Methods: Milk-sensitized BALB/c mice were treated or not with milk EPIT. Tregs (CD4+CD25+ T cells) from EPIT, Sham or non-sensitized groups and effector T cells (CD4+CD25-) from milk or peanut sensitized mice were sorted and co-cultured for 4 days at different ratios, using allergen stimulation and allergen-pulsed CD11c+ antigen-presenting cells. Anti CTLA-4, anti-TGF-b antibodies were also tested to determine whether EPIT-induced Tregs act via cytokines or by cell-contact dependent mediation. Suppression was analyzed by tracking divided CD4+CD25- with CFSE by flow cytometry. Supernatants were also collected to quantify cytokine secretion.
Results: Effector T cells proliferate up to 82.5%-92.5%, respectively with milk or peanut stimulation. Presence of EPIT-induced Tregs significantly inhibited effector T cells proliferation in specific or bystander conditions (68-71% of proliferation, i.e. 20%-25% proliferation inhibition) compared to Sham or non-sensitized Tregs. Interestingly, blocking CTLA-4 and TGF-b strongly abrogated suppression capacity of EPIT-induced Tregs in both conditions. IL-2 and TH2 cytokines were barely detectable in supernatants with EPIT Tregs.
Conclusions: EPIT-induced Tregs inhibited effector T cell proliferation with the same potency in specific and bystander conditions. Suppression induced by EPIT-induced Tregs might use 3 complementary pathways: a low availability of IL-2, TGF-b secretion and CTLA-4 cell contact mediation.