Contributions
Abstract: P575
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
Background: The role of B cells as key mediators of inflammatory processes in multiple sclerosis (MS) has been increasingly recognized in the recent years. This notion was substantiated by the recent success of pan B cell depletion by anti-CD20 monoclonal antibodies. This however, not only targets pathogenic B cells but can also affect regulatory B cell properties. An alternative strategy may be the therapeutic abrogation of pro-inflammatory B cell functions by Bruton´s tyrosine kinase (BTK) inhibition. BTK is centrally involved in B cell receptor signaling and subsequent activation and differentiation of B cells. BTK inhibition (BTKi) could thereby be a promising strategy to control pathogenic function, such as antigen presentation and release of pro-inflammatory cytokines, while leaving regulatory B cell properties unaffected.
Methods: Daily oral treatment in C57Bl/6 mice with the BTK inhibitor evobrutinib (M2951) at three doses or vehicle control started 7 days prior to immunization. Mice were immunized with conformational MOG 1-117 protein, a model of experimental autoimmune encephalomyelitis (EAE) in which B cells can directly recognize the immunogen. EAE severity was assessed daily using a standard scale (0-5). Histopathology was performed at day 60 after immunization. Flow cytometry of activation markers on B cells, T cells and myeloid cells as well as analysis of the B cell phenotype was performed at day 12 after immunization. Intra-cellular calcium flux analysis was performed in vitro or after 3 days of BTKi treatment ex vivo using Fluo-3 and Fura Red calcium-sensitive dyes and anti-IgM B cell receptor (BCR) stimulation.
Results: The intermediate and highest dose of BTKi showed an amelioration of EAE severity throughout the 60-day observation period. Supporting these findings, BTKi reduced CNS inflammation and demyelination. BTKi treatment led to an accumulation of naïve B cells with a corresponding reduction of antigen-activated B cells. The expression of activation markers CD80, CD86, CD69 and MHCII was significantly reduced on B cells treated with evobrutinib. B cell receptor stimulation lead to reduced calcium influx in BTKi treated B cells in vitro and ex vivo.
Conclusion: BTKi reduces the influx of excitatory calcium in B cells upon BCR stimulation preventing their activation and conversion from naïve to antigen-activated B cells. This translates into reduced CNS inflammation and clinical amelioration in a B cell-mediated EAE model.
Disclosure: Sebastian Torke: Nothing to disclose; Roland Grenningloh: Employee of EMD Serono; Ursula Boschert: Employee of EMD Serono; Martin S. Weber: Editor for PLoS One. Received travel funding and/or speaker honoraria from Biogen-Idec, EMD Serono, Novartis, Roche and Bayer. Receives research support from the National Multiple Sclerosis Society (NMSS; PP1660), the Deutsche Foreschungsgemeinschaft (DFG; WE 3547/4-1), from Novartis, EMD Serono, TEVA, Biogen-Idec, Roche and the ProFutura Programm of the Universitätsmedizin Göttingen
Abstract: P575
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
Background: The role of B cells as key mediators of inflammatory processes in multiple sclerosis (MS) has been increasingly recognized in the recent years. This notion was substantiated by the recent success of pan B cell depletion by anti-CD20 monoclonal antibodies. This however, not only targets pathogenic B cells but can also affect regulatory B cell properties. An alternative strategy may be the therapeutic abrogation of pro-inflammatory B cell functions by Bruton´s tyrosine kinase (BTK) inhibition. BTK is centrally involved in B cell receptor signaling and subsequent activation and differentiation of B cells. BTK inhibition (BTKi) could thereby be a promising strategy to control pathogenic function, such as antigen presentation and release of pro-inflammatory cytokines, while leaving regulatory B cell properties unaffected.
Methods: Daily oral treatment in C57Bl/6 mice with the BTK inhibitor evobrutinib (M2951) at three doses or vehicle control started 7 days prior to immunization. Mice were immunized with conformational MOG 1-117 protein, a model of experimental autoimmune encephalomyelitis (EAE) in which B cells can directly recognize the immunogen. EAE severity was assessed daily using a standard scale (0-5). Histopathology was performed at day 60 after immunization. Flow cytometry of activation markers on B cells, T cells and myeloid cells as well as analysis of the B cell phenotype was performed at day 12 after immunization. Intra-cellular calcium flux analysis was performed in vitro or after 3 days of BTKi treatment ex vivo using Fluo-3 and Fura Red calcium-sensitive dyes and anti-IgM B cell receptor (BCR) stimulation.
Results: The intermediate and highest dose of BTKi showed an amelioration of EAE severity throughout the 60-day observation period. Supporting these findings, BTKi reduced CNS inflammation and demyelination. BTKi treatment led to an accumulation of naïve B cells with a corresponding reduction of antigen-activated B cells. The expression of activation markers CD80, CD86, CD69 and MHCII was significantly reduced on B cells treated with evobrutinib. B cell receptor stimulation lead to reduced calcium influx in BTKi treated B cells in vitro and ex vivo.
Conclusion: BTKi reduces the influx of excitatory calcium in B cells upon BCR stimulation preventing their activation and conversion from naïve to antigen-activated B cells. This translates into reduced CNS inflammation and clinical amelioration in a B cell-mediated EAE model.
Disclosure: Sebastian Torke: Nothing to disclose; Roland Grenningloh: Employee of EMD Serono; Ursula Boschert: Employee of EMD Serono; Martin S. Weber: Editor for PLoS One. Received travel funding and/or speaker honoraria from Biogen-Idec, EMD Serono, Novartis, Roche and Bayer. Receives research support from the National Multiple Sclerosis Society (NMSS; PP1660), the Deutsche Foreschungsgemeinschaft (DFG; WE 3547/4-1), from Novartis, EMD Serono, TEVA, Biogen-Idec, Roche and the ProFutura Programm of the Universitätsmedizin Göttingen