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Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency  Jacob M. Rosenberg, ScB, Jordan.

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Presentation on theme: "Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency  Jacob M. Rosenberg, ScB, Jordan."— Presentation transcript:

1 Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency  Jacob M. Rosenberg, ScB, Jordan V. Price, PhD, Gabriela Barcenas-Morales, PhD, Lourdes Ceron-Gutierrez, BSc, MSc, Sophie Davies, BSc, Dinakantha S. Kumararatne, MBBS, FRCpath, DPhil, Rainer Döffinger, PhD, FRCPath, Paul J. Utz, MD  Journal of Allergy and Clinical Immunology  Volume 137, Issue 1, Pages e3 (January 2016) DOI: /j.jaci Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Protein microarrays for ACAA detection. A, Schematic representation of cytokine microarrays. Cytokines, chemokines, growth factors, and traditional autoantigens are printed onto a nitrocellulose-coated microscope slide. Arrays are probed with serum (green) and detected by using fluorophore-conjugated secondary antibody (red). B-D, Representative images of protein microarrays probed with serum from a healthy control subject (Fig 1, B), a patient with PAP (Fig 1, C), and a patient with APS-1 (Fig 1, D). A control IgG feature printed on the array is boxed in cyan, GM-CSF features are boxed in green, and IFN-α2a features are boxed in purple. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Unsupervised hierarchical clustering demonstrates 2 distinct groups of cytokine reactivity. Fifty-eight serum samples from patients with immunodeficiency and healthy control subjects were probed on protein microarrays. Samples were hierarchically clustered in an unsupervised fashion by using a Pearson correlation and plotted with samples along the x-axis, and autoantigens, cytokines, and chemokines were plotted along the y-axis. Clusters of high reactivity to GM-CSF (green box) and type I interferons (purple box) were identified. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 SAM identifies reactivities associated with ACAAs against type I interferons and GM-CSF. Three-class SAM was applied based on cytokine reactivity to type I interferons, GM-CSF, or neither. SAM identified 14 statistically significant differences. These targets include cytokines purchased from multiple vendors. GM-CSF reactivity and type I interferon–associated reactivity are boxed in green and purple, respectively, for emphasis. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Comparison of protein microarray performance with an established bead-based assay. A, Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and IFN-α, and Pearson correlation coefficients were determined. All P values were less than B, Receiver operating characteristic (ROC) curves were generated to assess the performance of protein microarrays, with a bead-based assay used as the gold standard. P values were less than .05 for all areas under the curve. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Sera from patients with ACAAs block cytokine signaling in vitro. A, U937 cells were incubated in the presence of sera from healthy control subjects or from patients with PAP and stimulated with LPS alone or LPS and GM-CSF. IL-6 concentrations were measured by the using Luminex analyzer. An IL-6 stimulation index was calculated as described in the text. B, Heterologous PBMCs were incubated in the presence of LPS alone or LPS and IFN-α. TNF-α concentration was measured by using a Luminex analyzer. A TNF-α stimulation index was calculated as described in the text. C, Heterologous PBMCs were incubated in the presence of medium, IL-12, or IL-12 and IL-18. IFN-γ levels in the supernatant were measured by ELISA and plotted. Given the limited sample size, statistical analysis of a stimulation index was not performed. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Protein microarray composition. Purified biomolecules, including cytokines, chemokines, growth factors, and autoantigens, are listed with their respective vendors. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 Clinical characteristics of the cohort. The clinical diseases and phenotypes of patients and healthy control subjects are listed. Patients with more than 1 disease or disorder are listed more than once under each applicable category. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 IL-23 ACAA validation. IL-23 binding was also measured by means of a Luminex analyzer, as described in the Methods section, to confirm IL-23 reactivity observed by using a cytokine microarray. MFI of samples 092B and 562B were compared with all others by using the unpaired t test. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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