Wisniewski et al.
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ONLINE REPOSITORY MATERIALS
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Julia A. Wisniewski, MD1,2*, Scott P. Commins, MD, PhD1,2*, Rachana Agrawal, PhD1, Kathryn
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E. Hulse3, Mingxi D. Yu, MD1, Julia Cronin, MD1, Peter W. Heymann, MD2, Anna Pomes,
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PhD4, Thomas Platts-Mills, MD PhD1, Lisa Workman, BS1, Judith A. Woodfolk, MBChB,
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PhD1.
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*J. Wisniewski and S. Commins contributed equally to this manuscript.
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Department of Medicine and 2Department of Pediatrics, University of Virginia Health System,
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Charlottesville, VA 22908, USA. 3Division of Allergy-Immunology, Feinberg School of
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Medicine, Northwestern University, Chicago, IL 60611, USA. 4Indoor Biotechnologies,
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Charlottesville, VA, 22903, USA.
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Corresponding author: Judith A. Woodfolk, MBChB, PhD, Allergy Division, PO Box 801355,
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University of Virginia Health System, Charlottesville, VA 22908-1355. Tel: 434-924-1293;
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Fax: 434-924-5779; e-mail: jaw4m@virginia.edu.
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Methods
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Oral Immunotherapy Regimen.
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Pre-measured doses of peanut flour (Item #RMF171P, Greer, Lenoir, NC; 2g flour = 1g
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peanut protein) were prepared as previously described.1 The highest tolerated dose (or 3mg) of
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peanut flour was used as the starting dose for the buildup phase and was given on the UVA
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Hospital Clinical Research Unit. Thereafter, subjects dosed at home for two weeks. Subjects
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returned every two weeks for approximately 22 weeks for dose escalations. Doses were
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increased by 50-100% until a dose of 75mg was attained. Doses were then increased by 25-33%
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until reaching maintenance dose (306mg roughly equivalent to 1 peanut).2 Three subjects
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withdrew during the dose escalation phase owing to gastrointestinal symptoms. After dose
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escalation, subjects were instructed to continue to ingest the maintenance dose daily for at least
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four months before returning for an open oral food challenge (OFC). During OFC, subjects
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ingested doses of peanut flour every 10-20 minutes up to a cumulative dose of 5000 mg of
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protein. Among subjects who experienced a decrease in IgE to peanut below 15kUA/L at the
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time of OFC, maintenance OIT was discontinued and an open OFC was administered at 1 month
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after cessation of therapy to assess sustained unresponsiveness. If subjects passed that challenge,
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they were then asked to keep peanut in their diet 2-3 times per week. No failures were observed
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in this study arm. All subjects with high IgE (>15kUA/L) passed OFC after 4 months on
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maintenance therapy, but continued on daily maintenance OIT (300-600mg) with re-testing of
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IgE to peanut every 6 months. Subjects selected for T-cell studies experienced no significant
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decrease in IgE to peanut during the pilot study.
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Antibodies and Other Reagents.
Purified natural Ara h 1, Ara h 2, and Fel d 1 were obtained from Indoor Biotechnologies,
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Inc. (Charlottesville, VA). The endotoxin content of each allergen was low (≤ 6.0 EU/g).
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Antibodies against CD3 (PerCP, clone UCHT1), CD25 (brilliant violet-711, clone BC96), IL-4
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(PerCP/Cy5.5, clone MP4-25D2), IFN- (Alexa 700, clone 4S.B3), IL-17A (APC Cy7, clone
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BL168), and IL-10 (PECy7, clone JES3-907), were obtained from Biolegend (San Diego, CA).
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Anti-Foxp3 (PE, clone 236A/E7), anti-IL-13 (FITC, clone PVM13-1), and Foxp3 Fix/Perm kit
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were purchased from eBioscience (San Diego, CA). Anti-IL-5 (APC, clone JES1-39D10) was
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purchased from Miltenyi Biotech. Anti-CD8 (PE-TR, clone 3B5), anti-CD14 (PE-TR, clone
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TüK4), anti-CD19 (PE-TR, clone SJ25-C1) and fixable Aqua Live/Dead Staining Kit were
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obtained from Invitrogen (Eugene, OR). Mouse IgG used to block Fc receptors prior to cell
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staining was obtained from Lampire Biological Laboratories (Pipersville, PA).
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References
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1. Vickery BP, Lin J, Kulis M, Fu Z, Steele PH, Jones SM, et al. Peanut oral immunotherapy
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modifies IgE and IgG4 responses to major peanut allergens. J Allergy Clin Immunol
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2013;131:128-34.
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2. Jones SM, Pons L, Roberts JL, Scurlock AM, Perry TT, Kulis M, et al. Clinical efficacy and
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immune regulation with peanut oral immunotherapy. J Allergy Clin Immunol. 2009;124:292-
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300.
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Figure Legends.
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Figure S1. Study Design for Children Receiving Peanut Oral Immunotherapy. Arrows
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denote time points of blood draws. All children received oral food challenge after 4 months of
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maintenance therapy. Grey shading denotes the group from which children were selected for T
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cell studies.
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Figure S2. Longitudinal Change in Serum IgE During Peanut Oral Immunotherapy.
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Changes in IgE to peanut and its major allergens were assessed in children who received OIT.
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Data is shown for serum samples corresponding to 18 children for whom sufficient sample was
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available for component analysis at baseline. IgE data was obtained on 16 of these subjects at
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the 12-24 month time point. Dashed line denotes cut-off of 15 kUA/L. NS, not significant.
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Figure S3. Analysis of IL-10+ T Cells in PBMC Cultures Stimulated with Major Peanut
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Allergens. (A) Percentage of IL-10+ T cells in cultures from PA children and those who
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received OIT for 12-24 months (n=7 per group). NS, not significant. (B) Representative scatter
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plots for one subject from each group.
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Figure S4. Analysis of Cytokine-Producing Subpopulations in Peanut-Stimulated Cultures.
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(A) Pie charts comparing the distribution of T cell subpopulations within IL-4+ and IFN-+
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T-cell subsets induced by Ara h 1 in OIT (n=7) and non-OIT (n=5) groups. (B) Effect of
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increased duration of OIT on the distribution of T-cell subpopulations within Ara h 2-induced
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IFN-+ T cells in cultures from 2 children.
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Figure S5. Foxp3 Expression Profiles in Cultures from PA Children. (A) Mean
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fluorescence intensity (MFI) of Foxp3 expression within T cell subtypes induced by peanut
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allergens. * p≤0.01 for IL-4+ T cells versus CD25neg, CD25+, IFN-+ and IL-17+ subsets.
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