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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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Evaluation of antibodies for phosphoflow in naïve and BM cell subpopulations. Example of surface staining for identification of B cells and their subpopulations. CD19 and CD20 are typically used to define B cell populations (A). Several monoclonal antibodies were evaluated to define B cells, as well as naïve and memory B cell subpopulations during the optimization of the staining technique for phosphoflow. The CD19 marker is lost when cells are treated for phosphoprotein determinations; however, CD20 (H1 clone) defines these cells appropriately and therefore was subsequently used in phosphoflow assays (B). Polyclonal anti-IgD sera (goat anti-human) used in a two step staining technique (see “Materials and Methods” for details) provided appropriate resolution of naïve and memory B cell subpopulations for phosphoflow staining (B). FSC, forward scatter.
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FA1: Evaluation of antibodies for phosphoflow in naïve and BM cell subpopulations. Example of surface staining for identification of B cells and their subpopulations. CD19 and CD20 are typically used to define B cell populations (A). Several monoclonal antibodies were evaluated to define B cells, as well as naïve and memory B cell subpopulations during the optimization of the staining technique for phosphoflow. The CD19 marker is lost when cells are treated for phosphoprotein determinations; however, CD20 (H1 clone) defines these cells appropriately and therefore was subsequently used in phosphoflow assays (B). Polyclonal anti-IgD sera (goat anti-human) used in a two step staining technique (see “Materials and Methods” for details) provided appropriate resolution of naïve and memory B cell subpopulations for phosphoflow staining (B). FSC, forward scatter.

Mentions: A staining technique that allowed appropriate resolution of naïve and BM cell subpopulations in conditions suitable to assay phosphorylation of signaling proteins was developed (see “Appendix Figure A1 and Materials and Methods”). To investigate whether this optimized staining technique allowed the evaluation of phosphoproteins associated with the BCR, overnight rested PBMC were stimulated with goat F (ab')2 anti-human IgM (5 μg/ml) for 5 min. Phosphorylation of Syk (pSyk—Y352), one of the phosphotyrosine kinases (PTK) associated with the BCR was then measured in naïve and Um B cells. As expected, Sm CD27+ and Sm CD27− B cells, most of which are IgM negative (Figures 1C,D), did not show significant pSyk (Figure 3A). To further validate this assay, PBMC were stimulated with anti-IgG and anti-IgA antibodies [goat F (ab')2 anti-human IgG or IgA; 5 μg/ml; 5 min]. Under these conditions pSyk was detected in Sm CD27+ and Sm CD27− cells, but not in naïve and Um B cells (Figures 3B,C). In subsequent experiments, FCB, which provides a unique fluorescent signature to cells in independent samples and allows flow cytometric multiplexing, was incorporated in the phosphoflow assay (Figure 4A). FCB enabled the simultaneous measurement of multiple time points in a single tube and provided additional consistency in the staining. Fluorescent-cell barcoded samples were later deconvoluted for analysis (Figure 4B). Syk phosphorylation (Y352) was examined in PBMC stimulated with anti-IgM at various time points (0, 1, 2, 5, 8, 10, 15, 20, and 30 min) (Figure 4C). Similar levels of pSyk were detected in resting naïve and Um B cells (Figure 4C—0 min). Phosphorylation was detected as early as 1 min after anti-IgM stimulation and the intensity of pSyk (MFI) was higher in Um than in naïve B cells at every time point measured. Furthermore, Um B cells remained phosphorylated for a longer period of time (Figure 4C). In order to determine if the differences in Syk phosphorylation were also due to dissimilar levels of this protein, total levels of Syk were evaluated. Um B cells had higher amounts (2-fold) of pan-Syk (phosphorylated and non-phosphorylated) than naïve B cells (Figure 4D).


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)

Evaluation of antibodies for phosphoflow in naïve and BM cell subpopulations. Example of surface staining for identification of B cells and their subpopulations. CD19 and CD20 are typically used to define B cell populations (A). Several monoclonal antibodies were evaluated to define B cells, as well as naïve and memory B cell subpopulations during the optimization of the staining technique for phosphoflow. The CD19 marker is lost when cells are treated for phosphoprotein determinations; however, CD20 (H1 clone) defines these cells appropriately and therefore was subsequently used in phosphoflow assays (B). Polyclonal anti-IgD sera (goat anti-human) used in a two step staining technique (see “Materials and Methods” for details) provided appropriate resolution of naïve and memory B cell subpopulations for phosphoflow staining (B). FSC, forward scatter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

FA1: Evaluation of antibodies for phosphoflow in naïve and BM cell subpopulations. Example of surface staining for identification of B cells and their subpopulations. CD19 and CD20 are typically used to define B cell populations (A). Several monoclonal antibodies were evaluated to define B cells, as well as naïve and memory B cell subpopulations during the optimization of the staining technique for phosphoflow. The CD19 marker is lost when cells are treated for phosphoprotein determinations; however, CD20 (H1 clone) defines these cells appropriately and therefore was subsequently used in phosphoflow assays (B). Polyclonal anti-IgD sera (goat anti-human) used in a two step staining technique (see “Materials and Methods” for details) provided appropriate resolution of naïve and memory B cell subpopulations for phosphoflow staining (B). FSC, forward scatter.
Mentions: A staining technique that allowed appropriate resolution of naïve and BM cell subpopulations in conditions suitable to assay phosphorylation of signaling proteins was developed (see “Appendix Figure A1 and Materials and Methods”). To investigate whether this optimized staining technique allowed the evaluation of phosphoproteins associated with the BCR, overnight rested PBMC were stimulated with goat F (ab')2 anti-human IgM (5 μg/ml) for 5 min. Phosphorylation of Syk (pSyk—Y352), one of the phosphotyrosine kinases (PTK) associated with the BCR was then measured in naïve and Um B cells. As expected, Sm CD27+ and Sm CD27− B cells, most of which are IgM negative (Figures 1C,D), did not show significant pSyk (Figure 3A). To further validate this assay, PBMC were stimulated with anti-IgG and anti-IgA antibodies [goat F (ab')2 anti-human IgG or IgA; 5 μg/ml; 5 min]. Under these conditions pSyk was detected in Sm CD27+ and Sm CD27− cells, but not in naïve and Um B cells (Figures 3B,C). In subsequent experiments, FCB, which provides a unique fluorescent signature to cells in independent samples and allows flow cytometric multiplexing, was incorporated in the phosphoflow assay (Figure 4A). FCB enabled the simultaneous measurement of multiple time points in a single tube and provided additional consistency in the staining. Fluorescent-cell barcoded samples were later deconvoluted for analysis (Figure 4B). Syk phosphorylation (Y352) was examined in PBMC stimulated with anti-IgM at various time points (0, 1, 2, 5, 8, 10, 15, 20, and 30 min) (Figure 4C). Similar levels of pSyk were detected in resting naïve and Um B cells (Figure 4C—0 min). Phosphorylation was detected as early as 1 min after anti-IgM stimulation and the intensity of pSyk (MFI) was higher in Um than in naïve B cells at every time point measured. Furthermore, Um B cells remained phosphorylated for a longer period of time (Figure 4C). In order to determine if the differences in Syk phosphorylation were also due to dissimilar levels of this protein, total levels of Syk were evaluated. Um B cells had higher amounts (2-fold) of pan-Syk (phosphorylated and non-phosphorylated) than naïve B cells (Figure 4D).

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