Oral Tolerance Induction with Antigen Conjugated to Cholera Toxin B Subunit Generates Both Foxp3+CD25+ and Foxp3−CD25− CD4+ Regulatory T Cells

Abstract

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 (Treg). We found that total Treg, KJ1–26+ Treg and CTLA-4+ Treg 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-β in serum. This could be abolished by coadministering cholera toxin or by treatment with anti-TGF-β mAb. CD25+ Treg, 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+ Treg from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ Treg. Furthermore, in Rag1−/− mice that had adoptively received highly purified Foxp3−CD25−CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ Treg cells, which expressed Foxp3 more strongly than naturally developing Treg and also had stronger ability to suppress effector OT-II 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-β and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ Treg together with the generation of both Foxp3+ and Foxp3−CD25− CD4+ Treg.

Secretory IgA-mediated protection againstV. choleraeand heat-labile enterotoxin-producing enterotoxigenicEscherichia coliby rice-based vaccine

“…In fact, rice seed containing CTB-fused allergen-specific T cell epitopes induces oral tolerance to allergen more efficiently than does rice expressing allergen-specific epitopes alone (34). Moreover, conjugation of an antigen to CTB can induce the proliferation of regulatory T cells (35,36); this may be the mechanism by which the above mentioned rice seed containing the CTB-fused epitopes effectively induces oral tolerance.…”

Important Role for FcγRIIB on B Lymphocytes for Mucosal Antigen-Induced Tolerance and Foxp3+ Regulatory T Cells

“…Previous studies showed that intragastric, s.l., or intranasal mucosal administration of OVA and other Ags in mice, given alone or with increased efficiency when linked to CTB, can induce peripheral T cell tolerance ("oral tolerance") along with Ag-specific Tregs (8,9). We examined whether FcgRIIB is required for such mucosal induction of tolerance, initially by comparing the effect on the development of DTH in FcgRIIB 2/2 mice and WT mice after s.l.…”

“…We (3)(4)(5) and other investigators (6)(7)(8) demonstrated that mucosal administration of various types of Ags coupled to cholera toxin B subunit (Ag/CTB) by either chemical conjugation or genetic fusion can more efficiently, and at .100-fold reduced dosages compared with Ag alone or Ag mixed with cholera toxin B subunit (CTB), induce peripheral T cell tolerance. Similar to other low-dose tolerance, this effect is associated with induction of Foxp3 + , as well as Foxp3 2 , Tregs (9). The coupling of Ag to CTB, which binds to GM1 ganglioside receptors present on many cell types, including all known APCs, strongly potentiates the mucosal tolerizing effect (10) by facilitating Ag transport across the mucosal barrier and, more importantly, increasing Ag uptake and presentation by APCs, such as dendritic cells (DCs), B cells, and macrophages (11,12).…”

“…Highly purified recombinant CTB was kindly provided by Crucell-Sweden (Stockholm, Sweden). OVA protein was chemically coupled to CTB using N-succininmidyl (3-[2-pyridyl]-dithio) propionate (Pierce Biotechnology, Rockford, IL) as a bifunctional coupling reagent, as described elsewhere (9). The OVA/CTB conjugate was purified by fast protein liquid chromatography gel filtration on a Superdex 200 16/60 column (Pharmacia Biotech) using the BioLogic Workstation FPLC System (Bio-Rad, Richmond, CA).…”

“…The OVA/CTB conjugate was purified by fast protein liquid chromatography gel filtration on a Superdex 200 16/60 column (Pharmacia Biotech) using the BioLogic Workstation FPLC System (Bio-Rad, Richmond, CA). The purified conjugate was analyzed by ganglioside-monosialic acid (GM1) ELISA and shown to have strong GM1-binding activity, as well as high reactivity with Abs to either OVA or CTB (9). The conjugate also had a strong capacity to induce OVA-specific T cell proliferation when tested on OT-II splenocytes; further, in the latter assays, the activity of the conjugate was not significantly inhibited by preincubation and coculture with polymyxin, but it was completely inhibited by preincubation and coculture with highly purified GM1 (a gift from the late Prof. Lars Svennerholm).…”