Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells

Abstract

Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.Cholera toxin B subunit (CTB) and the closely related Escherichia coli heat-labile enterotoxin B subunit (LTB) are highly efficient carrier molecules for the induction of mucosal antibody responses (10, 17, 27, 31) and oral tolerance (2, 17, 42). The therapeutic applications of CTB-mediated oral tolerance, as demonstrated in animal models, include the prevention and treatment of T-cell-mediated autoimmune diseases (7,44,49), immunoglobulin E (IgE)-mediated allergic reactions (37,47,55), and infection-induced pathological inflammatory conditions (32, 43). The mechanism behind CTB's or LTB's efficacy as a mucosal carrier molecule has not been fully defined but is believed to be associated with the strong binding of CTB or LTB to the GM1 receptor present on most cells in the body, including epithelial cells and leukocytes. Efficient binding to GM1 could potentially increase the uptake of antigen across the mucosa and lead to an enhanced presentation of the conjugated molecule to the immune system (17, 34). Another possibility is that CTB per se has immunomodulating properties. Indeed, it has been shown that CTB induces major histocompatibility complex (MHC) class II expression on B cells (14), enhances antigen presentation by macrophages (M) in the absence of enhanced MHC II expression (29), and blocks the development of diabetes in NOD mice through the development of regulatory cells (40).Mucosal antigen-specific antibody formation and tolerance induction share the requirement ...

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

“…treatment with OVA/CTB conjugate is probably largely explained by the effects of the conjugate on gut mucosal APC. Previous studies have shown that conjugation of Ag to CTB, a molecule that binds with high affinity to the GM1 ganglioside receptors present on most cells (30), greatly facilitates Ag uptake and MHC class II-restricted Ag presentation by CD11c ϩ DC as well as other types of APC, such as B cells and macrophages (23). One possibility is that mucosally administered Ag/CTB conjugate preferentially binds to and is taken up by tolerogenic subsets of mucosal DC or other APC.…”

“…To test the regulatory function of isolated CD4 ϩ T cells or separated CD25 ϩ CD4 ϩ or CD25 Ϫ CD4 ϩ T cells from mice with different treatments, these cells were cocultured in different numbers with 1 ϫ 10 5 DO11.10 T eff cells together with 5 ϫ 10 3 of bone marrow-derived dendritic cells (DC) prepared as described in Ref. 23 and with 1 g/ml OVA 323-339 peptide. These tests were performed using 96-well round-bottom plates (Nunc) for 3 days with [ 3 H]thymidine added during the last 16 h. IL-2 production by cultured cells were assayed in 2-day culture supernatants using the cytokine cytometric bead array method (BD Biosciences).…”

Recombinant Cholera Toxin B Subunit Activates Dendritic Cells and Enhances Antitumor Immunity

“…Molecular mechanism of adjuvant property of rCTB is not understood completely although binding to GM 1 gangliosides is required for rCTB to act as an adjuvant (21). Like other bacterial components, rCTB stimulates immune system by inducing maturation of APC (8). Furthermore, rCTB would work as a carrier molecule for APC to deliver extracellular antigens, because strong binding of pentameric rCTB to GM 1 gangliosides would enhance antigen uptake by DC (8).…”

“…Like other bacterial components, rCTB stimulates immune system by inducing maturation of APC (8). Furthermore, rCTB would work as a carrier molecule for APC to deliver extracellular antigens, because strong binding of pentameric rCTB to GM 1 gangliosides would enhance antigen uptake by DC (8).…”

Autoantigens Plus Interleukin-10 Suppress Diabetes Autoimmunity

“…28,29 In addition, inoculation with autoantigens was shown to generate partial diabetes suppression in patients. [30][31][32] Mucosal delivery of the cholera toxin B subunit (CTB) stimulated dendritic cell-mediated induction of IL-4 secretion by T cells and exerted a variety of distinct anti-inflammatory effects resulting in immunological suppression. [33][34][35] Oral delivery of CTB conjugated with specific autoantigens demonstrated protection of mice against several Th1 cell-mediated autoimmune diseases, including autoimmune encephalomyelitis, 36,37 autoimmune chondritis, 38 and uveitis.…”