Modelled temporal changes in gene expression.

<p><b>A</b>. Heat map showing modelled changes in expression of the significant gene transcripts in TBM patients from the time of diagnosis (0) to 180 days. Green represents lower transcript abundance, red represents higher transcript abundance and black represents no difference in expression as compared to healthy children with a past history of TB sampled at least one year after diagnosis and treatment. The relative degree of transcript abundance is indicated by the colour intensity derived from the fitted mean expression levels over time (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185973#sec002" target="_blank">methods</a>). Genes showing similar temporal patterns of expression have been clustered together. The apparent linear change in colour is derived from the statistical model that interpolates the observed time points and can therefore be represented as a continuum. <b>B and C</b>. Example plots of two significantly differentially expressed gene transcripts. Expression levels for each TBM patient (red circles n = 9) are shown from diagnosis (time 0) to day 180. Blue circles are expression levels for healthy children (n = 9) with a past history of TB sampled at least one year after diagnosis and treatment. M = “minus” and denotes the log₂ ratio of the red and green channels. The line represents the fitted mean gene expression level over time, from linear mixed-effects model (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185973#sec002" target="_blank">methods</a>). 1b = <i>TARP</i>; 1c = <i>IL1R2</i>.</p

Gene expression of T-cell receptor signalling pathway and validation.

<p><b>A</b>. Transcripts that were SDE in TBM patients at admission compared to the 6 month time point that mapped to the T-cell receptor signalling pathway. After activation of the T-cell receptor, a cascade of signalling events is initiated leading to gene induction. Gene products highlighted green are significantly less abundant in TBM patients at admission compared to the 6 month time point. Corrected <i>p</i> value on Ingenuity Pathways Analysis = 1.47E^-11. Gene list provided in Tables D and E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185973#pone.0185973.s002" target="_blank">S1 File</a>. <b>B</b>. Validation of T-cell signalling pathway genes by RT-PCR in TBM patients (cohort 1). Selected genes in the T-cell signalling pathway were validated by RT-PCR including seven that were significantly less abundant at admission compared to post treatment (TRA, ZAP70, CD3G, CD3D, LAT, LCK, NFATC2) and one showing no change (NFATC3). Two genes were also included that were more abundant at admission compared to post treatment (AREG, SLC7A5) that acted as the positive controls. Fold change between TBM patients at admission and post treatment (n = 8) are shown relative to Beta actin control. Boxes show 25^th and 75^th percentile. Whiskers show lowest and highest data point and horizontal lines show medians. * <i>p</i><0.05, ** <i>p</i><0.01 shows significance using paired Wilcoxon rank test.</p

Confirmation of significantly differentially expressed genes from Cohort 1 in Cohort 2.

<p><b>A</b>. Average log fold change in the SDE transcripts identified in the time-course study (cohort 1) and their corresponding log fold change in the single time-point study (cohort 2). 140/262 transcripts identified in cohort 1 were measured in cohort 2. 129 transcripts followed the same regulation pattern (purple crosses); and 11 showed opposite regulation (represented by red crosses, annotated by gene symbol). Correlation coefficient was r2 = 0.78, 95% CI = [0.71, 0.82] p<2x10-16. The y-axis shows log fold change of SDE gene transcripts in cohort 1 relative to cohort 1 Healthy Controls (HC), and the x-axis shows their log fold change in cohort 2 relative to cohort 2 HC. <b>B</b>. Average log fold change in TBM patients relative to cohort 2 HC (x-axis) plotted against average log fold change in PTB patients relative to cohort 2 HC (y-axis) of the significant transcripts (140) that were identified in cohort 1 and common to both cohorts. Least-squares fitted line is shown in dashes. Correlation coefficient was <i>r</i>^<i>2</i> = 0.71, 95% CI = [0.62, 0.79] <i>p</i><2x10^-16. <b>C.</b> Heat map showing almost complete discrimination between TBM cases from cohort 1 and cohort 2 and healthy controls (cohort 2) using 129 transcripts significantly differentially expressed in both cohorts. Gene list is provided in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185973#pone.0185973.s002" target="_blank">S1 File</a>. Hierarchical clustering was performed by the complete linkage method to identify similar clusters. Solid red bar (top) shows cases, green bar shows controls. Intensity of colour indicates degree of reduced (green) or elevated (red) abundance of each transcript relative to healthy controls. White indicates no expression.</p

Functional T-cell responses in cohort 3.

<p><b>A.</b> Adjusted T-cell proliferative responses (cpm) to PHA in acute TB (TBM n = 19, EPTB n = 29, PTB n = 27) and healthy Mantoux positive controls n = 26. Normalised proliferative responses were determined by deducting the value for the unstimulated well from that of the PHA well. Means are shown by horizontal bars together with standard error of the mean. Asterisk denotes significant differences in corrected p values. PTB vs HC * <i>p</i> = 0.018, TBM vs HC ** <i>p</i> = 0.001, EPTB vs HC *** <i>p<</i>0.0003. <b>B.</b> IFNγ production in response to PHA in acute TB (TBM n = 36, other EPTB n = 57, PTB n = 55) and healthy Mantoux positive controls (HC) n = 75. Medians are shown by horizontal bars together with their interquartile ranges. Asterisk denotes significant difference in corrected p value between TBM and controls * <i>p</i><0.0003.</p