Reply Journal of Allergy and Clinical Immunology

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Reply Journal of Allergy and Clinical Immunology Eszter Herczenik, PhD, Simon D. van Haren, PhD, Aleksandra Wroblewska, MSc, Paul Kaijen, MSc, Maartje van den Biggelaar, PhD, Alexander B. Meijer, PhD, Anja ten Brinke, PhD, Jan Voorberg, PhD Journal of Allergy and Clinical Immunology Volume 129, Issue 4, Pages 1174-1175 (April 2012) DOI: 10.1016/j.jaci.2011.12.1004 Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Proliferation of a FVIII C2 domain–specific CD4+ T-cell clone is mannan insensitive. A, Monocytes were isolated from a matched HLA DRB1∗0101/DRB1∗1301 and cultured with 1000 U/mL of GM-CSF and 800 U/mL of IL-4 for 4-7 days as described.1 DCs were first pretreated for 15 minutes with 1 mg/mL of mannan and incubated with 100 nM of FVIII for 1 hour to allow endocytosis. Then the cells were washed twice, resuspended at a concentration of 5 × 103/100 μL, and combined with 5 × 104/100 μL of carboxyfluorescein succinimidyl ester–labeled CD4+ T-cell clone 32A-18, which was derived from a DRB1∗0101-positive hemophilia A patient.2,3 After 6 days of culture, the mean fluorescent intensity of the resuspended CD4+ T cells was measured and the data were analyzed with FlowJo 7.6.4 software (Tree Inc, Ashland, Ore). B, The glycosylation site N2118 in the C1 domain remains distant from the KM33 epitope that includes residues K2092 and F2093. The carbohydrate structure depicted is a core-fucosylated high-mannose structure that was modeled by using the glycam Webserver (http://glycam.ccrc.uga.edu/ccrc/). The image was generated by using the PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC (New York, NY). FITC, Fluorescein isothiocyanate. Journal of Allergy and Clinical Immunology 2012 129, 1174-1175DOI: (10.1016/j.jaci.2011.12.1004) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions