Scientific background A functionally important subset of B cells, known as regulatory B cells (Breg), are specialised to control immune response and immunological diseases such as autoimmunity. Breg cells are best characterised by their inducible production of IL-10 and can appear in different phenotypic forms. Various activation signals, including Toll-like receptors (TLRs), B cell antigen receptor (BCR) or CD40 stimulation can trigger the regulatory transformation of B cells, although it is not clear which of these signals are the most effective and which B cells have the most susceptible phenotype.

We have studied the effect of the BCR and TLR9 stimulation on three populations of B cells in mouse spleen (Transitional-2-Marginal Zone Precursors (T2-MZP), Marginal Zone (MZ) and Follicular (FO) B cells) to induce differentiation into Bregs.

Materials and methods We set up in vitro cultures using sorted B cells from T2-MZP, MZ or FO pools from the spleen of adult mice and stimulated them via the BCR and/or TLR9. The transformation to Breg was monitored by following IL-10 production using gene expression analysis, IL-10-specific ELISA or intracellular IL-10 staining measured by FACS.

Results Based on our experimental set up, we found that the BCR signal alone was not sufficient to induce IL-10 production, while TLR9 stimulation mediated the regulatory transformation of all B cell types with different extent. Comparing the three populations, we found that B cells from the marginal zone responded more efficiently for TLR9 stimulation. We detected the highest level of secreted IL-10 and the highest number of induced IL-10 positive cells among MZ B cells. The results at protein level were confirmed by gene expression analysis, additionally, at mRNA level a time-dependent synergistic crosstalk through the BCR and TLR9 was observed.

Conclusion We concluded that MZ B cells were the most inducible B cell population and TLR9 signal alone was efficient to trigger IL-10 production. Using the most sensitive B cell population with the efficient stimulus will help us to study the signalling characteristics of Bregs and their important role in autoimmune diseases.

This project was supported by OTKA NK 104846 and by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences.