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Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist‐induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract. [ABSTRACT FROM AUTHOR]

Summary Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8 + T cells in L. monocytogenes -infected mice. While reacting to the spliced epitopes, these CD8 + T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8 + T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design. [ABSTRACT FROM AUTHOR]

Regulation of the proteasome system, which is responsible for the generation of most MHC class I-bound peptides, occurs through the interaction of the 20S proteasome with several regulatory proreins. One of these is PI31, which acts in vitro as an inhibitor of proteasome activity. Here, we demonstrate that, rather than inhibiting proteasome function, PI31 acts as a selective modulator of the proteasome-mediated steps in MHC class I antigen processing. Overexpression of PI31 in mouse embryonic cells has no impact on proteasome-mediated proteolysis. Instead, PI31, which localizes at the nuclear envelope/endoplasmic reticulum membrane, selectively interferes with the maturation of immunoproteasome precursor complexes. Consequently, overexpression of PI31 abrogates MHC class I presentation of an immunoproteasome-dependent cytotoxic T lymphocyte epitope and reduces the surface MHC class I levels on IFN-γ-treated mouse embryonic cells. Thus, PI31 represents a cellular regulator of proteasome formation and of proteasome-mediated antigen processing. [ABSTRACT FROM AUTHOR]