M. Caliste, E. Prediletto, E. Corsiero, L. Jagemann, C. Pitzalis, M. Bombardieri
2022 Annals of the Rheumatic Diseases  
BackgroundRheumatoid Arthritis (RA) is characterized by the formation of ectopic lymphoid structures (ELS) in the synovial tissue, which can promote B cells activation and local production of autoantibodies. B cells exert an essential role in RA immunopathogenesis, as demonstrated by therapeutical effects of Rituximab (1). We previously showed that ELS in the RA joints frequently accumulate Epstein Barr virus (EBV)-infected B cells displaying evidence of both latent (LMP2A) and early lytic
more » ... reactivation in locally differentiated plasma cells (PCs)(2). RA synovial fibroblasts (SFs) can sustain B cells activation, proliferation and maturation into high affinity antibodies producing cells, mimicking B cells physiological differentiation in germinal centres (3). Whether RASFs can also promote preferential selection of naturally-occurring EBV+ B cells is currently unknown.ObjectivesHere, we aim to a) demonstrate SFs role in EBV+ B cells selection b) phenotypically characterize B cells after co-culture with SFs c) dissect the molecular mechanisms behind the B cells SFs crosstalk.MethodsLong-term in vitro B cells SFs co-cultures have been established, followed by phenotypical characterization of B cells in flowcytometry. Supernatant were then screened by ELISA at different timepoints, to measure IgG, IgM and IgA production. EBV infection status on B cells were analysed by qRT-PCR after gDNA extraction. Single cells RNA sequencing was finally performed at 28 days of co-culture.ResultsPreliminary results confirmed RASFs role in sustaining B cells activation and maturation, showing B cells survival up to 90 days, production of IgG and an increased IgG/IgM ratio overtime. Interestingly, we identified a particular B cells phenotype occurring in long term in vitro co-cultures, characterized by CD38 expression and the subdivision into two functional subsets, CD58+/CD23high and CD58+/CD23low. These two subpopulations - previously described by Megyola et al. in in vitro EBV infected B cells - are characterized by two different functional states: an highly proliferating (CD58+/CD23high) population and an IL-6 producers (CD58+/CD23low) one(4). We also observed that RASFs preferentially support EBV+ clones expansion, showing a preferential expression of EBV markers in CD58+/CD23high cells. The high proliferation rate of these B cells allowed – on a specific experiment - the establishment of a cell line, named "Carejavi", that we are currently employing as tool for functional investigation of RASFs primed EBV+ B-cells. Finally, the transcriptomic analysis revealed the selection of a relatively small number of clonotype at the VDJ analysis at the end of co-culture. In addition, we observed the upregulation of genes related to GC formation (such as EBI3, LTA and LTB), B cells proliferation (mki67) and viral oncogenic transformation (MYC).ConclusionHere, we demonstrated that RA SFs not only support B cells maturation and activation in local autoantibodies producing cells, but they are also able to preferentially induce selection and proliferation of EBV+ clones, characterized by a peculiar expression of CD58 and CD23. The molecular mechanisms behind this phenomenon are currently under investigation.References[1]Cohen SB et al. Rituximab for rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: Results of a multicenter, randomized, double-blind, placebo-controlled, phase III trial evaluating primary efficacy and safety at twenty-four weeks. Arthritis Rheum 2006[2]Croia C, et al. Epstein-Barr virus persistence and infection of autoreactive plasma cells in synovial lymphoid structures in rheumatoid arthritis. Ann Rheum Dis 2013.[3]Bombardieri M, et al. A BAFF/APRIL-dependent TLR3-stimulated pathway enhances the capacity of rheumatoid synovial fibroblasts to induce AID expression and Ig class-switching in B cells. Ann Rheum Dis 2011.[4]Megyola C et al. Identification of a sub-population of B cells that proliferates after infection with epstein-barr virus. Virol J 2011Disclosure of InterestsNone declared.
doi:10.1136/annrheumdis-2022-eular.4387 fatcat:sm5y5btrajanhl5kisv6lfi4ze