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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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Naïve and BM cell subpopulations defined by IgD and CD27 markers in PBMC. Four B cell (CD19+ CD3−) subpopulations are defined by expression of IgD and CD27: Naïve (IgD+ CD27−), Um (IgD+ CD27+), Sm CD27+ (IgD− CD27+), and Sm CD27− (IgD− CD27−) (A). The percentages of these B-cell subpopulations showed little variation in 8 healthy adult volunteers (B). An example of surface Ig isotype (IgM, IgD, IgA, and IgG) distribution in B cell subpopulations in a representative volunteer is shown (C). Dotted lines indicate where the gates were set to determine the percentage of each Ig class. (D) Compiled data of the percentages of the Ig isotypes on the surface of each B-cell subpopulation (n = 8).
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Figure 1: Naïve and BM cell subpopulations defined by IgD and CD27 markers in PBMC. Four B cell (CD19+ CD3−) subpopulations are defined by expression of IgD and CD27: Naïve (IgD+ CD27−), Um (IgD+ CD27+), Sm CD27+ (IgD− CD27+), and Sm CD27− (IgD− CD27−) (A). The percentages of these B-cell subpopulations showed little variation in 8 healthy adult volunteers (B). An example of surface Ig isotype (IgM, IgD, IgA, and IgG) distribution in B cell subpopulations in a representative volunteer is shown (C). Dotted lines indicate where the gates were set to determine the percentage of each Ig class. (D) Compiled data of the percentages of the Ig isotypes on the surface of each B-cell subpopulation (n = 8).

Mentions: B cells were identified in PBMC of healthy volunteers as CD19+ and CD3− cells. Subsequently, naïve and BM subpopulations were defined using IgD and CD27 as previously reported (Sanz et al., 2008). These markers show four distinct populations of B cells: naïve (IgD+ CD27−), Um (IgD+ CD27+), Sm CD27+ (IgD− CD27+) and Sm CD27− (IgD− CD27−) (Figure 1A). In PBMC from 8 screened volunteers, naïve B cells were the most abundant population (mean 71.7%; SE ± 2.4), followed by Um (mean 15.9%; SE ± 2.7), Sm CD27+ (mean 8.2%; SE ± 1.4) and Sm CD27− (mean 2.5%; SE ± 0.6) (Figure 1B). Expression of surface Ig isotypes was evaluated within each of these subpopulations (Figures 1C,D). Naïve as well as Um cells expressed both IgM and IgD, while Sm CD27+ and Sm CD27− B cells expressed mainly IgA and IgG. Interestingly, a small percentage of Sm CD27+ cells also expressed IgM (mean 17%; SE ± 3) (Figures 1C,D).


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)

Naïve and BM cell subpopulations defined by IgD and CD27 markers in PBMC. Four B cell (CD19+ CD3−) subpopulations are defined by expression of IgD and CD27: Naïve (IgD+ CD27−), Um (IgD+ CD27+), Sm CD27+ (IgD− CD27+), and Sm CD27− (IgD− CD27−) (A). The percentages of these B-cell subpopulations showed little variation in 8 healthy adult volunteers (B). An example of surface Ig isotype (IgM, IgD, IgA, and IgG) distribution in B cell subpopulations in a representative volunteer is shown (C). Dotted lines indicate where the gates were set to determine the percentage of each Ig class. (D) Compiled data of the percentages of the Ig isotypes on the surface of each B-cell subpopulation (n = 8).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Naïve and BM cell subpopulations defined by IgD and CD27 markers in PBMC. Four B cell (CD19+ CD3−) subpopulations are defined by expression of IgD and CD27: Naïve (IgD+ CD27−), Um (IgD+ CD27+), Sm CD27+ (IgD− CD27+), and Sm CD27− (IgD− CD27−) (A). The percentages of these B-cell subpopulations showed little variation in 8 healthy adult volunteers (B). An example of surface Ig isotype (IgM, IgD, IgA, and IgG) distribution in B cell subpopulations in a representative volunteer is shown (C). Dotted lines indicate where the gates were set to determine the percentage of each Ig class. (D) Compiled data of the percentages of the Ig isotypes on the surface of each B-cell subpopulation (n = 8).
Mentions: B cells were identified in PBMC of healthy volunteers as CD19+ and CD3− cells. Subsequently, naïve and BM subpopulations were defined using IgD and CD27 as previously reported (Sanz et al., 2008). These markers show four distinct populations of B cells: naïve (IgD+ CD27−), Um (IgD+ CD27+), Sm CD27+ (IgD− CD27+) and Sm CD27− (IgD− CD27−) (Figure 1A). In PBMC from 8 screened volunteers, naïve B cells were the most abundant population (mean 71.7%; SE ± 2.4), followed by Um (mean 15.9%; SE ± 2.7), Sm CD27+ (mean 8.2%; SE ± 1.4) and Sm CD27− (mean 2.5%; SE ± 0.6) (Figure 1B). Expression of surface Ig isotypes was evaluated within each of these subpopulations (Figures 1C,D). Naïve as well as Um cells expressed both IgM and IgD, while Sm CD27+ and Sm CD27− B cells expressed mainly IgA and IgG. Interestingly, a small percentage of Sm CD27+ cells also expressed IgM (mean 17%; SE ± 3) (Figures 1C,D).

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