Figure 1.
(A) Selection of populations containing single cell CD4 or CD8 T cells. Each subsequent panel shows only the population of interest that has been selected from the gate on the previous plot. (B) Selection of CD4 T cells producing the cytokines of interest shown in an unstimulated and a BCG stimulated sample from one individual. The same gating strategy was used for CD8 T cells (not shown). Boolean gating was used to calculate proportions of polyfunctional T cells.
Figure 2.
Proportion of cytokine-producing T cells in children before (pre, n = 13) and 10 weeks after (post, n = 13) BCG immunisation.
Whole blood was incubated for 12 hours with the antigen BCG to measure proportions of IFN-γ, IL-2 and TNF-α producing CD4 T cells (A) and CD8 T cells (C). Boolean analysis was used to calculate proportions of multifunctional cells producing two or all three Th1 cytokines simultaneously (B, D). Bars indicate medians. Statistical differences with p-values <0.05 are shown. Significant changes after Bonferroni correction are shown in red.
Figure 3.
Proportion of cytokine-producing T cells in adults before (pre, n = 16) and 10 weeks after (post, n = 13) BCG immunisation. Panels A to D as for Figure 2.
Figure 4.
Box plots (with lower quartile, median and upper quartile, whiskers 1.5 IQR) showing median change in the proportion of cytokine-producing subpopulations of CD4 T cells 10 weeks after BCG immunisation in children (n = 13) and adults (n = 13).
The proportion of Th1-cytokine-producing subpopulations in CD4 and CD8 T cells was measured following in vitro stimulation with the antigens BCG and PPD, in whole blood taken before and after BCG immunisation. The median change for (A) CD4 and (B) CD8 T cells was calculated by subtracting results before BCG immunisation from results after BCG immunisation. Differences were compared using a Mann-Whitney test.
Figure 5.
The fraction of single-, double-, and triple- cytokine-producing CD4 T cells relative to the total population of Th1-cytokine producing cells in children (n = 13) and adults (n = 13) following in vitro stimulation with antigens (A) BCG and (B) PPD.
Differences were compared using a Mann-Whitney test.
Figure 6.
Fluorescence intensity of CD4 T cells following in vitro stimulation with the antigen BCG.
(A) Fluorescence intensity of single-(yellow), double-(blue), or triple-(red) cytokine-producing CD4 T cells in a representative child 10 weeks after BCG immunisation. Events are plotted on a logarithmic scale. Cells in the top right hand corner are those producing the greatest amount of both cytokines. (B, C) Median fluorescence intensity (MFI) for IFN-γ-, IL-2- and TNF-α-producing CD4 T cell subpopulations following in vitro stimulation with the antigen BCG for (B) children (n = 13) and (C) adults (n = 13) 10 weeks after BCG immunisation. White bars show total cytokine-producing populations. Whiskers indicate IQR. Bars with * are based on one value only. Overall comparison was done using a Kruskal-Wallis test and comparisons of two populations were done using a Mann-Whitney test. The numbers above the horizontal bars represent the p-values for comparisons. ns = not significant (p>0.05).
Figure 7.
Comparison of the changes in cytokines/chemokines between 13 children (circles) and 13 adults (triangles).
Concentrations were determined using xMAP technology following in vitro stimulation with the antigens BCG and PPD in samples taken before and 10 weeks after BCG immunisation. Statistical differences were analysed using a using a Mann-Whitney test and p-values <0.05 are shown. Bars indicate medians.