In vitro induction of functional allergen-specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity

Abstract

Background: Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. Objective: We have investigated whether the suppressive function of CD4+ CD25high cells is impaired in IBH-affected horses and possible ways to restore it. Methods: CD4+ CD25- cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or tetanus toxoid as control antigen, in the presence of CD4+ CD25high cells. Furthermore, Culicoides-specific CD4+ CD25high regulatory cells were expanded or induced from CD4+ CD25- cells in vitro in the presence of a combination of rIL-2 and rTGF-β1 (rIL-2/rTGF-β1) or of retinoic acid and rapamycin (RetA/Rapa). Proliferation was determined by [3H] thymidine incorporation and cytokine production measured by flow cytometry. Results: The ability of Culicoides- but not tetanus-stimulated CD4+ CD25high cells to suppress proliferation of CD4+ CD25- cells was significantly lower in IBH-affected horses (28%) than in healthy controls (86%). The decreased suppression in IBH-affected horses was associated with a significantly higher proportion of IL-4+ cells and a lower percentage of FoxP3+ IL-10+ compared to controls. Addition of rIL-2/rTGF-β1 or of RetA/Rapa to Culicoides-stimulated CD4+ CD25high cells from IBH-affected horses significantly increased the proportion of FoxP3+ IL-10+ cells. We also found that RetA/Rapa induced a more significant decrease in the frequency of IL-4+ cells than rIL-2/rTGF-β1. Moreover, the suppressive activity of Culicoides-stimulated CD4+ CD25high cells was significantly restored by both rIL-2/rTGF-β1and RetA/Rapa, albeit in an antigen-unspecific manner. In contrast, in vitro induced Culicoides-specific CD4+ CD25high cells suppressed proliferation of CD4+ CD25- cells in an antigen-specific manner. Conclusion and Clinical Relevance: The in vitro induction of functional allergen-specific Treg cells in IBH-affected horses suggests a potential therapeutic use of these cells in allergy. © 2013 John Wiley & Sons Ltd.