tnf jejunum, the inflamed ileum of Tnf mice showed significantly increased levels of IL-12 and IFN-gamma. Conclusions: Our in vivo findings indicate that a certain subset of late endosomes, the MVLE, might be crucially involved in class II-associated antigen processing and peptide loading within IEC. A33 labeling on internal vesicles of MVLE further suggests that these compartments are most likely the origin of MHC class II/peptide-loaded exosomes released from IEC. OVA targeting and accumulation in MVLE seems to be a uniform process within the small bowel and independent of mucosal inflammation. Thus, further studies need to address modulations of antigen handling within MVLE which might be responsible for differences in the IEC's capacity of antigen presentation to CD4+ T cells.