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The adjuvant double mutant Escherichia coli heat labile toxin enhances IL-17A production in human T cells specific for bacterial vaccine antigens - PubMed

The adjuvant double mutant Escherichia coli heat labile toxin enhances IL-17A production in human T cells specific for bacterial vaccine antigens

Susannah Leach et al. PLoS One. 2012.

Abstract

The strong adjuvant activity and low enterotoxicity of the novel mucosal adjuvant double mutant Escherichia coli heat labile toxin, LT(R192G/L211A) or dmLT, demonstrated in mice, makes this molecule a promising adjuvant candidate. However, little is known about the mechanisms responsible for the adjuvant effect of dmLT or whether dmLT also has an adjuvant function in humans. We investigated the effect of DMLT on human T Cell responses to different bacterial vaccine antigens: the mycobacterial purified protein derivative (PPD) antigen, tested in individuals previously vaccinated with Bacillus Calmette-Guérin, the LT binding subunit (LTB), evaluated in subjects immunised with oral inactivated whole cell vaccines against enterotoxigenic Escherichia coli, and Streptococcus pneumoniae whole cell vaccine antigens, tested in subjects naturally exposed to pneumococci. We found that dmLT enhanced the production of IL-17A by peripheral blood mononuclear cells in response to all antigens tested. dmLT had comparable effects on IL-17A responses to PPD as the single mutant LT(R192G) adjuvant, which has demonstrated clinical adjuvant activity in humans. Neutralisation of IL-1β and IL-23, but not IL-6, suppressed the IL-17A-enhancing effect of dmLT. Furthermore, CD4+ T cells produced higher levels of IL-17A when stimulated with monocytes pulsed with PPD and dmLT compared to PPD alone, supporting an important role of antigen presenting cells in enhancing IL-17A responses. dmLT also potentiated mitogen-induced IL-17A and IL-13 production. However, dmLT had variable influences on IFN-γ responses to the different stimuli tested.Our demonstration of a potent ability of dmLT to enhance human Th17 type T cell responses to bacterial vaccine antigens encourages further evaluation of the adjuvant function of dmLT in humans.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. dmLT enhances IL-17A responses to PPD in BCG immunised individuals.

PBMCs from BCG vaccinated volunteers (n = 9) were stimulated with PPD in combination with increasing concentrations of dmLT (0, 0.1, 1 and 10 µg/ml). Proliferation (A), and concentration of IL-17A (B) and IFN-γ (C) in culture supernatants were determined. Responses to medium alone and dmLT alone (10 µg/ml) are shown by the leftmost and rightmost bars in each graph. Bars represent mean + SEM. Results shown are from 6 independent experiments. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05 and *** P<0.001; compared to cells stimulated with PPD alone.

Figure 2
Figure 2. dmLT enhances IL-17A and IL-13 responses to PHA stimulation.

PBMCs from volunteers (n = 9) were stimulated with PHA, in combination with increasing concentrations of dmLT (0, 1 and 10 µg/ml) and production of IL-17A (A) and IL-13 (B) were determined. Responses to medium alone and dmLT alone (10 µg/ml) are shown by the leftmost and rightmost bars in each graph. Bars represent mean + SEM. Results shown are from 6 independent experiments. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05, ** P<0.01 and *** P<0.001; compared to cells stimulated with PHA alone.

Figure 3
Figure 3. dmLT enhances IL-17A and IL-13 responses in CD4+ T cells.

PBMCs and PBMCs depleted of CD4+ T cells isolated from BCG vaccinated volunteers were stimulated with PPD (A, n = 4), or PHA (B–C, n = 3), in combination with 10 µg/ml dmLT, and the IL-17A production was determined. (D–E) CD4+ T cells from another set of volunteers (n = 6) were stimulated with beads coated with anti-CD3/CD28 antibodies and increasing concentrations (0, 1 and 10 µg/ml) of dmLT, and the IL-17A (D) and IL-13 (E) concentration in culture supernatants were determined. Bars represent mean + SEM. Results shown are from 2 (A–C) or 3 (D–E) independent experiments. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05 and ** P<0.01; compared to cells stimulated with anti-CD3/CD28 beads alone.

Figure 4
Figure 4. dmLT enhances IL-17A production from T cells via soluble factors and monocytes.

(A) PBMCs from BCG vaccinated volunteers (n = 7) were stimulated with PHA in the presence of supernatants (Sup) derived from PBMCs stimulated with PPD, dmLT or PPD + dmLT, and the IL-17A production was determined. Results shown are from 3 independent experiments. (B) PBMCs from BCG vaccinated volunteers (n = 6) were stimulated with PPD and dmLT in the presence and absence of neutralising antibodies (Abs) against IL-1β, IL-6 and IL-23, and the IL-17A production was determined. Results shown are from 3 independent experiments. (A and B) Bars represent mean + SEM. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05, ** P<0.01 and *** P<0.001; compared to cells stimulated with PHA alone (A) or PPD plus dmLT (B). In (B), indicated differences were also significant (P<0.05) compared to treatments with isotype control antibodies. (C) CD14+ monocytes isolated from BCG vaccinated volunteers (n = 4) were pulsed with PPD alone or together with 10 µg/ml dmLT, and then washed. CD4+ T cells were then added to the monocytes, and the resulting IL-17A production was determined. Results shown are from 2 independent experiments.

Figure 5
Figure 5. dmLT enhances IL-17A responses to components of novel ETEC and pneumococcal vaccines.

(A and B) PBMCs from 20 volunteers collected pre and post-vaccination with oral inactivated whole cell vaccines against ETEC containing CTB or LCTBA, were stimulated with 10 µg/ml LTB with and without 1 µg/ml dmLT, and the resulting IL-17A (A) and IFN-γ (B) production was determined. Statistical analysis was performed using the Wilcoxon signed rank test. Results shown are from 6 independent experiments. (C and D) PBMCs from 8 volunteers were stimulated with WCA and increasing concentrations (0, 1 and 10 µg/ml) of dmLT, and the resulting IL-17A (C) and IFN-γ (D) production was determined. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. Results shown are from 6 independent experiments. (A–D) Bars represent mean + SEM. * P<0.05, ** P<0.01; compared to cells stimulated with LTB (A and B) or WCA (C and D) alone. In A and B, separate comparisons of post- versus pre-vaccination responses are also indicated.

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