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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

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          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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          Most cited references67

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          MAP kinases in the immune response.

          MAP kinases are among the most ancient signal transduction pathways and are widely used throughout evolution in many physiological processes. In mammalian species, MAP kinases are involved in all aspects of immune responses, from the initiation phase of innate immunity, to activation of adaptive immunity, and to cell death when immune function is complete. In this review, we summarize recent progress in understanding the function and regulation of MAP kinase pathways in these phases of immune responses.
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            Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire.

            The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive, immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) "memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis, complementarity-determining region 3 (CDR3) spectratyping during a T-independent response, and gene-expression profiling of the different blood and splenic B-cell subsets, we show here that blood IgM(+)IgD(+)CD27(+) cells correspond to circulating splenic marginal zone B cells. Furthermore, analysis of this peripheral subset in healthy children younger than 2 years shows that these B cells develop and mutate their immunoglobulin receptor during ontogeny, prior to their differentiation into T-independent antigen-responsive cells. It is therefore proposed that these IgM(+)IgD(+)CD27(+) B cells provide the splenic marginal zone with a diversified and protective preimmune repertoire in charge of the responses against encapsulated bacteria.
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              Effector and regulatory B cells: modulators of CD4+ T cell immunity.

              B cells are essential for humoral immunity, but the role that they have in regulating CD4(+) T cell responses remains controversial. However, new data showing that the transient depletion of B cells potently influences the induction, maintenance and reactivation of CD4(+) T cells, with the recent identification of antibody-independent functions of B cells, have reinvigorated interest in the many roles of B cells in both infectious and autoimmune diseases. In this Review, we discuss recent data showing how effector and regulatory B cells modulate CD4(+) T cell responses to pathogens and autoantigens.
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                Author and article information

                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Inf. Microbio.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                17 October 2012
                2012
                : 2
                : 128
                Affiliations
                [1] 1Department of Medicine, Center for Vaccine Development, University of Maryland Baltimore, MD, USA
                [2] 2Department of Pediatrics, Center for Vaccine Development, University of Maryland Baltimore, MD, USA
                Author notes

                Edited by: Lisa A. Morici, Tulane University School of Medicine, USA

                Reviewed by: Lisa A. Morici, Tulane University School of Medicine, USA; Hui Wu, University of Alabama at Birmingham, USA

                *Correspondence: Marcelo B. Sztein, Department of Medicine, Center for Vaccine Development, University of Maryland, 685 West Baltimore Street, HSF-1 Rm480, Baltimore, MD 21201, USA. e-mail: msztein@ 123456medicine.umaryland.edu
                Article
                10.3389/fcimb.2012.00128
                3473368
                23087912
                254a46e3-3695-4973-b55e-92316a9d4830
                Copyright © 2012 Toapanta, Bernal and Sztein.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.

                History
                : 04 August 2012
                : 01 October 2012
                Page count
                Figures: 13, Tables: 0, Equations: 0, References: 79, Pages: 17, Words: 11993
                Categories
                Microbiology
                Original Research Article

                Infectious disease & Microbiology
                salmonella typhi,phosphoflow,igm memory b cells,cell signaling,naïve b cells,streptococcus pneumoniae,fluorescent-cell barcoding,vaccines

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