Multicolor flow-cytometric analysis of milk allergen-specific T-helper type 2 cells revealed coexpression of interleukin-4 with Foxp3

Background Allergen-specific T-helper type 2 (T_H2) cells play an important role in the development of allergic inflammation; however, investigations of the properties of allergen-specific T cells have been challenging in humans. Despite clear evidence that forkhead box p3 (Foxp3) is expressed in conventional effector T cells, its function has remained unknown. Objective To characterize allergen-specific T_H2 cells in milk allergy, with particular focus on the expression of Foxp3. Methods Twenty-one children with milk allergy and 11 children without milk allergy were studied. Peripheral blood mononuclear cells from subjects were stimulated with milk allergen for 6 hours and analyzed using multicolor flow cytometry to identify CD154⁺ allergen-specific T-helper cells. Simultaneously, the expression of intracellular cytokines and Foxp3 was analyzed. Results The milk allergy group had significantly larger numbers of milk allergen-specific interleukin (IL)-4- and IL-5-producing CD4⁺ T cells than the control group. Subjects in the milk allergy group had significantly more CD154⁺CD4⁺ IL-10-producing cells and CD154⁺Foxp3⁺CD4⁺ cells than those in the control group. In addition, the number of milk allergen-specific CD154⁺Foxp3⁺CD4⁺ cells strongly correlated with that of CD154⁺IL4⁺CD4⁺ cells. Bcl-2 expression in CD154⁺IL-4⁺Foxp3⁺ T-helper cells was significantly lower compared with that in total CD4 cells. Conclusion Increased numbers of IL-4-producing allergen-specific T-helper cells were found in patients with milk allergy. In addition, Foxp3 was coexpressed with IL-4 in allergen-specific T_H2 cells from patients. This coexpression was associated with lower Bcl-2 levels and could contribute to the phenotype and function of T_H2 cells.