Effect of cholera toxin on BCG immunogenicity and <i>M.tb</i> challenge outcome.

<p>(<b>A</b>) Cytokine gating in spleens and lungs, unstimulated vs. Ag85A peptide pool-stimulated. (<b>B</b>) Balb/c mice received 4×10⁵ CFU BCG <i>i.n.</i> or <i>i.d.</i> followed 10 weeks later by 1×10⁶ PFU MVA85A <i>i.d.</i> At weeks 10, 11 and 14 post-BCG, lungs were examined for cytokine production by ICS following Ag85A peptide pool stimulation in the presence of Brefeldin A and GolgiStop. (<b>C</b> & <b>D</b>) Balb/c mice received 4×10⁵ CFU BCG±2 µg CT <i>i.n</i>. After 10 weeks, lungs and spleen were dissected and stimulated with PPD in the presence of Brefeldin A and GolgiStop. Percentages of CD4⁺ T cells producing IFN-γ and IL-17 were calculated following ICS on lung cells (<b>C</b>) and splenocytes (<b>D</b>). Mice receiving BCG only are plotted with closed circles and those receiving CT have open circles. P values were calculated using a Mann Whitney test (n = 5). (<b>E</b>) Ten weeks post-BCG, mice received ∼100 CFU <i>M.tb</i> via aerosol and four weeks later lungs and spleen were homogenised and plated out for CFU quantitation. Statistical analysis was performed using a one way ANOVA and post-hoc tests on the vaccinated groups (n = 8).</p

Immunogenicity and protective efficacy of BCG + CT – MVA85A.

<p>Balb/c mice received BCG±CT <i>i.n.</i> followed by 1×10⁶ CFU MVA85A 10 weeks later. Lungs (<b>A</b>) and spleen (<b>B</b>) were taken at 10 (black circles), 11 (dark grey circles) and 14 (light grey circles) weeks post-BCG and cytokine-producing cells responding to an Ag85A peptide pool quantified using ICS. Responses from animals receiving BCG – MVA85A (closed circles) were compared with those receiving BCG + CT followed by MVA85A (open circles). Statistical analysis was performed using a Mann Whitney test. n = 10, five each from two experiments. (<b>C</b>) Balb/c mice were vaccinated as above. Control groups included unvaccinated and BCG <i>i.d.</i> A group receiving BCG <i>i.n.</i> was included to compare BCG – MVA85A <i>i.n.</i> to BCG <i>i.n</i>. Animals were exposed to ∼100 CFU <i>M.tb</i> via aerosol four weeks post-MVA85A. Four weeks post-challenge, lungs and spleen were homogenised and plated for CFU quantitation. (<b>D</b>) Balb/c mice were vaccinated and challenged as described above. Groups receiving BCG – MVA85A and BCG + CT – MVA85A received an anti-IL-17 blocking antibody (MAB421; R&D Systems) administered <i>i.p.</i> every three days post-challenge. One group receiving BCG – MVA85A received an IgG2a isotype control antibody (MAB006; R&D Systems) on the same regimen. Mice were culled four weeks post-challenge and lung CFU quantitated as described above. Statistical analysis was performed using a one way ANOVA and post-hoc tests on the vaccinated groups (n = 8–16).</p

Histological analysis of <i>M.tb</i>-exposed lungs.

<p>Four Balb/c mice from each group shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078312#pone-0078312-g002" target="_blank">Figure 2C</a> had one lung lobe inflated with 10% NBF and dissected. Lungs were embedded in paraffin, sectioned, mounted onto slides and stained with haematoxylin and eosin. Images were cropped so that no external white space was visible (<b>A</b>). Analysis was performed using a k means clustering algorithm in MATLAB (<b>B</b>). (<b>C</b>) Representative sections from each group. (<b>D</b>) Percentages of the image represented by one of three colours (purple, red or white) were calculated and plotted, n = 2-4.</p

Characterization of the human Treg population.

<p>(A) CD4⁺CD25⁻, CD4⁺CD25^int and CD4⁺CD25^hi cells were identified as shown. CD4⁺CD25^hi cells are those where CD25 expression was higher than that on the CD4⁻ population. (B) FOXP3 expression within each population was assessed by intracellular staining. (C) Freshly isolated CD4⁺CD25⁻ T cells (2×10⁴ cells/well) were cultured alone (CD25-/low) or with CD4⁺CD25^hi T cells at various ratios, and stimulated with plate-bound anti-CD3 and soluble anti-CD28. Proliferation was assessed by (³H)-thymidine incorporation. The results represent the average of triplicate wells with standard deviation, from a representative assay.</p

CRC patients are able to mount CD4⁺ T cell responses to the recall antigens PPD and HA.

<p>Whole PBMC from 11 age-matched controls and 14 CRC patients were assessed for responses to the antigens PPD and HA, measured by IFN-γ ELISPOT. Purified PBMC were added at 3.5×10⁵ cells/well, and incubated for 18 hours in the presence of either 1 µg/ml PPD, 1 µg/ml HA or with no antigen. Wells were set up in duplicate. Fewer than 5 spot forming cells/10⁶ PBMC was considered negative.</p