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.

FIG. 1

Construction of the genetic CTB fusion proteins. (A) Addition of the 17-amino-acid OVA peptide to the C terminus of CTB. (B) Insertion of the 13-amino-acid HA peptide into the CTB molecule, replacing residues 56 to 64 of the mature protein and resulting in a protein of 107 instead of 103 amino acids. The first residue of the peptide and residue 55 of CTB are both serine. Additionally, the last residue of the peptide and residue 63 of CTB are both lysine. Open rectangles, HA or OVA peptide; solid rectangles, mature CTB protein. The serine residue at position 55 and the lysine residue at position 63 of CTB that are shared with the peptide sequence in CTB55-64HA are boxed.

FIG. 2

Conjugation of antigen to CTB enhances antigen presentation in vitro. Spleen cells were incubated with antigen in free form or conjugated to CTB together with purified antigen-specific TCR-transgenic T cells. Data are expressed as the proliferative responses obtained in response to free or CTB-conjugated whole OVA (A), OVA peptide (B), or HA peptide (C). (A) APC and OVA-specific TCR-transgenic T cells were incubated with 10⁻⁸ M OVA or OVA-CTB or 10⁻⁸ M OVA plus 6 × 10⁻⁹ M CTB. (B) APC and OVA-specific TCR-transgenic T cells were incubated with graded amounts of OVA peptide (□) or with CTB-conjugated OVA peptide (■). (C) APC and HA-specific TCR-transgenic T cells were incubated with graded amounts of free HA peptide (□), CTB-conjugated HA peptide (●), or the equivalent amounts of unconjugated HA peptide and CTB (○).

FIG. 3

Conjugation of antigen to CTB enables the use of Mφ as APC. DC, B cells, and Mφ were pulsed with free antigen or with CTB-coupled antigen and incubated with antigen-specific TCR-transgenic T cells. (A through C) Data are expressed as the SI obtained with different concentrations of HA peptide (□), chemically conjugated CTB-HA peptide (■), and genetically coupled CTH-HA peptide (●) using either DC (A), B cells (B), or Mφ (C) as APC. (D through F) Data are expressed as the SI obtained with different concentrations of OVA (◊), CTB-OVA (⧫), OVA peptide (▿), and CTB-OVA peptide (▾) using either DC (D), B cells (E), or Mφ (F) as APC. Data represent one of two experiments using 10⁵ purified T cells with 10⁴ APC.

FIG. 4

CTB-conjugated antigens induce enhanced IFN-γ secretion in vitro. (A through C) IFN-γ production by HA-specific TCR-transgenic T cells following incubation with HA peptide or HA-CTB-treated APC. Data are expressed as the concentrations of IFN-γ in 48-h culture supernatants obtained with different concentrations of HA peptide (□), chemical CTB-HA peptide (■), and genetic CTB-HA peptide (●) using DC (A), B cells (B), or Mφ (C) as APC. (D through F) IFN-γ production by OVA-specific TCR-transgenic T cells following incubation with antigen-pulsed DC (D), B cells (E), or Mφ (F). Data are expressed as the concentrations of IFN-γ in 48-h culture supernatants using different concentrations of OVA (◊), CTB-OVA (⧫), OVA peptide (▿), and CTB-OVA peptide (▾). Data represent one of two experiments using 10⁵ purified T cells with 10⁵ APC.

FIG. 5

CTB-conjugated antigens induce enhanced IL-12p40 secretion in vitro. Shown is IL-12p40 production by antigen-specific TCR-transgenic T cells following incubation with antigen-pulsed APC. (A through C) Data are expressed as concentrations of IL-12p40 in 48-h culture supernatants obtained with different concentrations of HA peptide (□), chemical CTB-HA peptide (■), and genetic CTB55-63HAp (●) using DC (A), B cells (B), or Mφ (C) as APC. (D through F) IL-12p40 production by OVA-specific TCR-transgenic T cells following incubation with antigen-pulsed DC (D), B cells (E), or Mφ (F). Data are expressed as concentrations of IL-12p40 in 48-h culture supernatants obtained with different concentrations of OVA (◊), CTB-OVA (⧫), OVA peptide (▿), and CTB::OVAp (▾). Data represent one of two experiments using 10⁵ purified T cells with 10⁵ APC.

FIG. 6

Mφ incubated with CTB-coupled antigens up-regulate their cell surface expression of CD40 and CD86 in vitro. Mφ were pulsed with OVA, CTB-OVA, OVA peptide, or CTB-coupled OVA peptide, or were left untreated, and then incubated for 24 h with OVA-specific TCR-transgenic T cells. The Mφ cell surface levels of MHC II, CD40, CD80, and CD86 were then evaluated by FACS analysis. Data are presented as fluorescent intensities and were recorded for unpulsed (shaded), OVA-pulsed (red line), and CTB-OVA-pulsed (green line) Mφ (A) and for unpulsed (shaded), OVA peptide-pulsed (orange line), and CTB-OVA peptide-pulsed (blue line) Mφ.