Generation of a B Cell Model to Investigate the Function of FcRL4 in the Context of RA

Background: FcRL4 is an inhibitory receptor primarily expressed on memory B cells located in mucosal associated lymphoid tissue and in the case of RA, synovial fluid (SF). FcRL4 defines a RANKL-producing B cell, can act as a low affinity IgA receptor and captures IgA IC in inflamed tissue. The objectives of my thesis are to investigate consequences of IgA binding on signal transduction and antigen presentation. To investigate this, a B cell model is useful as cell numbers in SF are relatively low and samples are finite. Inducing continuous expression of FcRL4 in B cell lines leads to growth inhibition so I have designed a model using the Tet-On system, that induces expression of FcRL4 upon doxycycline treatment.

Methods: The FcRL4 gene was cloned and inserted into the Tet-On lentiviral vector to generate a doxycycline-inducible vector. The Priess B cell line was transfected and selected using puromycin. FcRL4 expression was induced with doxycycline, cultured overnight and FcRL4+ cells were isolated. FcRL4+ cells were cultured overnight with a citrullinated enolase peptide and HLA-DR4+ T cell hybridoma. Supernatants were collected and IL-2 production measured as a readout for antigen presentation.

Results: Transfected Priess cells were able to stably express FcRL4 and its expression had no effect on expression of other co-stimulatory molecules including CD80, CD86 and HLA-DR. Co-culture with the citrullinated enolase peptide resulted in similar levels of IL-2 production by T cells compared to FcRL4- Priess. We are now investigating whether FcRL4 & IgA IC can be internalised using pHrodo avidin and detected in internal compartments.

Conclusion: The TetOn lentiviral vector system is suitable for expression of FcRL4 and investigating antigen presentation of a citrullinated peptide.