Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions.

Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed-type hypersensitivity (DTH) responses? To what extent can s.l.-induced tolerance be explained by the generation of Foxp3+CD25+CD4+ regulatory T cells (T(reg))? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40-mug doses of OVA/CTB conjugate suppressed T-cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag-specific T(reg) in CLN, MLN and spleen and also transforming growth factor-beta (TGF-beta) levels in serum. The CLN and splenic cells from OVA/CTB-treated BALB/c mice efficiently suppressed OVA-specific T-cell receptor (TCR) transgenic (DO11.10) CD25-CD4+ effector T-cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA-specific DTH responses in vivo and T-cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA-specific (KJ1-26+) CD4+ T cells from OVA/CTB-treated mice. Thus, s.l. administration of CTB-conjugated Ag can efficiently induce peripheral T-cell tolerance associated with strong increases in serum TGF-beta levels and in Ag-specific Foxp3+CD25+CD4+ T(reg) cells.