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

<p>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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> 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.</p

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

<p>(A and B) PBMCs from 20 volunteers collected pre and post-vaccination with oral inactivated whole cell vaccines against ETEC containing CTB or LCTB<i>A</i>, 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> 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.</p

IL-17A production in response to PPD and increasing concentrations (1 and 10 µg/ml) of ADP-ribosylating toxins, detoxified mutants or subunits.

a<p>IL-17A concentration (pg/ml) in supernatants from cells isolated from BCG vaccinated volunteers (n = 6) expressed as medians (range). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 3 independent experiments.</p>b<p>Fold rises in IL-17A concentration as compared to stimulation with PPD alone.</p>*<p>P<0.05 and **P<0.01; compared to cells stimulated with PPD alone.</p

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

<p>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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> 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.</p

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

<p>(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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 2 independent experiments.</p

Presentation_1_T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.pptx

BackgroundInfection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.MethodsTh cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.ResultsETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.ConclusionsOur results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.</p

Table_1_T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.docx

BackgroundInfection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.MethodsTh cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.ResultsETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.ConclusionsOur results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.</p

Table_2_T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.docx

BackgroundInfection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.MethodsTh cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.ResultsETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.ConclusionsOur results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.</p

Polyclonal IL-17 and IFN-γ responses in PBMCs after PHA stimulation.

<p>IL-17 (A) and IFN-γ (B) production from PBMCs isolated from infants (n = 20), children (n = 10) and adults (n = 15) was analyzed after PHA stimulation. Filled symbols represent <i>H. pylori</i> infected participants (Hp+) and open symbols represent <i>H. pylori</i> uninfected participants (Hp-). Responses to medium alone have been subtracted from the values shown. Horizontal lines represent median values (*<i>P</i><0.05, **<i>P</i><0.01***<i>P</i><0.001).</p

Polyclonal cytokine responses in CD4⁺ T cells after anti-CD3/CD28 stimulation.

<p>Production of IL-17 (A), IFN-γ (B), IL-13 (C), IL-5 (D) and IL-4 (E) by CD4⁺ T cells isolated from <i>H. pylori</i> infected infants (n = 9) and adults (n = 8) was analyzed after stimulation with beads coated with anti-CD3 and anti-CD28 antibodies. Horizontal lines represent median values (*<i>P</i><0.05).</p