Background: Rheumatoid Arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures, where we previously showed accumulation of Epstein Barr virus (EBV)-infected B-cells. RA synovial fibroblasts (RA-SFs) can sustain B-cells activation and maturation. In more recent work, we showed that RA-SFs activate Interferon type I signalling (IFN-I) upon B-cells co-culture and preferentially support EBV+B-cells clones expansion.

Objectives: 1) to characterize B-cells after RA-SFs co-culture 2) to dissect the molecular mechanisms behind B-cells-SFs crosstalk.

Methods: RA B-cells were co-cultured with RA-SFs for 28 days and then characterized through flowcytometry for CD58 and CD23 expression. STING (stimulator of interferon genes) activation/expression in B cells were evaluated through RT-PCR, immunoblot and immunocytofluorescence. RA synovium was characterized by immunofluorescence. CRISPR-Cas9 was employed to generate stable STING-KO EBV+CD58/CD23+ B-cells.

Results: After RA-SFs co-culture, B-cells were organized in two subpopulations: CD58+/CD23 ^high and CD58+/CD23 ^low cells, previously described in EBV-infected B-cells. EBV+CD58/CD23+ B-cells express STING, physiologically absent in B-cells. The analysis of STING alternative RNA splicing isoforms revealed the expression of IFN-I inactive variants in B-cells, while EBV+CD58/CD23+ B-cells express the fully active protein. STING activation in EBV+CD58/CD23+ B-cells promotes the phosphorylation/activation of TBK1 and IRF3, and subsequent INFα and IFNβ production. CD58/CD23+ B-cells are expanded in RA synovium, showing STING expression and different levels of CD23 among aggregates. STING activation promotes the accumulation of CD58+/CD23 ^low cells. STING-KO EBV+CD58/CD23+ B-cells confirms reduced percentages of CD58+/CD23 ^low at basal level and upon STING activation.

Conclusion: We demonstrated that RA-SFs are able to preferentially induce the selection/proliferation of EBV+CD58/CD23+ B-cells, with dysregulation of alternative RNA splicing mechanisms, promoting the expression of STING. These cells can be identified in the synovium of RA patient. STING expression and activation in EBV+CD58/CD23+ B-cells not only regulate IFN-I cytokines production, but are involved in CD23 and CD58 expression and in the organization of two functional subpopulations.

REFERENCES: [1] 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.

[2] 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.

[3] Megyola C et al. Identification of a sub-population of B cells that proliferates after infection with epstein-barr virus. Virol J 2011.

Acknowledgements: NIL.

Disclosure of Interests: None declared.