Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and intracellular cytokine staining

Abstract

Background: The vaccine efficacy reported following Mycobacterium bovis Bacillus Calmette Guerin (BCG) administration to UK adolescents is 77% and defining the cellular immune response in this group can inform us as to the nature of effective immunity against tuberculosis. The aim of this study was to identify which cytokines and lymphocyte populations characterise the peripheral blood cellular immune response following BCG vaccination.

Results: Diluted blood from before and after vaccination was stimulated with Mycobacterium tuberculosis purified protein derivative for 6 days, after which soluble biomarkers in supernatants were assayed by multiplex bead array. Ten out of twenty biomarkers measured were significantly increased (p < 0.0025) 1 month after BCG vaccination when compared to paired samples (n = 12) taken prior to vaccination (IFNgamma, TNFalpha, IL-1alpha, IL-2, IL-6, IL-10, IL-17, GM-CSF, MIP1alpha, IP-10). All of these remained detectable by multiplex bead array in samples taken 12 months after BCG vaccination of a partially overlapping adolescent group (n = 12). Intracellular cytokine staining after 24 hour Mycobacterium tuberculosis purified protein derivative stimulation of PBMC samples from the 12 month group revealed that IFNgamma expression was detectable in CD4 and CD8 T-cells and natural killer cells. Polyfunctional flow cytometry analysis demonstrated that cells expressing IFNgamma alone formed the majority in each subpopulation of cells. Only in CD4 T-cells and NK cells were there a notable proportion of responding cells of a different phenotype and these were single positive, TNFalpha producers. No significant expression of the cytokines IL-2, IL-17 or IL-10 was seen in any population of cells.

Conclusions: The broad array of biomarker responses detected by multiplex bead array suggests that BCG vaccination is capable, in this setting, of inducing a complex immune phenotype. Although polyfunctional T-cells have been proposed to play a role in protective immunity, they were not present in vaccinated adolescents who, based on earlier epidemiological studies, should have developed protection against pulmonary tuberculosis. This may be due to the later sampling time point available for testing or on the kinetics of the assays used.

Figure 1

Mtb PPD-specific cytokine/chemokine biomarker responses 1 month post BCG vaccination of UK adolescents. Plots representing concentrations of indicated biomarkers (see y-axis legends) are shown as measured by multiplex bead array assay on supernatants from 6 day diluted whole blood assays. Blood samples were donated from individuals (n = 12) prior to or 1 month following BCG vaccination. The dotted line indicates the lower limit of detection of the assay (3.2 pg/ml) and each data point represents the concentration measured in PPD-stimulated sample minus that measured in unstimulated sample. The Wilcoxon Signed Rank test was used to calculate p-values between pre and post-vaccination sample groups.

Figure 2

Mtb PPD-specific cytokine expression in CD4 T-cells, CD8 T-cells and NK cells 12 months post BCG vaccination of UK adolescents. Box plots representing the proportion of A) CD4 T-cells, B) CD8 T-cells and C) NK cells that express the indicated cytokine (see x-axis labels) are shown as determined by intracellular cytokine staining of Mtb PPD-stimulated PBMC. D) CD4 T-cell cytokine expression in response to the positive control stimulant SEB. Cells were isolated from blood samples from individuals (n = 12) 12 months following BCG vaccination. The horizontal line represents the median response, the box represents the interquartile range and the whiskers represent the overall range.

Figure 3

Polyfunctional analysis of Mtb PPD-specific cytokine expression in CD4 T-cells and NK cells 12 months post BCG vaccination of UK adolescents. A) Dot plots from one individual donor are shown comparing simultaneous expression of IFNγ (x-axis) and TNFα (y-axis). Previous gating was used to isolate singlet, live cells that were either CD3⁺CD4⁺ (CD4 T-cells) or CD3^-CD56⁺ (NK cells) as indicated for each plot. Responses in unstimulated and Mtb PPD-stimulated samples are shown. Values indicate the proportion (as a percentage) of the indicated subset present in that quadrant. B) Boolean gating and Spice analysis was used to determine the extent of polyfunctionality within the 12 month post-BCG vaccination group (n = 12). Bar charts represent, for either CD4 T-cells or NK cells, the median percentage (and interquartile range) of each subset expressing the indicated cytokine combination (x-axis labels). Pie segments represent the average proportion, across the group, of cells expressing the indicated cytokine combination (pie chart legend) within the total population of cells expressing either IFNγ or TNFα.