Background

B cells are a crucial part of the adaptive immune system and play an important role in protection against pathogens. B cells can sense pathogens by innate receptors such as Toll-like receptors (TLRs). The most important receptor for the development and activation of B cells, however, is the B-cell antigen receptor (BCR). The genes encoding the immunoglobulin heavy and light chains that comprise the BCR are assembled during B cell development in the bone marrow through a stepwise process called V(D)J recombination (Tonegawa, 1983). The functionality and autoreactivity of the BCR are checked during three crucial checkpoints in B cell development: (i) productive heavy chain rearrangement is monitored at the pre-B stage by surface deposition of the heavy chain protein together with pre-existing surrogate light chain as the pre-BCR complex; (ii) productive light chain recombination enables surface expression of the BCR, which is checked for auto-reactivity at the immature B cell stage and (iii) upon bone marrow egress when transitional B cell progress to mature, naive B cells in peripheral lymphoid tissues. When expression of (pre-)BCR signaling molecules is altered, B cell development in the bone marrow is severely affected (Martensson et al., 2010; Pieper et al., 2013). Balanced BCR signaling is also crucial for appropriate selection, survival and activation of mature B cells and its distortion is a key factor in the development of autoimmune disease or leukemia (Nishizumi et al., 1995; Kil et al., 2012; Rip et al., 2018).

Upon BCR engagement by antigen, Src family kinases phosphorylate the intracellular tyrosine-based activation motif (ITAM) of CD79a (Ig-α) and CD79b (Ig-β), which are the transmembrane proteins that form a complex with the BCR, as well the cytoplasmic tail of co-receptor CD19. Phosphorylation of the CD79a/b ITAM in turn activates spleen tyrosine kinase (SYK), which subsequently activates the SH2 domain-containing leukocyte protein of 65 kDa (SLP-65) linker protein. Simultaneously, phosphorylated CD19 will activate phosphoinositide 3-kinase (PI3K), which in turn phosphorylates and converts phosphatidylinositol (4,5)-bisphosphate (PIP₂) to phosphatidylinositol (3,4,5)-triphosphate (PIP₃). Signaling molecules containing a pleckstrin-homology domain, such as Bruton’s tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2), are recruited to the cell membrane by the newly formed PIP₃ (Saito et al., 2001). After BTK recruitment to the cell membrane, SLP-65 mediates SYK-dependent activation of BTK, which in turn will phosphorylate PLCγ2 (Wang et al., 2000). The activation of this signaling complex eventually results in activation of protein kinase B (PKB/AKT) signaling (Craxton et al., 1999), NF-κB translocation to the cell nucleus (Bajpai et al., 2000), calcium mobilization (Fluckiger et al., 1998) and activation of the mitogen-activated protein kinase (MAPK) pathway (Jiang et al., 1998). The complex containing BTK, SLP-65, PLCγ2 together with other PI3K-derived signals is essential for B cell survival, proliferation and differentiation. BCR signal transduction is regulated by the equilibrium of kinase activity and negative regulation of signaling through recruitment of tyrosine phosphatases including SHP-1 (Franks and Cambier, 2018).

Western blotting used to be the method of choice for detection and quantification of phosphorylation of the BCR signaling molecules described above. The main drawbacks of Western blotting are the large number of purified cells that are required to perform the analysis and that quantification is not sensitive enough to detect subtle differences between samples. Phosphoflow is a flow-cytometry based technique that allows for quantification of multiple subpopulations within a single sample without prior purification and requires relatively few cells. In addition, phosphoflow cytometry is less time-consuming compared to Western blotting. Although for the quantification of protein phosphorylation phosphoflow appeared to be superior to Western blotting, the fixation and permeabilization procedure of phosphoflow often affected cellar epitopes or features of the monoclonal antibodies (mAbs) that were required to identify the immune cell population of interest. Due to recent advances in phosphoflow procedures, it is now possible to combine the analysis of phosphorylation of signaling proteins with a comprehensive staining for B cell subpopulations. In addition, more and more mAbs become available for phosphoflow analysis.

Here, we present our step-by-step phosphoflow protocol that is complementary to our original research paper (Rip et al., 2020). The phosphoflow protocol allows for sensitive detection of phosphorylation of numerous BCR signaling molecules in human and murine B cell subpopulations. Next to a description of phosphoflow targets as published in the original paper (Rip et al., 2020), we also present additional BCR signaling targets that can be sensitively quantified using this phosphoflow protocol.