Diverse phosphorylation patterns of B cell receptor-associated signaling in naïve and memory human B cells revealed by phosphoflow, a powerful technique to study signaling at the single cell level

Abstract

Following interaction with cognate antigens, B cells undergo cell activation, proliferation, and differentiation. Ligation of the B cell receptor (BCR) leads to the phosphorylation of BCR-associated signaling proteins within minutes of antigen binding, a process with profound consequences for the fate of the cells and development of effector immunity. Phosphoflow allows a rapid evaluation of various signaling pathways in complex heterogenous cell subsets. This novel technique was used in combination with multi-chromatic flow cytometry (FC) and fluorescent-cell barcoding (FCB) to study phosphorylation of BCR-associated signaling pathways in naïve and memory human B cell subsets. Proteins of the initiation (Syk), propagation (Btk, Akt), and integration (p38MAPK and Erk1/2) signaling units were studied. Switched memory (Sm) CD27+ and Sm CD27− phosphorylation patterns were similar when stimulated with anti-IgA or -IgG. In contrast, naïve and unswitched memory (Um) cells showed significant differences following IgM stimulation. Enhanced phosphorylation of Syk was observed in Um cells, suggesting a lower activation threshold. This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels, and enhanced susceptibility to phosphatase inhibition. All other signaling proteins evaluated also showed some degree of enhanced phosphorylation in Um cells. Furthermore, both the phospholipase C-γ2 (PLC-γ2) and phosphatidylinositol 3-kinase (PI3K) pathways were activated in Um cells, while only the PI3K pathway was activated on naïve cells. Um cells were the only ones that activated signaling pathways when stimulated with fluorescently labeled S. Typhi and S. pneumoniae. Finally, simultaneous evaluation of signaling proteins at the single cell level (multiphosphorylated cells) revealed that interaction with gram positive and negative bacteria resulted in complex and diverse signaling patterns. Phosphoflow holds great potential to accelerate vaccine development by identifying signaling profiles in good/poor responders.

Increased BCR responsiveness in B cells from patients with chronic GVHD

“…21 We found similar results in our antigen-experienced CD27 1 B cells also express IgM (44-49%) with no significant differences between patient phenotypes found ( Figure 4). …”

“…21 Intracellular staining was performed according to the manufacturer's instructions (BD Biosciences). Cells were fixed (BD cytofix buffer), permeabilized (BD Perm Buffer III), and stained using the following antibodies from BD Biosciences: BLNK (clone 2B11) or Syk (clone 4D10).…”

Enhanced Tyrosine Phosphatase Activity Underlies Dysregulated B Cell Receptor Signaling and Promotes Survival of Human Lupus B Cells

“…Since it has been shown that naive and memory B cells have different BCR activation thresholds , further analyses were performed using conventional CD27− naive and CD27+ memory B cells. Of note, the recently published memory‐like CD27−Syk++ B cell subset was excluded from the CD27− naive B cell compartment and analyzed separately (Supplementary Figure , online at http://onlinelibrary.wiley.com/doi/10.1002/art.39559/abstract).…”

Naïve and memory B cells exhibit distinct biochemical responses following BCR engagement

“…IgM + memory B cells express approximately twice as many BCRs on their surface than naïve B cells (Figure 5a). 12 , 13 , 26 , 55 To test the possibility that the significant differences in Ca 2+ mobilization between naïve B cells and IgM + memory B cells (Figures 3c) resulted from differences in the amounts of surface IgM, naïve and IgM + memory B cells were stimulated with different concentrations of F(ab’) 2 anti‐Igμ and Ca 2+ mobilization was then tracked. The level of Ca 2+ mobilization increased in a dose‐dependent manner in both naïve and IgM + memory B cells.…”

“…Many studies have utilized immortalized B‐cell lines from different species, 4 , 31 , 39 primary B cells from transgenic mice 37 , 46 , 51 or syngeneic or heterologous overexpression systems 39 , 57 . More recently, some studies have addressed BCR signaling events in primary human B cells 40 , 47 , 55 . These have revealed the complexities of BCR signal transduction and documented the influence of the Ig isotype on these processes with respect to the kinetics and magnitude of the response 4 , 32 , 33 , 41 .…”