CFP-10/ESAT-6-specific CD8 T cell phenotype is associated with mycobacterial antigen load.

<p>Phenotypic analysis of CFP-10 and ESAT-6-specific IFN-γ⁺ CD8 T cells was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094949#pone-0094949-g001" target="_blank">Figure 1</a>. Cells were analyzed prior to initiating anti-TB treatment (Pre-TB Tx), and 2 months and 6 months after initiation of treatment. (<b>A</b>) Flow cytometry data indicating Ki67 and Bcl-2 expression by IFN-γ⁺ ESAT-6-specific CD8 T cells from a TB diseased patient at two time points: prior to treatment and 6 months after initiation of treatment, corresponding to the end of the treatment period. (<b>B</b>) Representative histogram overlays indicating expression of Bcl-2, CD127 and CD95 within CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells. Black lines indicate expression prior to treatment; grey lines indicate expression 6 months after initiation of treatment. The numbers in the upper right corner of the histograms indicate the median fluorescence intensity (MFI) at each time point (black: pre-treatment MFI; grey: 6-month TB treatment MFI). Data shown in panels A and B are gated on VIVID^l°CD3⁺CD8⁺IFN-γ⁺ cells. (<b>C</b>) Summary data comparing the expression of Ki67, Bcl-2, CD127 and CD95 on CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells from the same individual at two time points: prior to treatment (Pre-TB Tx) and at the end of the 6-month treatment period (6 mo. TB Tx). Data are shown from 7 individuals who maintained positive CFP-10/ESAT-6-specific CD8 T cell responses throughout the 6-month duration of treatment. Differences between time points were assessed using the Wilcoxon matched pairs test. § indicates p values that did not retain statistical significance after applying the Bonferroni correction for multiple comparisons. The proportion of CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells that express Ki67 (<b>D</b>), CD95 (<b>E</b>), Bcl-2 (<b>F</b>), and CD127 (<b>G</b>) are shown for individuals with LTBI (n = 6), and TB diseased patients prior to treatment (n = 13; Pre-TB Tx), and 2 months (n = 10; 2 mo. TB Tx) and 6 months (n = 8; 6 mo. TB Tx) following initiation of treatment. Differences in panels D-G were first assessed using a Kruskal-Wallis test, followed by a Dunn's post-test to correct for multiple comparisons; § indicates p values that did not remain significant following correction with the Dunn's post-test.</p

CFP-10/ESAT-6-specific CD8 T cells in patients with TB have a short-lived, pro-apoptotic phenotype.

<p>PBMCs from individuals with LTBI (n = 18) and patients with TB disease (n = 20) were stimulated for 6 hours with CFP-10 and ESAT-6 peptide pools; intracellular IFN-γ production was measured in CD8 T cells by flow cytometry. (<b>A</b>) Frequencies of CFP-10 and ESAT-6-specific IFN-γ⁺ CD8 T cells in persons with LTBI and patients with TB disease. Horizontal lines represent the median. Data are shown after subtraction of background cytokine production in the negative control condition. The dotted line separates individuals who met the criteria for a positive CD8 T cell response for further phenotypic analyses (above dotted line: n = 6 LTBI; n = 13 TB disease), and individuals in whom the frequency of IFN-γ⁺ CD8 T cells was too low to meet the criteria for further phenotypic analyses (below dotted line). (<b>B</b>) Representative flow cytometry data from an individual with LTBI and a patient with TB disease, following stimulation of PBMCs with an ESAT-6 peptide pool. Grey cells indicate the total CD8 T cell population (gated on VIVID^l°CD3⁺CD8⁺ cells); black cells indicate ESAT-6-specific CD8 T cells (gated on VIVID^l°CD3⁺CD8⁺IFN-γ⁺ cells). (<b>C</b>) Summary data of the percentage of specific IFN-γ⁺ CD8 T cells expressing Ki67, Bcl-2, CD127, CD57 and CD95 in individuals with LTBI and TB disease. Differences were assessed using the Mann-Whitney test. § indicates p values that did not remain significant after applying the Bonferroni correction for multiple comparisons. (<b>D</b>) Comparison of the expression of intracellular Ki67 and Bcl-2 between the total CD8 T cell population and specific IFN-γ⁺ CD8 T cells within the same individual. Only individuals meeting the criteria for a positive CD8 T cell response in the ICS assay were included in this paired analysis (n = 6 LTBI; n = 13 TB disease). Differences were assessed using the Wilcoxon matched-pairs test.</p

Lack of restoration of CFP-10/ESAT-6-specific CD4 and CD8 T cell proliferative capacity following completion of treatment.

<p>The proliferative capacity of CFP-10/ESAT-6-specific CD4 and CD8 T cells was measured following a 6-day stimulation of freshly isolated PBMCs with CFP-10 and ESAT-6 peptide pools. (A) Flow cytometry data of the proliferative capacity of antigen-specific CD8 T cells from a TB diseased patient analyzed at 4 time points: prior to treatment (Pre-TB Tx), 2 months of treatment (2 mo. TB Tx), 6 months of treatment (6 mo. TB Tx, corresponding to the end of the treatment period), and 6 months after completion of treatment (12 mo. TB Tx). The cells shown are gated on VIVID^l°CD3⁺CD8⁺ lymphocytes. The percentage on each plot indicates the percentage of proliferating CD8 T cells after subtraction of background proliferation in the negative control condition. (B, D) Longitudinal analysis of the proliferative capacity of CFP-10 and ESAT-6-specific CD8 (panel B) and CD4 (panel D) T cells in a subset of TB diseased patients who were followed for up to one year after TB diagnosis (n = 19 with at least 3 time points available for analysis). There were no significant differences in the frequency of proliferating antigen-specific CD8 or CD4 T cells over time (Kruskal-Wallis test). (C, E) Cross-sectional comparison of the CFP-10 and ESAT-6-specific CD8 (panel C) and CD4 (panel E) T cell proliferative capacity between individuals with LTBI (n = 34) and active TB disease at 4 time points (pre-TB tx: n = 34; 2 months of TB tx: n = 19; 6 months of TB Tx [corresponding to the end of the treatment period]: n = 25; 12 months post initiation of TB tx [corresponding to 6 months after the end of the treatment period]: n = 10). Data are shown after subtraction of background proliferation in the negative control condition. Differences in panels C and E were first assessed using a Kruskal-Wallis test, followed by a Dunn's post-test to correct for multiple comparisons. All comparisons remained significant following correction with the Dunn's post-test.</p

Characteristics of study population.

a<p>The remaining 8 individuals with TB were sputum smear-negative, culture positive for <i>M. tuberculosis</i>.</p>b<p>Values denote median number of days on anti-TB treatment at first sample collection (range).</p>c<p>Values denote median (range).</p>d<p><i>p</i><0.05, compared with LTBI.</p><p>N/A, not applicable.</p

Differential co-expression patterns of Bcl-2, CD57 and CD95 by CFP-10/ESAT-6-specific CD8 T cells distinguish individuals with LTBI and patients with TB.

<p>Boolean analysis was performed to determine co-expression patterns of Bcl-2, CD57, and CD95 by CFP-10 and ESAT-6-specific CD8 T cells detected by ICS, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094949#pone-0094949-g001" target="_blank">Figure 1</a>. (A) Co-expression patterns of Bcl-2, CD57, and CD95 by specific IFN-γ⁺ CD8 T cells detectable in individuals with LTBI (open bars; n = 6) and patients with TB disease (grey bars; n = 13). Cells were gated on VIVID^l°CD3⁺CD8⁺IFN-γ⁺ cells. Differences were assessed using the Mann-Whitney test. § indicates p values that did not retain statistical significance after applying the Bonferroni correction for multiple comparisons. Pie graphs indicate the median proportion of each population contributing to the total specific IFN-γ⁺ CD8 T cell response. (B) Comparison of the proportion of Bcl-2⁺CD57⁻CD95⁺ cells contributing to the total CFP-10 and ESAT-6-specific CD8 T cell responses in individuals with LTBI and patients with TB disease. Horizontal lines represent the median. Differences were assessed using the Mann-Whitney test. The dotted line indicates the cut-off (18.7%) that distinguishes individuals with LTBI and TB disease, with 100% specificity and 100% sensitivity. (C) Receiver operator characteristic (ROC) curve indicating the sensitivity and specificity of the proportion of CFP-10/ESAT-6-specific CD8 T cells that are Bcl-2⁺CD57⁻CD95⁺ to distinguish individuals with LTBI and TB disease. An area under the ROC curve (AUC) analysis was performed to further evaluate the performance of this phenotypic expression profile in distinguishing individuals with LTBI and TB disease.</p

Mycobacteria-specific cytokine expression by innate cells in whole blood from adults and mycobacterial growth inhibition.

<p>(<b>A</b>) Absolute numbers of total cytokine expressing innate cells per mL of whole blood plotted against <i>M</i>.<i>tb</i> H37Rv growth. R and p values were calculated using Spearman’s correlation analysis. (<b>B</b>) Numbers of BCG-specific mDCs, monocytes and neutrophils co-expressing IL-6, IL-12 and/or TNF-α in whole blood from QFT+ (red) and QFT- (blue) adults. Medians are represented by the horizontal line, interquartile ranges by the boxes, and ranges by the whiskers. The Mann-Whitney test was used to assess differences between QFT+ and QFT- adults and none were found to be different.</p

Detection of GFP-expressing BCG by innate cells and association between absolute numbers of innate cells and mycobacterial growth inhibition.

<p>(<b>A</b>) Representative flow cytometry plot of IL-6, IL-12 and TNF-α cytokine expression by myeloid dentritic cells (mDCs), monocytes and neutrophils, measured in whole blood stimulated for 6 hours with BCG, BCG-GFP (shown) or LPS, relative to an unstimulated control sample. (<b>B</b>) Representative histograms indicating proportions of innate cells that phagocytosed BCG-GFP (green). (<b>C</b>) Absolute numbers of innate cell subsets per milliliter of unstimulated whole blood plotted against <i>M</i>.<i>tb</i> H37Rv growth. R and p values were calculated using Spearman’s correlation. (<b>D</b>) Absolute numbers of BCG-GFP-positive mDCs, monocytes or neutrophils per mL of whole blood in adult individuals, stratified by QFT status. The inclusion of TruCount beads during the cell staining steps of the innate whole blood assay allowed determination of the absolute number of each subset of cells per mL of whole blood. The red and blue circles represent QFT+ and QFT- adults, respectively. Horizontal lines represent medians and whiskers, the interquartile range. Differences in absolute counts of BCG-GFP-positive innate cells between the groups were evaluated with the Mann-Whitney test (shown <i>P</i> values). The pie charts show relative proportions of BCG-GFP-positive cells among each innate cell subset.</p

Mycobacteria-specific T cells in whole blood from adults and mycobacterial growth inhibition.

<p>(<b>A</b>) Absolute numbers of BCG-specific CD4, CD8 and γδ T cell subsets co-expressing IL-2, IFN-γ, TNF-α and/or IL-17, in whole bood from QFT+ (red) and QFT- (blue) adults. Medians are represented by the horizontal lines, interquartile ranges by the boxes, and ranges by the whiskers. The Mann-Whitney test was used to assess differences between QFT+ and QFT- adults and none were found to be different. (<b>B</b>) <i>M</i>.<i>tb</i> H37Rv growth plotted against frequencies of BCG-specific CD4, CD8 or γδ T cells expressing IFN-γ, or CD4 T cells expressing IL-17 in adults. R and p values were calculated using Spearman’s correlation analysis.</p

<i>In vitro</i> mycobacterial growth inhibition in <i>M</i>.<i>tb</i>-infected (QFT+) and uninfected (QFT-) individuals.

<p>Inhibition of <i>M</i>.<i>tb</i> H37Rv growth by whole blood from QFT+ and QFT- adults (<b>A</b>), young adults (<b>B</b>) and children (<b>C</b>) assessed using a mycobacterial growth inhibition assay (MGIA). The growths of <i>M</i>. <i>bovis</i> BCG, <i>M</i>.<i>tb</i> H37Rv, <i>M</i>.<i>tb</i> isolate CDC1551 and <i>M</i>.<i>tb</i> isolate HN878 in the whole blood of adults (<b>D</b>) and young adults (<b>E</b>), respectively were compared. Spearman’s correlation analyses of the growth of different mycobacterial strains (<b>F</b> and <b>G</b>). The growth of BCG (<b>H</b>), HN878 and CDC1551 (<b>I</b> and <b>J</b>), was measured in the whole blood of adults and young adults, respectively. The red and blue circles represent QFT+ and QFT- individuals, respectively while the green and orange circles represent children and young adults respectively. The horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the range. Differences in mycobacterial growth inhibition between both groups of individuals were evaluated with the Mann-Whitney test (shown <i>P</i> values).</p

Application of a whole blood mycobacterial growth inhibition assay to study immunity against <i>Mycobacterium tuberculosis</i> in a high tuberculosis burden population

<div><p>The determinants of immunological protection against <i>Mycobacterium tuberculosis (M</i>.<i>tb)</i> infection in humans are not known. Mycobacterial growth inhibition assays have potential utility as <i>in vitro</i> surrogates of <i>in vivo</i> immunological control of <i>M</i>.<i>tb</i>. We evaluated a whole blood growth inhibition assay in a setting with high burden of TB and aimed to identify immune responses that correlate with control of mycobacterial growth. We hypothesized that individuals with underlying <i>M</i>.<i>tb</i> infection will exhibit greater <i>M</i>.<i>tb</i> growth inhibition than uninfected individuals and that children aged 4 to 12 years, an age during which TB incidence is curiously low, will also exhibit greater <i>M</i>.<i>tb</i> growth inhibition than adolescents or adults. Neither <i>M</i>.<i>tb</i> infection status, age of the study participants, nor <i>M</i>.<i>tb</i> strain was associated with differential control of mycobacterial growth. Abundance and function of innate or T cell responses were also not associated with mycobacterial growth. Our data suggest that this assay does not provide a useful measure of age-associated differential host control of <i>M</i>.<i>tb</i> infection in a high TB burden setting. We propose that universally high levels of mycobacterial sensitization (through environmental non-tuberculous mycobacteria and/or universal BCG vaccination) in persons from high TB burden settings may impart broad inhibition of mycobacterial growth, irrespective of <i>M</i>.<i>tb</i> infection status. This sensitization may mask the augmentative effects of mycobacterial sensitization on <i>M</i>.<i>tb</i> growth inhibition that is typical in low burden settings.</p></div