P035 The transcriptional co-activator bob.1 prevents terminal differentiation and induces costimulatory capacity of B cells in GC-like environment
Authors: MJ Levels, CM Fehres, NOP Van Uden, AQ Bakker, H Spits, DL Baeten, NG Yeremenko
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Introduction</h3> Recently we identified the transcriptional co-activator BOB.1 as specifically overexpressed in RA synovium, where its levels strongly correlated with the presence of germinal centres (GCs). In accordance with human data, mice lacking functional BOB.1 failed to mount GC response and were resistant to the experimental model of RA. <h3>Objectives</h3> In this study we investigated whether increased levels of BOB.1 impact the phenotype and function of B cells in a GC-like environment. <h3>Methods</h3> Expression levels of BOB.1 were assessed by qPCR and immunofluorescence. DNA encoding BOB.1 was introduced into B cells by retroviral-mediated gene transfer. The phenotype, activation and BCR signalling were analysed by FACS. Expression of CCL3 was measured by qPCR. Impact of high levels of BOB.1 in B cells on proliferative responses of autologous T<sub>FH</sub> cells was assessed by FACS after 5 days of co-culture with SEB-pulsed B cells. <h3>Results</h3> Immunofluorescence staining of inflamed RA synovium demonstrated the presence of high numbers of BOB.1-positive cells clustered in the lymphoid follicles in a pattern similar to the normal tonsillar tissue. Further analysis revealed that expression of BOB.1 in RA synovial B cells was significantly higher than in tonsillar B cells. To investigate how high levels of BOB.1 impact phenotype and function of B cells in a GC-like environment we overexpressed BOB.1 in human primary B cells cultured with CD40L and IL-21. In these conditions B cells rapidly differentiate into antibody-producing plasma cells, a process that is accompanied by a decrease in expression of CD20 and increase in CD38 and CD27. In contrast to the control, cells transduced with BOB.1 retained CD20 expression while expressed intermediate levels of CD27 and CD38. Accordingly, the percentage of plasmablasts was significantly lower in BOB.1-overexpressing cells, confirming that high levels of BOB.1 suppress plasma cell differentiation. Further analysis revealed that BOB.1-transduced memory B cells expressed high levels of costimulatory receptors CD40, CD80 and PD-L2 involved in B-T cell interactions and showed more rapid Ca<sup>2+</sup>mobilisation and increased production of T-cell-recruiting chemokine CCL3 following BCR stimulation. Accordingly, T<sub>FH</sub> cells co-cultured with BOB.1-transduced B cells exhibit a higher rate of proliferation as compared to T<sub>FH</sub> cells co-cultured with B cells transduced with the control retrovirus. <h3>Conclusions</h3> These data suggest that increased levels of BOB.1 in B cells during T cell-dependent responses in GCs suppress their terminal differentiation and enhance expression of costimulatory molecules and BCR signalling strength, which is translated into their greater help to T<sub>FH</sub> cells. Whether this is sufficient to drive/accelerate autoimmune disease will be evaluated in a B cell-specific BOB.1 tg mouse model. <h3>Disclosure of interest</h3> None declared