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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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Time course of signaling proteins and pathways associated with the BCR in naïve and Um B cells. Two multicolor panels were used to explore simultaneous phosphorylation of signaling proteins and pathways associated with the BCR at various time points. The data shown is from one representative volunteer and is presented as mean fluorescence intensity (MFI). Um demonstrated enhanced phosphorylation potential for all the phosphoproteins assayed compared to naïve B cells (A to E) following anti-IgM stimulation. Interestingly, Um and naïve B cells demonstrated phosphorylation of Syk, Btk, and Akt (A,B, and C), which suggest that the PI3K pathway is activated in both cell populations. However, p38MAPK and pERK1/2 (D and E) are phosphorylated only in the Um subset, suggesting that the PLC-γ2 pathway is also activated in these cells. Additionally, different phosphoproteins have different kinetics. For example, the peak of Syk phosphorylation was detected at 5 min after stimulation (A), and then slowly declined; meanwhile, Akt, p38MAPK, and Erk1/2 showed a biphasic behavior (C,D, and E), which is also evident, but less prominent in Btk (B).
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Figure 5: Time course of signaling proteins and pathways associated with the BCR in naïve and Um B cells. Two multicolor panels were used to explore simultaneous phosphorylation of signaling proteins and pathways associated with the BCR at various time points. The data shown is from one representative volunteer and is presented as mean fluorescence intensity (MFI). Um demonstrated enhanced phosphorylation potential for all the phosphoproteins assayed compared to naïve B cells (A to E) following anti-IgM stimulation. Interestingly, Um and naïve B cells demonstrated phosphorylation of Syk, Btk, and Akt (A,B, and C), which suggest that the PI3K pathway is activated in both cell populations. However, p38MAPK and pERK1/2 (D and E) are phosphorylated only in the Um subset, suggesting that the PLC-γ2 pathway is also activated in these cells. Additionally, different phosphoproteins have different kinetics. For example, the peak of Syk phosphorylation was detected at 5 min after stimulation (A), and then slowly declined; meanwhile, Akt, p38MAPK, and Erk1/2 showed a biphasic behavior (C,D, and E), which is also evident, but less prominent in Btk (B).

Mentions: Anti-IgM stimulated PBMC (similar time points as described before) were assayed for phosphorylation of proteins associated with the BCR signaling pathways (Syk-Y352, Btk-Y551, Akt-S473, p38MAPK-T180/Y182, and Erk1/2-T202/Y204) in two separate panels. These proteins allow the simultaneous evaluation of the initiation (Syk), propagation (Btk, Akt), and integration signaling units (p38MAPK-T180/Y182 and Erk1/2-T202/Y204). Furthermore, the selected proteins can also provide some information regarding whether the PLCγ2, PI3K, or both BCR-associated signaling pathways are activated. All evaluated proteins showed enhanced phosphorylation in Um B cells when compared to naïve B cells (Figures 5A–E). As described above, Syk, which represents a protein from the initiation signaling subunit, showed higher phosphorylation in Um than in naïve B cells (Figure 5A). Interestingly, the phosphorylation peak was observed at 5 min, followed by a slow dephosphorylation slope (Figures 4B, 5A). Among proteins of the integration subunit, Btk and Akt were also evaluated. Naïve and Um cells showed an early peak of Btk phosphorylation (Y551) 1–2 min after stimulation, followed by a plateau established around minute 10 which persisted until the last time point collected (30 min). Akt was phosphorylated in naïve and Um cells; however, it was more prominent in the latter cells (Figure 5C). Interestingly, two phosphorylation peaks were identified, one between minutes 5–8 and a second one at 15–20 min. Following the second peak, pAkt-S473 levels returned to the resting state in both B cell populations. Among the proteins of the integration signaling subunit, p38MAPK (T180/Y182) and Erk1/2 (T202/Y204) were evaluated (Figures 5D,E). Phosphorylation of both proteins was identified in Um, but not in naive B cells. Furthermore, phosphorylation of these proteins also showed a biphasic phase with peaks at 2–3 and 8–10 min.


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 signaling proteins and pathways associated with the BCR in naïve and Um B cells. Two multicolor panels were used to explore simultaneous phosphorylation of signaling proteins and pathways associated with the BCR at various time points. The data shown is from one representative volunteer and is presented as mean fluorescence intensity (MFI). Um demonstrated enhanced phosphorylation potential for all the phosphoproteins assayed compared to naïve B cells (A to E) following anti-IgM stimulation. Interestingly, Um and naïve B cells demonstrated phosphorylation of Syk, Btk, and Akt (A,B, and C), which suggest that the PI3K pathway is activated in both cell populations. However, p38MAPK and pERK1/2 (D and E) are phosphorylated only in the Um subset, suggesting that the PLC-γ2 pathway is also activated in these cells. Additionally, different phosphoproteins have different kinetics. For example, the peak of Syk phosphorylation was detected at 5 min after stimulation (A), and then slowly declined; meanwhile, Akt, p38MAPK, and Erk1/2 showed a biphasic behavior (C,D, and E), which is also evident, but less prominent in Btk (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 5: Time course of signaling proteins and pathways associated with the BCR in naïve and Um B cells. Two multicolor panels were used to explore simultaneous phosphorylation of signaling proteins and pathways associated with the BCR at various time points. The data shown is from one representative volunteer and is presented as mean fluorescence intensity (MFI). Um demonstrated enhanced phosphorylation potential for all the phosphoproteins assayed compared to naïve B cells (A to E) following anti-IgM stimulation. Interestingly, Um and naïve B cells demonstrated phosphorylation of Syk, Btk, and Akt (A,B, and C), which suggest that the PI3K pathway is activated in both cell populations. However, p38MAPK and pERK1/2 (D and E) are phosphorylated only in the Um subset, suggesting that the PLC-γ2 pathway is also activated in these cells. Additionally, different phosphoproteins have different kinetics. For example, the peak of Syk phosphorylation was detected at 5 min after stimulation (A), and then slowly declined; meanwhile, Akt, p38MAPK, and Erk1/2 showed a biphasic behavior (C,D, and E), which is also evident, but less prominent in Btk (B).
Mentions: Anti-IgM stimulated PBMC (similar time points as described before) were assayed for phosphorylation of proteins associated with the BCR signaling pathways (Syk-Y352, Btk-Y551, Akt-S473, p38MAPK-T180/Y182, and Erk1/2-T202/Y204) in two separate panels. These proteins allow the simultaneous evaluation of the initiation (Syk), propagation (Btk, Akt), and integration signaling units (p38MAPK-T180/Y182 and Erk1/2-T202/Y204). Furthermore, the selected proteins can also provide some information regarding whether the PLCγ2, PI3K, or both BCR-associated signaling pathways are activated. All evaluated proteins showed enhanced phosphorylation in Um B cells when compared to naïve B cells (Figures 5A–E). As described above, Syk, which represents a protein from the initiation signaling subunit, showed higher phosphorylation in Um than in naïve B cells (Figure 5A). Interestingly, the phosphorylation peak was observed at 5 min, followed by a slow dephosphorylation slope (Figures 4B, 5A). Among proteins of the integration subunit, Btk and Akt were also evaluated. Naïve and Um cells showed an early peak of Btk phosphorylation (Y551) 1–2 min after stimulation, followed by a plateau established around minute 10 which persisted until the last time point collected (30 min). Akt was phosphorylated in naïve and Um cells; however, it was more prominent in the latter cells (Figure 5C). Interestingly, two phosphorylation peaks were identified, one between minutes 5–8 and a second one at 15–20 min. Following the second peak, pAkt-S473 levels returned to the resting state in both B cell populations. Among the proteins of the integration signaling subunit, p38MAPK (T180/Y182) and Erk1/2 (T202/Y204) were evaluated (Figures 5D,E). Phosphorylation of both proteins was identified in Um, but not in naive B cells. Furthermore, phosphorylation of these proteins also showed a biphasic phase with peaks at 2–3 and 8–10 min.

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