An integrated framework using high-dimensional mass cytometry and fluorescent flow cytometry identifies discrete B cell subsets in patients with red meat allergy.

BACKGROUND: B cells play a critical role in the development and maintenance of food allergy by producing allergen-specific IgE. Despite the importance of B cells in IgE-mediated food allergy, the identity of sIgE-producing human B cells and how IgE is regulated are poorly understood. OBJECTIVE: To identify the immunophenotypes of circulating B cells associated with the production of galactose-alpha-1,3-galactose-specific IgE production in patients with red meat allergy. METHODS: B cells in PBMC samples obtained from 19 adults with physician-diagnosed red meat allergy and 20 non-meat allergic healthy controls were assessed by mass cytometry along with a bioinformatics analysis pipeline to identify discrete B cell phenotypes that associated with serum sIgE. Fluorescent flow cytometry was then applied to sort purify discrete B cell subsets, and B cells were functionally evaluated on an individual cell level for the production of sIgE by ELISPOT. RESULTS: Discrete B cell phenotypes abundant in meat allergic subjects compared to non-meat allergic controls were found in peripheral blood that do not share typical characteristics of classical isotype-switched memory B cells that express high levels of CD27. These B cell subsets shared higher IgD and lower IgM expression levels coupled with CXCR4, CCR6 and CD25 expression. In vitro polyclonal stimulation of purified B cell subsets from meat allergic subjects demonstrated that these subsets were enriched for cells induced to secrete sIgE. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating B cells display increased abundance of discrete B cell subsets in meat allergic subjects. This observation, coupled with the capacity of individual B cell subsets to produce sIgE following activation, implicates these novel B cell phenotypes in promoting IgE in meat allergy.

Flow cytometry imaging identifies rare T(H)2 cells expressing thymic stromal lymphopoietin receptor in a "proallergic" milieu.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is expressed at sites of allergic inflammation, including eczematous skin. This cytokine has been reported to exert its T(H)2-inducing properties through dendritic cells. Expression of TSLP receptor on the surface of activated T(H)2 cells could amplify T(H)2 responses at inflamed sites through the direct actions of TSLP. OBJECTIVE: To test rigorously whether T(H)2 cells induced by "proallergic" factors express TSLP receptor and characterize these cells using an experimental platform that combines flow cytometry with microscopic capabilities. METHODS: CD4(+) T cells isolated from patients with atopic dermatitis or normal healthy controls were cocultured with autologous dendritic cells in the presence of T(H)2-promoting stimuli (TSLP ± allergen and staphylococcal enterotoxin B ± TSLP). Surface expression of TSLP receptor was analyzed by image-based flow cytometry, and responsiveness of purified T cells to TSLP was assessed by phosphorylation of signal transducer and activator of transcription-5 and cytokine secretion. RESULTS: T(H)2-promoting stimuli induced a robust population of activated T(H)2 cells (CD25(+)IL-4(+)). Regardless of the nature of the stimulus, flow cytometry imaging confirmed that T cells expressing TSLP receptor were rare, constituting a minor fraction of the IL-4(+) T cell pool; however, TSLP responsiveness was nonetheless detectable. Analysis of cell size and nuclear morphology revealed preferential expression of TSLP receptor on IL-4-expressing cells undergoing mitosis. Analysis of lesional skin in atopic dermatitis supported the view that rare IL-4(+) T cells expressing TSLP receptor are present at inflamed sites. CONCLUSION: In a "proallergic" milieu, TSLP receptor is preferentially expressed on rare actively dividing T(H)2 cells. The direct action of TSLP on T cells could amplify T(H)2 responses at sites of allergic inflammation.