E Holen1, B Bolann, S Elsayed. 1. Allergy Research Group, Department of Clinical Biochemistry, University Hospital, Haukeland sykehus, Bergen, Norway.
Abstract
BACKGROUND: Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. OBJECTIVES: The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. METHODS: Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. RESULTS: Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61-74, had the ability to bind IgE. Another peptide, OM 101-114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41-56, OM 71-84, OM 131-144 and OM 171-186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-gamma, IL-6, IL-4, IL-13, IL-10 and TNF-alpha in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-gamma, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71-84 and 51-64) when TCLs from two non-allergic donors were used. CONCLUSIONS: One novel B cell epitope (OM 61-74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1-14 to 71-84, the overlapping peptide-pairs OM 121-134 and OM 131-144 and peptides OM 161-174 and 171-186. Peptides OM 1-14 and 171-186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11-24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-gamma and particularly IL-6.
BACKGROUND:Chickenovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. OBJECTIVES: The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. METHODS: Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. RESULTS: Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61-74, had the ability to bind IgE. Another peptide, OM 101-114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41-56, OM 71-84, OM 131-144 and OM 171-186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-gamma, IL-6, IL-4, IL-13, IL-10 and TNF-alpha in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-gamma, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71-84 and 51-64) when TCLs from two non-allergic donors were used. CONCLUSIONS: One novel B cell epitope (OM 61-74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1-14 to 71-84, the overlapping peptide-pairs OM 121-134 and OM 131-144 and peptides OM 161-174 and 171-186. Peptides OM 1-14 and 171-186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11-24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-gamma and particularly IL-6.
Authors: Anna M Ehlers; Henny G Otten; Eva Wierzba; Ulrike Flügge; Thuy-My Le; André C Knulst; Waltraud Suer Journal: Clin Exp Allergy Date: 2020-09-24 Impact factor: 5.018