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, 4 (5), e5590

Identification of T-cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

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Identification of T-cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

Sebastian D Schuck et al. PLoS One.

Abstract

Background: T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans.

Methodology/principal findings: We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNgamma response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI.

Conclusions: Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. IFNγ-expressing CD4+ CD45RO+ T cells after 16 h and 7 days restimulation with immunodominant and latency-associated antigens from LTBI and TST-negative controls.
Intracellular cytokine expression after 16 h restimulation in PBMC from LTBI (A), and 7 days – including two rounds of in vitro restimulation – in PBMC from LTBI (B, upper graph) and TST-negative donors (B, lower graph) are shown. Scatter plots indicate mean and standard deviation. Percentages of IFNγ-expressing CD4+ CD45RO+ memory T cells are indicated on the y-axis for SEB, PPD from M. tuberculosis, and tested antigens (x-axes). Background values of non-stimulated controls were subtracted for each individual donor. The most promising candidate Rv3407 is underlined. PPD: purified protein derivative of M. tuberculosis; SEB: Staphylococcus enterotoxin B.
Figure 2
Figure 2. IFNγ ELISA analyses after restimulation with immunodominant and latency-associated antigens of PBMC from LTBI and TST-negative donors.
Analyses of IFNγ in the culture supernatant by ELISA after 7 days and two rounds of in vitro restimulation in PBMC from LTBI (A) and TST-negative donors (B) are shown. Scatter plots indicate mean and standard deviation. Background values of non-stimulated controls were subtracted. IFNγ concentrations in the supernatant are indicated on the y-axis for stimulation with SEB, PPD from M. tuberculosis, and tested antigens (x-axes). The most promising candidate Rv3407 is underlined.
Figure 3
Figure 3. Comparison of IFNγ-expressing CD4+ T cells specific for immunodominant and latency-associated M. tuberculosis antigens between patients with TB, LTBI, and TST-negative donors.
(A). Percentages of IFNγ-expressing CD4+ CD45RO+ memory T cells are shown for stimulation with SEB, PPD from M. tuberculosis, and 11 latency-associated antigens after 7 days and two rounds of in vitro restimulation. T-cell responses from TST-negative donors are indicated as green circles, LTBI are indicated as blue squares, and TB patients are indicated as red triangles. Two-sided p-values for the Mann-Whitney U-test are indicated as follows: * P<0.05, ** P<0.01; and *** P<0.001. (B) Classification of TB patients and LTBI based on random forest analysis using 11 latency-associated antigens as well as ESAT6_CFP-10, and PPD. Results from the cross validation are shown in a bar chart. Each bar represents an individual donor. TB patients are shown on the left (red bars), LTBI on the right side (blue bars). The y-axis indicates the prediction threshold calculated by random forest analysis. Negative bars predict a TB patient, positive bars an LTBI. The prediction probability is represented as the bar height. (C) Mean decrease of class impurity over all trees measured as Gini index (y-axis) indicates the relative importance of each factor (x-axis) for classification. PPD: purified protein derivative of M. tuberculosis; SEB Staphylococcus enterotoxin B.
Figure 4
Figure 4. Overlapping peptide pools of latency-associated protein Rv3407 stimulate IFNγ-expressing CD4+ T cells after 7 days and two rounds of restimulation.
PBMC from six LTBI (A–F) were restimulated with 15-mer synthetic peptide pools of Rv3407 for 7 days including two rounds of in vitro restimulation. IFNγ-expressing CD4+ CD45RO+ T cells are shown for stimulation with peptide pools 1 to 6 (grey bars) and pools 7 to 11 (black bars). Each peptide is constituent of one pool within pools 1 to 6 and of one pool within pools 7 to 11. Peptides inducing the most prominent responses are indicated for donors A–D. The horizontal line indicates the threshold for positive responses (0.2%). Background values of non-stimulated controls were subtracted.
Figure 5
Figure 5. Gating procedures of flow cytometry analyses to determine protein candidate specific T cell proportions.
Representative analyses from a patient with Tb (A) and an LTBI (B) are shown. Open red circles and dot plot connected by red arrows indicate the sequence of analysis steps. First, lymphocytes were gated using size (forward scatter; FSC) and granularity (side scatter, SSC). These cells were then analyzed for CD4 expression. CD4+ T cells were analyzed for IFNγ CD45RO expression for each stimulation (without stimulus, w/o; proteine 3; protein 11; SEB). Proportions of CD45ROhigh IFNγ expressing CD4+ T cells (upper right quadrants) were determined. The background of non-stimulated T cells (w/o) was subtracted for analyses.

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