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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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Related in: MedlinePlus

Time course of phosphorylation of pSyk in Sm CD27+ and Sm CD27− B cells following anti-IgA and anti-IgG stimulation. PBMC from healthy volunteers were stimulated with either anti-IgA or anti-IgG and phosphorylation of Syk was evaluated at different time points (1, 2, 5, 8, 10, 15, 20, and 30 min) in Sm CD27+ and Sm CD27− B cell populations (A). Samples were fluorescently barcoded for multiplexing. Displayed are detailed histograms of pSyk following anti-IgA (B) and -IgG (C) at each time point.
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FA2: Time course of phosphorylation of pSyk in Sm CD27+ and Sm CD27− B cells following anti-IgA and anti-IgG stimulation. PBMC from healthy volunteers were stimulated with either anti-IgA or anti-IgG and phosphorylation of Syk was evaluated at different time points (1, 2, 5, 8, 10, 15, 20, and 30 min) in Sm CD27+ and Sm CD27− B cell populations (A). Samples were fluorescently barcoded for multiplexing. Displayed are detailed histograms of pSyk following anti-IgA (B) and -IgG (C) at each time point.

Mentions: Anti-IgA and anti-IgG stimulated PBMC were evaluated for phosphorylation of proteins associated with the BCR (Figures 7, A2) in Sm CD27+ and Sm CD27− B cells. Syk phosphorylation was similar between Sm CD27+ and CD27− B cells following stimulation with anti-IgA (Figure 7A—left panel and Figure A2B); the phosphorylation peak was detected between 5–10 minutes. Btk phosphorylation induced by anti-IgA increased slowly in Sm CD27+ and Sm CD27− cells, showing a late peak at 15 min. A quicker dephosphorylation in Sm CD27− B cells was recorded (Figure 7A—center panel). Anti-IgA induced slightly higher phosphorylation of Akt in Sm CD27+ cells than Sm CD27− cells. However, the phosphorylation/dephosphorylation patterns were similar in both populations (Figure 7B—right panel). In contrast, anti-IgG stimulation induced similar Syk, Btk, and Akt phosphorylation intensity and kinetics in both Sm CD27+ and Sm CD27− B cells (Figure 7B and Figure A2C). Phosphorylation peaks of Syk and Btk were observed at 5 min (Figure 7B—left and center panels), while Akt showed a biphasic phosphorylation peak (5 and 15 min) (Figure 7B—right panel). Phosphorylation of p38MAPK and Erk1/2 were also evaluated in Sm CD27+ and Sm CD27− B cells following anti-IgA and -IgG stimulation; however, no phosphorylation was detected in our assay. Finally, inhibition of phosphatases in Sm CD27+ and Sm CD27− B cells was also evaluated using 6 mM H2O2 as described above. The results showed that Akt and p38MAPK were similarly phosphorylated (Figures 8A,B). Although slight differences were detected in Syk phosphorylation (Figures 8A,B, left panels) these changes were not as intense as those detected between naïve and Um B cells (Figures 6A,B, left panels).


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)

Time course of phosphorylation of pSyk in Sm CD27+ and Sm CD27− B cells following anti-IgA and anti-IgG stimulation. PBMC from healthy volunteers were stimulated with either anti-IgA or anti-IgG and phosphorylation of Syk was evaluated at different time points (1, 2, 5, 8, 10, 15, 20, and 30 min) in Sm CD27+ and Sm CD27− B cell populations (A). Samples were fluorescently barcoded for multiplexing. Displayed are detailed histograms of pSyk following anti-IgA (B) and -IgG (C) at each time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

FA2: Time course of phosphorylation of pSyk in Sm CD27+ and Sm CD27− B cells following anti-IgA and anti-IgG stimulation. PBMC from healthy volunteers were stimulated with either anti-IgA or anti-IgG and phosphorylation of Syk was evaluated at different time points (1, 2, 5, 8, 10, 15, 20, and 30 min) in Sm CD27+ and Sm CD27− B cell populations (A). Samples were fluorescently barcoded for multiplexing. Displayed are detailed histograms of pSyk following anti-IgA (B) and -IgG (C) at each time point.
Mentions: Anti-IgA and anti-IgG stimulated PBMC were evaluated for phosphorylation of proteins associated with the BCR (Figures 7, A2) in Sm CD27+ and Sm CD27− B cells. Syk phosphorylation was similar between Sm CD27+ and CD27− B cells following stimulation with anti-IgA (Figure 7A—left panel and Figure A2B); the phosphorylation peak was detected between 5–10 minutes. Btk phosphorylation induced by anti-IgA increased slowly in Sm CD27+ and Sm CD27− cells, showing a late peak at 15 min. A quicker dephosphorylation in Sm CD27− B cells was recorded (Figure 7A—center panel). Anti-IgA induced slightly higher phosphorylation of Akt in Sm CD27+ cells than Sm CD27− cells. However, the phosphorylation/dephosphorylation patterns were similar in both populations (Figure 7B—right panel). In contrast, anti-IgG stimulation induced similar Syk, Btk, and Akt phosphorylation intensity and kinetics in both Sm CD27+ and Sm CD27− B cells (Figure 7B and Figure A2C). Phosphorylation peaks of Syk and Btk were observed at 5 min (Figure 7B—left and center panels), while Akt showed a biphasic phosphorylation peak (5 and 15 min) (Figure 7B—right panel). Phosphorylation of p38MAPK and Erk1/2 were also evaluated in Sm CD27+ and Sm CD27− B cells following anti-IgA and -IgG stimulation; however, no phosphorylation was detected in our assay. Finally, inhibition of phosphatases in Sm CD27+ and Sm CD27− B cells was also evaluated using 6 mM H2O2 as described above. The results showed that Akt and p38MAPK were similarly phosphorylated (Figures 8A,B). Although slight differences were detected in Syk phosphorylation (Figures 8A,B, left panels) these changes were not as intense as those detected between naïve and Um B cells (Figures 6A,B, left panels).

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