Abstract
Up to 30% of the population suffers from immunoglobulin E (IgE)-mediated allergies. Despite current stepwise gating approaches, the unambiguous identification of human IgE-producing cells by flow cytometry and immunohistology remains challenging. This is mainly due to the scarcity of these cells and the fact that IgE is not only expressed in a membrane-bound form on the surface of IgE-producing cells in form of the B cell antigen receptor (BCR), but is more frequently found on various cell types bound to the low and high affinity receptors, CD23 and FcϵRI, respectively. Here we sought to develop a sequential gating strategy for unambiguous detection of cells bearing the IgE BCR on their surface. To that aim we first tested the monoclonal anti-IgE antibody omalizumab for its ability to discriminate between IgE BCR and receptor-bound IgE using cells producing IgE or bearing IgE bound to CD23 as well as basophils exhibiting FcϵRI receptor-bound IgE. Using flow cytometry, we demonstrated that omalizumab recognized IgE producing cells with a high sensitivity of up to 1 IgE+ cell in 1000 human peripheral blood mononuclear cells (PBMCs). These results were confirmed by confocal microscopy both in cell suspensions as well as in nasal polyp tissue sections. Finally, we established a consecutive gating strategy allowing the clear identification of class-switched, allergen-specific IgE+ memory B cells and plasmablasts/plasma cells in human PBMCs. Birch pollen specific IgE+ memory B cells represented on average 0.734% of total CD19+ B cells in allergic patients after allergen exposure. Thus, we developed a new protocol for exclusive staining of non-receptor bound allergen-specific IgE+ B cell subsets in human samples.
Highlights
Allergy, a worldwide disease affecting up to 30% of the world population, is characterized by immunoglobulin E (IgE) production specific to the culprit allergens [1]
In order to test if the humanized monoclonal anti-human IgE antibody omalizumab fulfilled the requirements of reliably detecting IgE as present in the IgE B cell antigen receptor (BCR) but not receptorbound IgE we performed fluorescent staining in three different cell types: (i) In the human IgE myeloma cell line U266, an IgEproducing cell line that has previously been used as a surrogate for BCR-bearing and IgE-producing cells [32, 33] (ii) In an Epstein barr virus (EBV)-transformed B cell line (EBV B cells) expressing high levels of CD23 incubated with humanized birch pollen specific IgE prior to staining [15, 34] and (iii) in human basophils enriched from blood of donors and bearing IgE bound to their high affinity receptor FcεRI
We have shown that the anti-human IgE antibody omalizumab recognizes IgE B cell antigen receptor whilst omitting cells bearing IgE bound to CD23 or FcεRI both in cellular suspension as well as in tissue using flow cytometry and confocal microscopy
Summary
A worldwide disease affecting up to 30% of the world population, is characterized by immunoglobulin E (IgE) production specific to the culprit allergens [1]. Though IgE is continuously produced and returns to baseline levels within few days after removal by extracorporeal immunoadsorption in sensitized patients [2], the location and the extent of contribution of IgE B cell antigen receptor (BCR) bearing memory B cells (MBCs) to human IgE production is not fully clarified [3, 4] This is mainly due to limited knowledge of these cells [5] as the characterization of human IgE-producing cells in blood by flow cytometry is challenging due to several reasons: Firstly, IgE BCR bearing cells are extremely rare in the blood. Especially CD23-bound IgE renders the detection of IgE+ BCR bearing cells difficult as many commonly used anti-human IgE antibodies are unable to discriminate between the membrane-expressed and receptor-bound form of IgE
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