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.

<p>(A). Percentages of IFNγ-expressing CD4⁺ CD45RO⁺ memory T cells are shown for stimulation with SEB, PPD from <i>M. tuberculosis</i>, and 11 latency-associated antigens after 7 days and two rounds of <i>in vitro</i> 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: * <i>P</i><0.05, ** <i>P</i><0.01; and *** <i>P</i><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 <i>M. tuberculosis</i>; SEB Staphylococcus enterotoxin B.</p

IFNγ ELISA analyses after restimulation with immunodominant and latency-associated antigens of PBMC from LTBI and TST-negative donors.

<p>Analyses of IFNγ in the culture supernatant by ELISA after 7 days and two rounds of <i>in vitro</i> restimulation in PBMC from LTBI (<i>A</i>) and TST-negative donors (<i>B</i>) 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 <i>M. tuberculosis</i>, and tested antigens (x-axes). The most promising candidate Rv3407 is underlined.</p

List of proteins candidates.

*<p><i>M. tuberculosis</i> dormancy-related antigens.</p

Design of Rv3407 peptide pools.

1<p>Peptide number according to the position within the primary sequence from C- to N-terminus.</p

Overlapping peptide pools of latency-associated protein Rv3407 stimulate IFNγ-expressing CD4⁺ T cells after 7 days and two rounds of restimulation.

<p>PBMC from six LTBI (A–F) were restimulated with 15-mer synthetic peptide pools of Rv3407 for 7 days including two rounds of <i>in vitro</i> 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 <i>A–D</i>. The horizontal line indicates the threshold for positive responses (0.2%). Background values of non-stimulated controls were subtracted.</p

Gating procedures of flow cytometry analyses to determine protein candidate specific T cell proportions.

<p>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 CD45RO_high IFNγ expressing CD4⁺ T cells (upper right quadrants) were determined. The background of non-stimulated T cells (w/o) was subtracted for analyses.</p