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Allison K. Martin, BS, Douglas G. Mack, PhD, Michael T

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1 Beryllium-specific CD4+ T cells in blood as a biomarker of disease progression 
Allison K. Martin, BS, Douglas G. Mack, PhD, Michael T. Falta, PhD, Margaret M. Mroz, MSPH, Lee S. Newman, MD, Lisa A. Maier, MD, Andrew P. Fontenot, MD  Journal of Allergy and Clinical Immunology  Volume 128, Issue 5, Pages e5 (November 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Frequency of Be-specific T cells in blood of healthy control subjects and patients with CBD. A, IFN-γ and IL-2 ELISpot analysis. Data are expressed as the mean SFUs, with median values indicated with solid lines. Values above the dotted line represent a positive cytokine response. B and C, ROC curves for IFN-γ and IL-2 SFUs in blood of patients with CBD (true-positive results) and control subjects (true-negative results). The area under the curve (AUC) and the chosen cutoff value for each ROC curve are shown. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Identification of a Be-induced adaptive immune response. A, Frequency of Be-specific T cells in blood, as determined by using the IFN-γ and IL-2 ELISpot assays. Data are expressed as the mean SFUs for each subject. Cytokine values above the dotted line represent a positive cytokine response. B, Be-induced proliferative response. An SI indicative of a positive response is defined as 2.5 or greater (dotted line). C, Correlation between the number of Be-responsive T cells in blood and peak SI. Subjects with confirmed CBD on clinical evaluation are shown as open circles. The rectangle surrounds subjects with a negative IFN-γ ELISpot response and BeLPT result. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Frequency of Be-responsive CD4+ T cells in blood and BAL of patients with CBD. A, Be-specific T cells in blood over time. Data are expressed as the mean SFUs per 5 × 105 cells per well. Open circles show 2 subjects who had a positive ELISpot response and a negative BeLPT result at first evaluation. B, Percentage of BAL CD4+ T cells that express IFN-γ (n = 11) and IL-2 (n = 10) after stimulation with 100 μmol/L BeSO4. Mean percentage of TH1 cytokines produced by BAL cells from patients with CBD is shown as a solid line. C, Correlation between the number of Be-responsive T cells in blood and the frequency of Be-responsive BAL CD4+ T cells expressing either IFN-γ (left panel) or IL-2 (right panel). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Frequency of Be-specific T cells in blood. A, Number of Be-responsive T cells in blood of BeS subjects and subjects whose disease status changed at the time of the clinical evaluation (Progressors). Data are expressed as the mean number of IFN-γ SFUs in response to 100 μmol/L BeSO4, and median values are indicated with solid lines. B, ROC curves for IFN-γ SFUs in blood of patients with CBD and BeS subjects. The area under the curve (AUC) and the chosen cutoff value are shown. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E1 Interassay variability in the IFN-γ ELISpot assay. PBMCs from 13 subjects were split, and IFN-γ ELISpot assays were performed on separate plates to assess interassay variability. Statistical comparisons were made with the paired t test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E2 Frequency of Be-specific T cells in the blood of subjects with and without expression of the genetic susceptibility marker HLA-DPB1 Glu69. Subjects were divided based on the presence (n = 80) or absence (n = 132) of HLA-DPB1 Glu69, and the number of IFN-γ SFUs was compared between the 2 groups. Values above the dotted line represent a positive cytokine response. Significance was determined with the Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E3 Representative examples of intracellular IFN-γ and IL-2 staining in BAL CD4+ T cells from a patient with CBD after 100 μmol/L BeSO4 stimulation. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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