The prevalence of allergic patients suffering from IgE-mediated food allergies has been increasing in recent decades. The immunological mechanisms causing IgE-mediated food allergy have been comprehensively investigated. In this context, B cells are the main source of allergen-specific IgE antibody production and markedly contribute to the development of IgE-mediated food allergy. Therefore, the aim of this study is to characterize the role of single allergen-specific IgE+ B cells in children with cow's milk allergy during oral immunotherapy (OIT).
Peripheral blood mononuclear cells (PBMC) from cow’s milk allergic children participating in clinical OIT were isolated and immortalized with a retroviral vector containing GFP, BCL6, and Bcl-xL. Cells were enriched by culturing with CD40L and IL-21. Total and specific IgE, IgG, and IgG subclass antibodies (IgG1, IgG2, IgG3, and IgG4) from the culture supernatants of immortalized B cells were measured by ELISA. Subsequently, the single IgE+ B cells were identified and sorted in flow cytometry with the combination of different surface markers such as IgM, IgD, IgG1, IgG2, IgG3, IgG4, and IgA. Next generation single cell RNA sequencing was performed for quantitative transcriptomics using state-of-art 10X genomics technology.
The production of specific IgE, IgG1, and IgG4 from the culture supernatants of the immortalized allergen-specific B cells pools from 7 patients before and 18-35 after OIT was significantly increased when compared with the non-specific population. Using the candidates immortalized B cell pools with the highest specific IgE production, we sorted the single allergen-specific IgE+ B cell along with their other immunoglobulin subclasses (IgG1, IgG2, IgG3, IgG4, and IgA) B cells and further processed them using 10X genomics’ single-cell RNA sequencing technology.
This study mainly focuses on the characterization of single allergen-specific IgE+ B cells from allergic individuals. Interestingly, allergen-specific immortalized B cells secreted an increased amount of specific IgE, IgG1 and IgG4. The differentially expressed genes of single allergen-specific IgE+, IgG1+, IgG2+, IgG3+, IgG4+, and IgA+ B cells need to be further investigated to uncover the immunological mechanisms that cause IgE-mediated food allergy.
Single IgE+ B cells, food allergy, oral immunotherapy (OIT), immunoglobulin subclasses, differentially expressed genes (DEGs)