Ambient particulate matter directs nonclassic dendritic cell activation and a mixed TH1/TH2-like cytokine response by naive CD4+ T cells Marc A. Williams,

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Ambient particulate matter directs nonclassic dendritic cell activation and a mixed TH1/TH2-like cytokine response by naive CD4+ T cells Marc A. Williams, PhD, Michael Porter, BS, Maureen Horton, MD, Jia Guo, MD, Jessica Roman, BA, D'Ann Williams, MS, Patrick Breysse, PhD, Steve N. Georas, MD Journal of Allergy and Clinical Immunology Volume 119, Issue 2, Pages 488-497 (February 2007) DOI: 10.1016/j.jaci.2006.10.022 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Flow-cytometric quantitation of the costimulatory molecules CD40 (A), CD80 (B), and CD86 (C) on the cell surface of resting and stimulated CD34+ PBPC-derived DCs. DCs were stimulated with APM (100 μg/mL), LPS (100 ng/mL), or CBs (100 μg/mL) for 48 hours as indicated. Data are recorded as geometric mean fluorescence intensity (FI) units ± SDs. The levels of significance shown are ∗P < .05 and ∗∗P < .01 compared with resting DCs. Journal of Allergy and Clinical Immunology 2007 119, 488-497DOI: (10.1016/j.jaci.2006.10.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Flow-cytometric quantitation of the cell-surface expression of MHC class II/HLA-DR (A) and CD1a (B) for resting and stimulated CD34+ PBPC-derived DCs as in Fig 1. Data are recorded as geometric mean fluorescence intensity (FI) units ± SDs. The levels of significance shown are ∗P < .05 and ∗∗P < .01 compared with resting DCs. Journal of Allergy and Clinical Immunology 2007 119, 488-497DOI: (10.1016/j.jaci.2006.10.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Flow-cytometric quantitation of the cell-surface expression of TLR2 and TLR4 for resting and stimulated DCs as in Fig 1. Data are recorded as geometric mean fluorescence intensity (FI) units ± SDs. The levels of significance shown are ∗∗P < .01 or ∗∗∗P < .001 compared with resting DCs. Journal of Allergy and Clinical Immunology 2007 119, 488-497DOI: (10.1016/j.jaci.2006.10.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Flow-cytometric quantitation of DC surface expression of CD206 (A) and time-dependent uptake of FITC-conjugated dextran (B). Data are recorded as geometric mean fluorescence intensity (FI) units ± SDs. The levels of significance shown are ∗P < .05 and ∗∗P < .01 compared with resting DCs. The functional uptake of FITC-dextran by CD34+ PBPC-derived DCs was also measured (B). Data are expressed as geometric mean FI units. Journal of Allergy and Clinical Immunology 2007 119, 488-497DOI: (10.1016/j.jaci.2006.10.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Dose-dependent stimulation of cytokines by naive alloreactive CD4+ T cells stimulated by DCs. T-cell–mediated secretion of IFN-γ (A), IL-5 (B), and IL-13 (C) is shown. In addition, we have shown (inset bar charts) cytokine production after prestimulation of DCs with high-dose LPS (5000 pg/mL) compared with APM 100 μg/mL and the corresponding concentration of LPS at 50 pg/mL found in that dose of APM for comparison. Data are presented as picograms secreted product per million naive CD4+ T cells ± 1 SD of the mean. The ratiometric analysis of IFN-γ to IL-5 and IFN-γ to IL-13 produced by these cocultures is also shown (D). Journal of Allergy and Clinical Immunology 2007 119, 488-497DOI: (10.1016/j.jaci.2006.10.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 APM and DC-stimulated proliferation of naive CD4+ alloreactive T cells. The dose-dependent effects of LPS-stimulated DCs (A) as well as the dose-dependent effects of APM-stimulated DCs on the proliferation of naive CD4+ T cells (B) is shown. LPS is titrated against DCs at doses (50 and 5 pg/mL) that were found in the appropriate concentrations of APM used to stimulate DCs, as well as 10-fold and 100-fold higher concentrations of LPS (500 and 5000 pg/mL LPS, respectively) for comparison. Data are recorded as mean OD from a bromodeoxyuridine (BrdU) DNA incorporation cell proliferation ELISA. Journal of Allergy and Clinical Immunology 2007 119, 488-497DOI: (10.1016/j.jaci.2006.10.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions