Limitations of EBV transformed human Raji B cells as a model for measuring canonical NF-κB activation

Autoimmune diseases, such as systemic lupus erythematosus (SLE), are underscored by dysregulated B cell function including the production of autoantibodies, skewed population ratios, and aberrant signaling. Given that the family of nuclear factor kappa B (NF-κB) transcription factors govern responses to stimuli, survival, differentiation, and so forth understanding the intricate regulatory network of NF-κB in B cell biology is paramount for unraveling treatments for B cell-linked autoimmune diseases. Here, we focus on a negative regulator of NF-κB signaling, A20 (TNFAIP3), that deactivates NF-κB transcription factor translocation through the ubiquitination and deubiquitination of target proteins. Haploinsufficiency in A20 results in an autoimmune phenotype and mutations to A20 have been associated with SLE, suggesting implications to B cell function. To investigate the role of A20 in NF-κB in human B cells, we generated a TNFAIP3 knockout (KO) Raji cell line. Cells were stimulated with either anti-IgM or Resiquimod (R848) to activate distinct NF-κB signaling pathways. Using qRT-PCR, western blotting, and flow cytometry, we assessed differences in gene expression, protein production, and NF-κB activation. We observed key limitations in using Epstein-Barr virus transformed B cell lines to model inducible NF-κB signaling.