Stimulation of naïve B cells with a fusion protein consisting of FlaA and Bet v 1 induces regulatory B cells ex vivo.

ABSTRACT

BACKGROUND: The experimental fusion protein rFlaABetv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaABetv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaABetv1 on naïve murine B cells. METHODS: Immune modulating capacity of rFlaABetv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaABetv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaABetv1-stimulated B cells was tested in mDCCD4+ T cellB cell triple cultures. RESULTS: Upon in vivo application of rFlaABetv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaABetv1. rFlaABetv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaABetv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaABetv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaABetv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. CONCLUSION: rFlaABetv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.