Three Complementary Pathways Characterize the Suppressive Properties of Epit-Induced Tregs

Rationale: Epicutaneous immunotherapy (EPIT) demonstrates clinical efficacy and induces significant Foxp3+ Tregs in mice. In addition, adoptive transfer of EPIT-induced Tregs protects mice from anaphylaxis and further sensitizations (bystander effect).  Nevertheless, mechanisms of action of EPIT-induced Tregs are not clearly elucidated. This study analyzed the suppressive properties of EPIT-induced Tregs in specific/bystander conditions.

 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.