Oral antigen exposure under co-stimulation blockade generates Treg cells to establish immune tolerance despite prior sensitization

Antigen-specific oral tolerance is effective in preventing harmful immune responses in antigen-non-sensitized animals but difficult to induce in those already antigen-primed. Here, we show in mice that feeding of antigen-containing diet generates in mesenteric lymph nodes and intestinal lamina propria peripherally derived regulatory T (pTreg) cells that exhibit a terminally differentiated effector phenotype as seen with tissue-resident thymus-derived Treg (tTreg) cells. Antigen feeding also enables the generated pTreg cells to acquire Treg-specific epigenomic changes in Treg signature genes including Foxp3 , hence the stability of Treg-specific function. Cessation of antigen feeding, however, results in decline in oral tolerance with diminution of pTreg cells. Transcriptomic comparison with tTreg cells has revealed that pTreg cells induced by antigen feeding predominantly express CD101. CD101 ⁺ Treg cells with a similar phenotype and epigenetic alterations can be generated in vitro from antigen-primed naïve CD4 ⁺ T cells by blockade of CD28-mediated co-stimulation during TGF-β-dependent Treg induction. Furthermore, in vivo CD28 signal blockade by CTLA-4-immunoglobulin fusion protein (CTLA4-Ig) before antigen feeding can establish oral tolerance in the mice precedingly antigen-sensitized by a non-oral route. The CTLA4-Ig treatment not only suppresses activation of antigen-reactive conventional T cells but also facilitates their differentiation into antigen-specific CD101 ⁺ pTreg cells. Thus, continuous oral antigen administration combined with CD28 co-stimulation blockade generates a specific type of antigen-specific functionally stable pTreg cells, which can establish long-term systemic antigen-specific immune tolerance even in antigen-pre-sensitized animals. This strategy for mucosal tolerance induction would be instrumental in treating chronic allergy and other immunological diseases in humans.