Oral tolerance induction with antigen conjugated to cholera toxin B subunit generates both Foxp3+CD25+ and Foxp3-CD25- CD4+ regulatory T cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25(+)CD4(+) regulatory T cells (T-reg). We found that total T-(reg), KJI-26(+) T-reg and CTLA-4(+) T-reg were all increased in Peyer's patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-beta in serum. This could be abolished by coadministering cholera toxin or by treatment with anti-TGF-beta mAb. CD25(+) T-reg but also CD25(-)CD4(+) T cells from OVA/ CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25(+) T-reg from OVA/CTB-treated mice, and treatment also markedly expanded CD25(+)Foxp3(+) T-reg. Furthermore, in Rag1(-/-) mice that had adoptively received highly purified Foxp3(-)CD25(-)CD4(+) OT-II T cells OVA/CTB feeding efficiently induced CD25(+) T-reg cells, which expressed Foxp3 more strongly than naturally developing T-reg and also had stronger ability to suppress effector OT-11 T cell proliferation. A remaining CD25(-) T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-beta and in both the frequency and suppressive capacity of Foxp3(+) and CTLA-4(+) CD25(+) T-reg together with the generation of both Foxp3(+) and Foxp3(-)CD25(-) CD4(+) T-reg. The Journal of Immunology, 2006,177: 7634-7644.