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Amplification of Toll-like receptor–mediated signaling through spleen tyrosine kinase in human B-cell activation  Shigeru Iwata, MD, PhD, Kunihiro Yamaoka,

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Presentation on theme: "Amplification of Toll-like receptor–mediated signaling through spleen tyrosine kinase in human B-cell activation  Shigeru Iwata, MD, PhD, Kunihiro Yamaoka,"— Presentation transcript:

1 Amplification of Toll-like receptor–mediated signaling through spleen tyrosine kinase in human B-cell activation  Shigeru Iwata, MD, PhD, Kunihiro Yamaoka, MD, PhD, Hiroaki Niiro, MD, PhD, Kazuhisa Nakano, MD, PhD, Sheau-Pey Wang, MS, Koichi Akashi, MD, PhD, Yoshiya Tanaka, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 129, Issue 6, Pages e2 (June 2012) DOI: /j.jaci Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Syk regulates proliferation and cell-cycle progression in B-cell subsets on BCR, CD40, and TLR9 stimulation. A, BCR-induced phosphorylation of Syk (15 minutes). B-D, Tritiated thymidine (3HTdr) incorporation of human B cells was measured during the last 18 hours of the 72-hour culture. The data are shown as means ± SDs. *P < .05. sCD40L, Soluble CD40 ligand. E, FACS histograms of nuclear DNA content in memory B cells 24 hours later. Unstim, Before stimulation; 3S, BCR, CD40, and TLR9 stimulation. Results are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Syk regulates expression of CD80 and CD86 in B-cell subsets on stimulation. Overlay histograms depict relative fluorescence intensities of human naive and memory B cells cultured for 72 hours. Unstim, Before stimulation; 3S, BCR, CD40, and TLR9 stimulation. Results are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Syk regulates cytokine production in B-cell subsets on stimulation. Human peripheral blood naive and memory B cells were cultured for 72 hours, and supernatants were harvested and assayed by using the cytometric bead array for IL-6, TNF-α, and IL-10 content. Data are shown as means ± SDs and are representative of 3 independent experiments. sCD40L, Soluble CD40 ligand. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Syk regulates B-cell differentiation on BCR, CD40, and TLR9 stimulation. Naive and memory B cells were cultured for 48 hours (AICDA and XBP-1 mRNA and Blimp-1) or for 5 days (IgG production). A, The level of AICDA and XBP-1 mRNA was measured by using real-time PCR. RQ, Relative quantity. B, Blimp-1 expression was measured by means of flow cytometry. Unstim, Before stimulation; 3S, BCR, CD40, and TLR stimulation. C, IgG in the supernatant was quantified by using ELISA. Data are shown as means ± SDs and are representative of 3 independent experiments. *P < .05. sCD40L, Soluble CD40 ligand. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 TLR9 and TRAF6 are key Syk-regulated molecules in B-cell subsets on stimulation. A, TLR9 mRNA was quantified by using real-time PCR (TaqMan PCR kit) 24 hours later. RQ, Relative quantity; sCD40L, soluble CD40 ligand. B and C, TRAF2, TRAF3, TRAF5, and TRAF6 levels (48 hours later) and NF-κB phosphorylation (p65; 12 hours later) were measured by means of flow cytometry (intracellular staining). Unstim, Before stimulation; 3S, combination of BCR, CD40, and TLR9 stimulation. Data are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Syk inhibitor exerts marked inhibitory action, even at an activated state of B cells. Raji cells were cultured with RPMI containing 2% FCS for 48 hours. A, TLR9 mRNA was quantified by means of real-time PCR. RQ, Relative quantity. B, Expression of Syk, phospho-Syk (Y348), TRAF6, and phospho-NF-κB (p65) was assessed by means of Western blotting. The intensity of bands was quantified and normalized with respect to those of corresponding β-actin. The resulting values were expressed as the percentage in reference to that of cells without Syk inhibitor IV. Data are shown as means ± SDs and are representative of 3 independent experiments. *P < .05. n.s., Not significant. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 Phenotypic analysis of B-cell subsets in human peripheral blood. B cells were obtained by means of negative selection from PBMCs. CD27+ memory B cells were then isolated by using positive selection from B cells with CD27 microbeads. The negative fraction of this isolation was assigned to CD27− naive B cells. The purity of naive and memory B cells was greater than 90% (x-axis, CD19; y-axis, CD27). Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 Syk provides survival signals for B cells after stimulation through all 3 receptors. B cells (2 × 105 per well) were cultured in triplicate in 96-well plates with anti-Igλ and anti-Igk antibodies (1 μg/mL), soluble CD40 ligand (sCD40L; 2 μg/mL), and CpG-ODN 2006 (2.5 μg/mL) with or without Syk inhibitor IV for 72 hours. The percentage of apoptotic B cells was assessed by means of double-staining with FITC–Annexin V and PI (x-axis, PI; y-axis, Annexin V). Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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