Abstract 456 Monitoring B cell activation by flow cytometric detection of phosphorylated Bruton’s Tyrosine Kinase (BTK)

Rationale: Atherosclerosis and myocardial infarction are driven by autoimmune responses that involves B cells activation and autoantibodies generation. Bruton’s tyrosine kinase (BTK) is a critical downstream mediator of B cell receptor (BCR) signalling, which leads to B cell activation, proliferation and differentiation. Here, we developed an assay to detect BTK phosphorylation in humans and mice by flow cytometry to monitor B cell activation. Human antigen-specific memory B cells were also detected by an ELISPOT assay.

Methods and results: Human PBMCs were stimulated with F(ab')2 anti-human IgG, IgM (10 μg/mL) at several time points to evaluate the dynamics of BTK phosphorylation at two phosphorylation sites, Tyr551 and Tyr223. The specific BTK inhibitor Ibrutinib was used at 0.1 nM to 10 µM to prevent BTK phosphorylation. Phosphorylation of BTK was assessed with phosphor-specific antibodies by intracellular flow cytometry. The mean fluorescence intensity (MFI) for Tyr223 and Tyr551 phosphorylation rose by up to 3.1- and 4.6-fold at 2 and 3.5 min after stimulation, respectively, and reached a plateau until 10 min. At 3.5 min, Ibrutinib (100 nM) inhibited both, Tyr551 and Tyr223 phosphorylation, by almost 50%. The Phosphorylation levels are different among different B cell subsets. Human PBMCs were pre-stimulated and then seeded in anti-IgG antibody and COVID S-protein pre-coated 96-well-plate. Of 77 memory B cells, 8 were COVID S-protein specific. In negatively isolated B cells from mice splenocytes, Tyr551 phosphorylation was also detected after F(ab')2 anti-mouse IgM (10 μg/mL) stimulation. Phosphorylation levels were significantly increased (p<0.05) in transitional 1 and 2, marginal zone, follicular, and plasma B cells, but not in memory B cells.

Conclusion: The detection of BTK phosphorylation upon B cell activation by flow cytometry provide a valuable method for monitoring B cell function in humans and mice. Identification of antigen-specific memory B cells by ELISPOT is a tool to screen for possible auto-antigens for B cells.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Monitoring B cell activation by flow cytometric detection of phosphorylated Bruton’s Tyrosine Kinase (BTK)
X. Li¹, T. Marchini², D. Wolf³
¹Innere Medizin III, Kardiologie und Angiologie, Albert- Ludwigs-Universität Freiburg, Freiburg im Breisgau; ²Klinik für Kardiologie und Angiologie I, Universitäts-Herzzentrum Freiburg - Bad Krozingen GmbH, Freiburg im Breisgau; ³Klinik für Kardiologie und Angiologie I, Universitäts-Herzzentrum Freiburg - Bad Krozingen, Freiburg im Breisgau;
Rationale: Atherosclerosis and myocardial infarction are driven by autoimmune responses that involves B cells activation and autoantibodies generation. Bruton’s tyrosine kinase (BTK) is a critical downstream mediator of B cell receptor (BCR) signalling, which leads to B cell activation, proliferation and differentiation. Here, we developed an assay to detect BTK phosphorylation in humans and mice by flow cytometry to monitor B cell activation. Human antigen-specific memory B cells were also detected by an ELISPOT assay. Methods and results: Human PBMCs were stimulated with F(ab')2 anti-human IgG, IgM (10 μg/mL) at several time points to evaluate the dynamics of BTK phosphorylation at two phosphorylation sites, Tyr551 and Tyr223. The specific BTK inhibitor Ibrutinib was used at 0.1 nM to 10 µM to prevent BTK phosphorylation. Phosphorylation of BTK was assessed with phosphor-specific antibodies by intracellular flow cytometry. The mean fluorescence intensity (MFI) for Tyr223 and Tyr551 phosphorylation rose by up to 3.1- and 4.6-fold at 2 and 3.5 min after stimulation, respectively, and reached a plateau until 10 min. At 3.5 min, Ibrutinib (100 nM) inhibited both, Tyr551 and Tyr223 phosphorylation, by almost 50%. The Phosphorylation levels are different among different B cell subsets. Human PBMCs were pre-stimulated and then seeded in anti-IgG antibody and COVID S-protein pre-coated 96-well-plate. Of 77 memory B cells, 8 were COVID S-protein specific. In negatively isolated B cells from mice splenocytes, Tyr551 phosphorylation was also detected after F(ab')2 anti-mouse IgM (10 μg/mL) stimulation. Phosphorylation levels were significantly increased (p<0.05) in transitional 1 and 2, marginal zone, follicular, and plasma B cells, but not in memory B cells. Conclusion: The detection of BTK phosphorylation upon B cell activation by flow cytometry provide a valuable method for monitoring B cell function in humans and mice. Identification of antigen-specific memory B cells by ELISPOT is a tool to screen for possible auto-antigens for B cells.
https://dgk.org/kongress_programme/jt2023/aP2242.html