Phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity in patients with inherited IFN-γR1 or IFN-γR2 deficiency Francesca Conti, MD,

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Phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity in patients with inherited IFN-γR1 or IFN-γR2 deficiency Francesca Conti, MD, PhD, Walmir Cutrim Aragão Filho, PhD, Carolina Prando, MD, PhD, Caroline Deswarte, MSc, Marjorie Hubeau, PhD, Peter E. Newburger, MD, Jean-Laurent Casanova, MD, PhD, Jacinta Bustamante, MD, PhD, Antonio Condino-Neto, MD, PhD Journal of Allergy and Clinical Immunology Volume 135, Issue 5, Pages 1393-1395.e1 (May 2015) DOI: 10.1016/j.jaci.2014.11.004 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 NADPH oxidase activity in human EBV-B cells, MDDCs, and MDMs. A, O2− generation of EBV-B cells from healthy controls (n = 31), patients with CGD (n = 18) and XR2-MSMD (n = 6), and patients with IFN-γR1/IFN-γR2 complete (c) deficiencies (n = 9 and 6, respectively), measured by cytochrome-c reduction test after 2 hours of PMA (400 ng/mL) activation. Each symbol represents an individual subject. B, Fluorimetric quatification of H2O2 release from EBV-B cells of healthy controls (n = 12), patients with CGD (n = 6) and XR2-MSMD (n = 6), and patients with cIFN-γR1/cIFN-γR2 deficiency (n = 9 and 6, respectively), measured by Amplex Red assay after 2 hours PMA (400 ng/mL) activation. C and D, Fluorimetric quatification of H2O2 release after 30 minutes from MDMs of healthy controls (n = 7), patients with CGD (n = 2), and patients with cIFN-γR1/cIFN-γR2 deficiency (n = 1 and 2, respectively), measured by Amplex Red assay and then left untreated (NS) or treated for 18 hours with IFN-γ (1 × 105 IU/mL), PPD (1 mg/mL), followed by no trigger or by treatment with PMA (400 ng/mL) activation. E. Release of H2O2 from MDDCs obtained from healthy controls (n = 18), patients with CGD (n = 3), patients with cIFN-γR1/cIFN-γR2 deficiency (n = 1 and 1, respectively), and patients with XR2-MSMD (n = 4), then left untreated (NS) or treated with LPS, followed by no trigger or by treatment with PMA (400 ng/mL). Each symbol represents an individual subject. Data are representative of 2 experiments (Fig 1, A and B; mean of duplicates) and mean of duplicates (Fig 1, C-E). PMA, Phorbol 12-myristate 13-acetate; PPD, purified protein derivative from M tuberculosis. Journal of Allergy and Clinical Immunology 2015 135, 1393-1395.e1DOI: (10.1016/j.jaci.2014.11.004) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 NADPH oxidase activity in human EBV-B cells. A, Monitoring of intracellular activity by luminol untreated or treated with PMA (400 ng/mL) using 350,000 EBV-B cells from healthy controls (n = 7), patients with CGD (n = 3), and patients with cIFN-γR1 (n = 5) and cIFN-γR2 (n = 3) deficiency. B, Monitoring of extracellular activity by isoluminol in response to PMA (400 ng/mL). We used 350,000 EBV-B cells from healthy controls (n = 7), patients with CGD (n = 3), and patients with cIFN-γR1 (n = 5) and cIFN-γR2 (n = 3) deficiency. PMA, Phorbol 12-myristate 13-acetate; RLU, relative light units. The kinetic data are mean of duplicates for 2 independent experiments. Journal of Allergy and Clinical Immunology 2015 135, 1393-1395.e1DOI: (10.1016/j.jaci.2014.11.004) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions