Defined breast milk EV subsets boost the immune response and skew the T-cell balance towards a regulatory phenotype

Abstract

Introduction: In the past years it has become clear that cell-derived extracellular vesicles (EV) are present in human breast milk and that these EV can play a role in the instruction of the immune system. Since breast milk impacts the development of the neonatal immune system by conveying environmental and maternal information to the child, we investigated the EV composition of human breast milk and evaluated the potential of these EV to modulate immune responses. Methods: For efficient and reliable recovery of naturally occurring EV from human breast milk, we applied a recently developed protocol, based on differential centrifugation and density gradient separation. Isolated EV were characterized by western blotting, EM, high-resolution flow cytometry, and lipidomics. For functional analysis of breast milk EV, removal of density gradient medium by column filtration was essential to avoid gradient medium-induced side-effects. EV-induced modulation of immune responses were analyzed in a T-cell stimulation assay with PBMC by profiling T-cell activation and cytokine release. Results: EV subsets were identified that differed in protein composition, light scattering, size and lipid composition. Furthermore, breast milk EV were also found to be highly enriched for several immune modulatory molecules, such as MHC class II, HSC/ HSP-70, MFG-E8, butyrophilin 1A1 and MUC-1. Addition of breast milk EV to a T-cell stimulation assay revealed that milk EV skew T cells towards a regulatory phenotype, while boosting the release of the pro- and anti-inflammatory cytokines, such as IFN-g, TNF-a, IL-17, IL-10 and IL-6. Summary/conclusion: These data indicate that breast milk EV have a broad potential to steer immune responses, and could be transporters of (antigen-specific) immune information. As such, these EV may be involved in inducing immunogenicity, as well as oral tolerance in the neonate via the gastrointestinal tract.