Flow cytometry imaging identifies rare T_H2 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_H2-inducing properties through dendritic cells. Expression of TSLP receptor on the surface of activated T_H2 cells could amplify T_H2 responses at inflamed sites through the direct actions of TSLP. Objective: To test rigorously whether T_H2 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 _H2-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_H2-promoting stimuli induced a robust population of activated T_H2 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 _H2 cells. The direct action of TSLP on T cells could amplify T _H2 responses at sites of allergic inflammation.