Toll-like receptor 9 suppression in plasmacytoid dendritic cells after IgE-dependent activation is mediated by autocrine TNF-α John T. Schroeder, PhD,

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Toll-like receptor 9 suppression in plasmacytoid dendritic cells after IgE-dependent activation is mediated by autocrine TNF-α John T. Schroeder, PhD, Kristin L. Chichester, MS, Anja P. Bieneman, BS Journal of Allergy and Clinical Immunology Volume 121, Issue 2, Pages 486-491 (February 2008) DOI: 10.1016/j.jaci.2007.09.049 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Time course for the secretion of TNF-α, IL-6, IL-10, and IFN-α by purified blood pDCs stimulated with goat antihuman IgE (5 μg/mL), goat IgG control (5 μg/mL), CpG-2216 (100 nmol/L), or medium alone. Values represent the average ± SEM of 4 experiment using cells from different subjects. Journal of Allergy and Clinical Immunology 2008 121, 486-491DOI: (10.1016/j.jaci.2007.09.049) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Representative experiment showing the loss of TLR9 expression in pDCs treated for the indicated times with anti-IgE antibody (5 μg/mL) or goat IgG isotype control (5 μg/mL). Western blot data (A) with TLR9/p85 densitometry depicted in the bar graph (B). Journal of Allergy and Clinical Immunology 2008 121, 486-491DOI: (10.1016/j.jaci.2007.09.049) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 TLR9 expression and functional responses to CpG in pDCs pretreated (18 hours) with different recombinant cytokines. A, Representative experiment showing the effect of recombinant cytokines (10 ng/mL) on TLR9 protein expression. B, Densitometry data from 4 individual experiments. C, Effect on TLR9 mRNA expression using cell preparations (n = 4) different from those shown in B. D, Effects on CpG-induced (100 nmol/L) IFN-α secretion. Values are normalized to percent of control (ie, pDCs pretreated in medium alone), which averaged 2033 ± 1110 pg/106, n = 4. ∗P < .05. Journal of Allergy and Clinical Immunology 2008 121, 486-491DOI: (10.1016/j.jaci.2007.09.049) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 The effect of neutralizing TNF-α activity during IgE-dependent activation on the expression of TLR9 in pDCs. Suspensions of pDCs prepared from 2 different subjects were treated as indicated for 18 hours. TLR9 protein expression was then analyzed by Western blot. Numbers in parentheses indicate relative densitometry (ie, TLR9 as a percentage of p85). Exp, Experiment. Journal of Allergy and Clinical Immunology 2008 121, 486-491DOI: (10.1016/j.jaci.2007.09.049) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 The effect of neutralizing TNF-α activity during IgE-dependent activation on the subsequent induction of IFN-α by CpG. pDC suspensions prepared from 6 different subjects were pretreated as indicated. After 18-hour incubation, CpG-2216 (or medium alone) was added for an additional 18 hours to induce IFN-α. Values, both individually (symbols) and averaged (bar), are reported as a percent of the CpG control, which was 6068 ± 2170 pg/106 pDCs for the 6 subjects. Journal of Allergy and Clinical Immunology 2008 121, 486-491DOI: (10.1016/j.jaci.2007.09.049) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions