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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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Fluorescently labeled bacteria bind preferentially to naïve and Um B cells. Heat-killed S. Typhi (gram negative) and S. pneumoniae (encapsulated gram positive) bacteria were fluorescently labeled with Alexa700 (A). Unstained bacteria (red closed histograms) and stained bacteria (continuous lines) are shown in panel A. PBMC were incubated with labeled bacteria at a MOI of 100:1 for 10 min at 37°C and the population of B cells recognizing the bacteria identified (B). A higher percentage of B cells recognizing S. pneumoniae than S. Typhi (B) was observed. A higher proportion of naïve than Um B-cell subpopulations was found to interact with the bacteria (C). B and C show data observed in a representative volunteer.
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Figure 9: Fluorescently labeled bacteria bind preferentially to naïve and Um B cells. Heat-killed S. Typhi (gram negative) and S. pneumoniae (encapsulated gram positive) bacteria were fluorescently labeled with Alexa700 (A). Unstained bacteria (red closed histograms) and stained bacteria (continuous lines) are shown in panel A. PBMC were incubated with labeled bacteria at a MOI of 100:1 for 10 min at 37°C and the population of B cells recognizing the bacteria identified (B). A higher percentage of B cells recognizing S. pneumoniae than S. Typhi (B) was observed. A higher proportion of naïve than Um B-cell subpopulations was found to interact with the bacteria (C). B and C show data observed in a representative volunteer.

Mentions: Heat-killed S. Typhi (gram negative) and S. pneumoniae (encapsulated gram positive) fluorescently labeled with Alexa700 (Figure 9A) were used to stimulate PBMC (MOI 100 to 1) (37°C; 10 min) from healthy volunteers. These bacteria were selected because they have different antigenic structures and it was of interest to determine whether they could induce different signaling cascades on B cells. Relatively small populations of B cells interacting with S. Typhi (0.23%) and S. Pneumoniae (1.9%) were identified (Figure 9B) and these cells corresponded mainly to naïve and Um B cells (Figure 9C). A higher percentage of naïve than Um B cells (ratio 2:1) interacted with both bacteria (Figure 9C). Phosphorylation of various B cells signaling proteins (Syk, Akt, and p38MAPK) was then assayed in these subpopulations (Figures 10A,B). Even though a higher percentage of naïve B cells interacted with both bacteria, these cells did not show phosphorylation of the assayed proteins (data not shown). On the other hand, phosphorylation of Syk, Akt, and p38MAPK was induced in Um B cells by both S. Typhi and S. pneumoniae (Figures 10A,B). The percentage of phosphorylated cells for each protein was consistently higher in cells stimulated with S. Typhi than with S. pneumoniae (Figures 10A,B). Finally, to better demonstrate the complexity of the signaling responses induced in Um B cells by bacteria we used this novel phosphoflow technique to determine the percentage of cells that showed single, double, or triple phosphorylation of the signaling proteins assayed, i.e., whether they are multi-phosphorylated (Figure 11). We observed, for the first time, the presence of multi-phosphorylated cells and determined that the signaling responses were different among cells stimulated with S. Typhi and S. pneumoniae. S. Typhi-stimulated cells showed a higher percentage of Um B cells that phosphorylated the three proteins (18%) assayed, followed by cells that showed only p38MAPK phosphorylation (12%) and finally 5% showing Akt and p38MAPK phosphorylation (Figure 11B). On the other hand, the percentages of Um B cells that phosphorylated proteins following S. pneumoniae stimulation were overall smaller. Um B cells that phosphorylated only p38MAPK (7%) were the predominant ones. Around 2% of Um cells showed single (Syk), double (Syk, p38MAPK, or Akt, p38MAPK) and triple (Syk, Akt, p38MAPK) phosphorylated proteins (Figure 11C).


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)

Fluorescently labeled bacteria bind preferentially to naïve and Um B cells. Heat-killed S. Typhi (gram negative) and S. pneumoniae (encapsulated gram positive) bacteria were fluorescently labeled with Alexa700 (A). Unstained bacteria (red closed histograms) and stained bacteria (continuous lines) are shown in panel A. PBMC were incubated with labeled bacteria at a MOI of 100:1 for 10 min at 37°C and the population of B cells recognizing the bacteria identified (B). A higher percentage of B cells recognizing S. pneumoniae than S. Typhi (B) was observed. A higher proportion of naïve than Um B-cell subpopulations was found to interact with the bacteria (C). B and C show data observed in a representative volunteer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 9: Fluorescently labeled bacteria bind preferentially to naïve and Um B cells. Heat-killed S. Typhi (gram negative) and S. pneumoniae (encapsulated gram positive) bacteria were fluorescently labeled with Alexa700 (A). Unstained bacteria (red closed histograms) and stained bacteria (continuous lines) are shown in panel A. PBMC were incubated with labeled bacteria at a MOI of 100:1 for 10 min at 37°C and the population of B cells recognizing the bacteria identified (B). A higher percentage of B cells recognizing S. pneumoniae than S. Typhi (B) was observed. A higher proportion of naïve than Um B-cell subpopulations was found to interact with the bacteria (C). B and C show data observed in a representative volunteer.
Mentions: Heat-killed S. Typhi (gram negative) and S. pneumoniae (encapsulated gram positive) fluorescently labeled with Alexa700 (Figure 9A) were used to stimulate PBMC (MOI 100 to 1) (37°C; 10 min) from healthy volunteers. These bacteria were selected because they have different antigenic structures and it was of interest to determine whether they could induce different signaling cascades on B cells. Relatively small populations of B cells interacting with S. Typhi (0.23%) and S. Pneumoniae (1.9%) were identified (Figure 9B) and these cells corresponded mainly to naïve and Um B cells (Figure 9C). A higher percentage of naïve than Um B cells (ratio 2:1) interacted with both bacteria (Figure 9C). Phosphorylation of various B cells signaling proteins (Syk, Akt, and p38MAPK) was then assayed in these subpopulations (Figures 10A,B). Even though a higher percentage of naïve B cells interacted with both bacteria, these cells did not show phosphorylation of the assayed proteins (data not shown). On the other hand, phosphorylation of Syk, Akt, and p38MAPK was induced in Um B cells by both S. Typhi and S. pneumoniae (Figures 10A,B). The percentage of phosphorylated cells for each protein was consistently higher in cells stimulated with S. Typhi than with S. pneumoniae (Figures 10A,B). Finally, to better demonstrate the complexity of the signaling responses induced in Um B cells by bacteria we used this novel phosphoflow technique to determine the percentage of cells that showed single, double, or triple phosphorylation of the signaling proteins assayed, i.e., whether they are multi-phosphorylated (Figure 11). We observed, for the first time, the presence of multi-phosphorylated cells and determined that the signaling responses were different among cells stimulated with S. Typhi and S. pneumoniae. S. Typhi-stimulated cells showed a higher percentage of Um B cells that phosphorylated the three proteins (18%) assayed, followed by cells that showed only p38MAPK phosphorylation (12%) and finally 5% showing Akt and p38MAPK phosphorylation (Figure 11B). On the other hand, the percentages of Um B cells that phosphorylated proteins following S. pneumoniae stimulation were overall smaller. Um B cells that phosphorylated only p38MAPK (7%) were the predominant ones. Around 2% of Um cells showed single (Syk), double (Syk, p38MAPK, or Akt, p38MAPK) and triple (Syk, Akt, p38MAPK) phosphorylated proteins (Figure 11C).

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