P052 Effects of hypoxia and activated human B cells on immunosuppressive and differentiation capacity of multipotent mesenchymal stromal cells

Introduction Multipotent mesenchymal stromal cells (MSCs) are a promising approach for the treatment of chronic inflammatory autoimmune diseases. However, little is known about whether their immunomodulatory, replicative and regenerative potential is influenced by the pathogenic environment. Objectives Activated lymphocytes and hypoxia are part of the cellular environment in chronic inflammatory diseases. The aim of the study was to investigate the influence of activated B cells and hypoxia on MSCs functions. Methods Bone marrow MSCs from healthy donors were cultured for various time periods under normoxia or hypoxia (3% O2) in presence or absence of B cells from healthy donors activated via B cell receptor cross-linking and IL-2. Alternatively, cell culture supernatant from B cells activated for three days with respective stimuli was used. Cell division and expression of surface markers were assessed by flow cytometry, cytokine secretion by ELISA and gene expression by qRT-PCR. For differentiation, MSCs were cultured for 21 days in established differentiation media in presence or absence of B cell culture supernatant. Osteogenesis and adipogenesis were assessed by fluorescence microscopy and quantified with ImageJ software. Results Significant increase of MSC IL-6 and IL-8 production over more than 100-fold in coculture with B cells revealed activation of MSCs by B cells both under normoxic and hypoxic conditions. In contrast, B cell specific TNF-α production was reduced by more than half, demonstrating suppression of B cell activation by activated MSCs in return. This was observed both in cocultures with direct contact and indirect contact upon separation by cell culture inserts, suggesting soluble factors were sufficient for observed effects. Both hypoxic conditions and coculture with MSCs significantly inhibited B cell division, without increasing apoptosis. B cell CD25 expression significantly decreased in cocultures, but remained higher under hypoxia. B cell supernatant increased MSC proliferation rate and delayed entry into senescence and this effect was more pronounced under hypoxia. Of note, B cell-derived factors increased MSC-specific expression of hypoxia induced transcription factor HIF-1α under normoxic conditions. B cell derived factors also supported osteogenic differentiation and inhibited adipogenesis both under normoxia and hypoxia. Conclusions These results suggest an important influence of the inflammatory environment on MSCs functions: In presence of B cells MSCs exert immunosuppressive properties, but also secrete high amounts of proinflammatory cytokines. In addition, the replicative potential of MSCs increases, which is further supported by hypoxia. In presence of activated B cells, MSCs differentiation is governed towards the osteogenic lineage and this effect is stable both under normoxia and hypoxia. Disclosure of interest None declared