BCR analysis of single-sorted, putative IgE⁺memory B cells in food allergy:an ephemeral existence?

Abstract

One Sentence Summary: The frequency of IgE + MBCs in the peripheral circulation of humans is orders of magnitude lower than previously reported and comparable between allergic and healthy donors, which cautions about the clinical utility of their assessment.Abstract: Immunoglobulin (Ig) E is the critical effector molecule in allergic reactions. Consequently, research efforts to understand the biology of IgE-expressing cells is of paramount importance. In particular, the role of IgE + memory B cells (MBCs) in the perpetuation of allergic reactivity has been the subject of intense research. Studies in mice have convincingly established that IgE + B cells are rare and transient and, therefore, an unlikely candidate to maintain allergic disease. In contrast, IgE + MBCs have been detected by flow cytometry in the sputum and peripheral blood of humans and have been proposed as a clinical marker of allergic disease. We established a method to genetically validate, at the single-cell level, the putative IgE + MBCs identified by flow cytometry from humans. We, then used this information to develop an enhanced flow cytometry protocol that more accurately identifies bona fide IgE + MBCs. We found that IgE + MBCs were detected in some patients with atopic dermatitis, but at a frequency that was ~100 times lower than previously reported. We also found that IgE + MBCs were undetectable in PBMCs from peanut allergic patients. These findings provide tools to identify bona fide IgE + MBCs, demonstrate their extreme rarity in circulation and are consistent with the lack of a central role for IgE + MBCs in the maintenance of allergic sensitivity. Main Text

A Distinct Phenotype of Polarized Memory B cell holds IgE Memory

“…A clear consensus has emerged that allergen-specific B cell memory is held by non-IgE MBCs (7)(8)(9). In both mice and humans, the allergen-specific MBC repertoire is dominated by IgG1, while IgE B cells are short-lived and IgE MBCs are exceptionally rare or absent (8,(10)(11)(12).…”

“…A clear consensus has emerged that allergen-specific B cell memory is held by non-IgE MBCs (7)(8)(9). In both mice and humans, the allergen-specific MBC repertoire is dominated by IgG1, while IgE B cells are short-lived and IgE MBCs are exceptionally rare or absent (8,(10)(11)(12). IgG1expressing MBCs become IgE-producing plasma cells (PC) during a memory response to allergen exposure, contributing to the persistence of IgE-mediated allergies (9,(13)(14)(15)(16).…”

Tracing Human IgE B Cell Antigen Receptor-Bearing Cells With a Monoclonal Anti-Human IgE Antibody That Specifically Recognizes Non-Receptor-Bound IgE

“…As proof of concept, we aimed to identify blood-derived allergenspecific MBCs and/or PBs/PCs bearing a surface IgE BCR in birch pollen allergic patients by employing a sequential gating strategy based on recent publications (6,7,9). To that aim, PBMCs were isolated from birch pollen allergic donors one week after controlled nasal allergen challenge as well as from nonallergic donors.…”

“…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. They are estimated to represent between 0.0019 and 0.3% of total B cells in allergic subjects (6)(7)(8)(9) and to contribute to 0.2% of the human serum IgE (4,10). Secondly, the IgE BCR is expressed at much lower levels than BCRs composed of other immunoglobulins such as IgG or IgM (7).…”

“…The compromise of excluding cells double positive for IgE and CD23 in the flow cytometer comprises the danger of accidentally removing true IgE + B cells having both free as well as CD23-bound IgE (20). Thus, more recent approaches to circumvent this issue applied stepwise gating for IgE + memory B cells and plasmablasts (PBs)/plasma cells (PCs) firstly using anti-CD19 and anti-CD38, followed by sequential exclusion of IgM + , IgD + , IgA + and IgG + cells (6,7,21) or intracellular staining for IgE for identification of IgE-producing cells (5,8). Nevertheless, these strategies identify IgE + cells only indirectly by stepwise exclusion of other cells and may also miss IgE + B cells having IgG bound to their FcgRIIb receptor (22)(23)(24).…”