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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.

Toapanta FR, Bernal PJ, Sztein MB - Front Cell Infect Microbiol (2012)

Bottom Line: This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels, and enhanced susceptibility to phosphatase inhibition.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for Vaccine Development, University of Maryland Baltimore, MD, USA.

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.

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Phosphatases in Um B cells are more susceptible to inhibition by H2O2. PBMC were treated with 6 mM of H2O2 and phosphorylation of proteins associated with the BCR were measured in naïve and Um B cells (A and B) after 5 min of stimulation at 37°C. Displayed are overlaps of unstimulated (1% BSA) (red) and stimulated (H2O2 without anti-IgM) (purple) naïve B cells (A). Similar data display was used for Um B cells (B). Shown are percentages (red letter/numbers = negative controls; purple letter/numbers = H2O2 stimulation) as well as fold changes in mean fluorescence intensity (MFI) of stimulated compared to unstimulated cells. Um cells showed enhanced phosphorylation of Syk and p38MAPK (B), compared to naïve B cells (A). Akt phosphorylation was similar in both cell populations (A and B).
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Figure 6: Phosphatases in Um B cells are more susceptible to inhibition by H2O2. PBMC were treated with 6 mM of H2O2 and phosphorylation of proteins associated with the BCR were measured in naïve and Um B cells (A and B) after 5 min of stimulation at 37°C. Displayed are overlaps of unstimulated (1% BSA) (red) and stimulated (H2O2 without anti-IgM) (purple) naïve B cells (A). Similar data display was used for Um B cells (B). Shown are percentages (red letter/numbers = negative controls; purple letter/numbers = H2O2 stimulation) as well as fold changes in mean fluorescence intensity (MFI) of stimulated compared to unstimulated cells. Um cells showed enhanced phosphorylation of Syk and p38MAPK (B), compared to naïve B cells (A). Akt phosphorylation was similar in both cell populations (A and B).

Mentions: Initiation of antigen receptor signaling not only requires activation of kinases; most importantly, it requires inhibition of phosphatases (e.g., Phospho-Tyrosine-Phosphatases), which have 100–1000-times higher turnover rate than kinases. H2O2 is a universal phosphatase inhibitor, which at high doses induces phosphorylation of proteins associated with the BCR-signaling pathway mimicking antigen stimulation (Reth, 2002; Singh et al., 2005; Tonks, 2005). To determine if phosphatases of naïve and Um B cells were equally susceptible to inhibition by H2O2 and if inhibition of these proteins contributed to the enhanced phosphorylation detected in Um B cells, PBMC were treated with 6 mM H2O2 (in the absence of anti-IgM) or 1% BSA (negative control of stimulation) (5 min; 37°C). This dose of H2O2 was chosen based on previous reports from Irish et al. (2006) and our own titration experiments (not shown), which demonstrated that doses lower than 5 mM did not inhibit phosphatases to the point of inducing spontaneous phosphorylation of proteins associated with the BCR. Phosphorylation of Syk, Akt, and p38MAPK was subsequently evaluated and detected only in cells stimulated with H2O2 (Figures 6A,B). Syk and p38MAPK phosphorylation in Um was more intense than in naïve B cells (percentage and fold changes over unstimulated controls) (Figures 6A,B). Interestingly, phosphorylation of Akt was similar in both populations.


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.

Toapanta FR, Bernal PJ, Sztein MB - Front Cell Infect Microbiol (2012)

Phosphatases in Um B cells are more susceptible to inhibition by H2O2. PBMC were treated with 6 mM of H2O2 and phosphorylation of proteins associated with the BCR were measured in naïve and Um B cells (A and B) after 5 min of stimulation at 37°C. Displayed are overlaps of unstimulated (1% BSA) (red) and stimulated (H2O2 without anti-IgM) (purple) naïve B cells (A). Similar data display was used for Um B cells (B). Shown are percentages (red letter/numbers = negative controls; purple letter/numbers = H2O2 stimulation) as well as fold changes in mean fluorescence intensity (MFI) of stimulated compared to unstimulated cells. Um cells showed enhanced phosphorylation of Syk and p38MAPK (B), compared to naïve B cells (A). Akt phosphorylation was similar in both cell populations (A and B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473368&req=5

Figure 6: Phosphatases in Um B cells are more susceptible to inhibition by H2O2. PBMC were treated with 6 mM of H2O2 and phosphorylation of proteins associated with the BCR were measured in naïve and Um B cells (A and B) after 5 min of stimulation at 37°C. Displayed are overlaps of unstimulated (1% BSA) (red) and stimulated (H2O2 without anti-IgM) (purple) naïve B cells (A). Similar data display was used for Um B cells (B). Shown are percentages (red letter/numbers = negative controls; purple letter/numbers = H2O2 stimulation) as well as fold changes in mean fluorescence intensity (MFI) of stimulated compared to unstimulated cells. Um cells showed enhanced phosphorylation of Syk and p38MAPK (B), compared to naïve B cells (A). Akt phosphorylation was similar in both cell populations (A and B).
Mentions: Initiation of antigen receptor signaling not only requires activation of kinases; most importantly, it requires inhibition of phosphatases (e.g., Phospho-Tyrosine-Phosphatases), which have 100–1000-times higher turnover rate than kinases. H2O2 is a universal phosphatase inhibitor, which at high doses induces phosphorylation of proteins associated with the BCR-signaling pathway mimicking antigen stimulation (Reth, 2002; Singh et al., 2005; Tonks, 2005). To determine if phosphatases of naïve and Um B cells were equally susceptible to inhibition by H2O2 and if inhibition of these proteins contributed to the enhanced phosphorylation detected in Um B cells, PBMC were treated with 6 mM H2O2 (in the absence of anti-IgM) or 1% BSA (negative control of stimulation) (5 min; 37°C). This dose of H2O2 was chosen based on previous reports from Irish et al. (2006) and our own titration experiments (not shown), which demonstrated that doses lower than 5 mM did not inhibit phosphatases to the point of inducing spontaneous phosphorylation of proteins associated with the BCR. Phosphorylation of Syk, Akt, and p38MAPK was subsequently evaluated and detected only in cells stimulated with H2O2 (Figures 6A,B). Syk and p38MAPK phosphorylation in Um was more intense than in naïve B cells (percentage and fold changes over unstimulated controls) (Figures 6A,B). Interestingly, phosphorylation of Akt was similar in both populations.

Bottom Line: This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels, and enhanced susceptibility to phosphatase inhibition.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for Vaccine Development, University of Maryland Baltimore, MD, USA.

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.

Show MeSH
Related in: MedlinePlus