Antigen-Specific IFN-γ Responses Correlate with the Activity of M. tuberculosis Infection but Are Not Associated with the Severity of Tuberculosis Disease

IFN-γ is a key cytokine in antituberculosis (TB) defense. However, how the levels of its secretion affect M. tuberculosis (Mtb) infection is not clear. We have analyzed associations between IFN-γ responses measured in QuantiFERON®-TB Gold In-tube (QFT) assay, TB disease severity, and Mtb infection activity. TB severity was evaluated based on the results of radiological, microbiological, and clinical examinations. Antigen-driven IFN-γ secretion did not correlate with TB severity. Mitogen-induced IFN-γ secretion correlated inversely with the form of pulmonary pathology and the area of affected pulmonary tissue; the levels of spontaneous IFN-γ secretion correlated with patients’ age (r = 0.395, p = 0.001). Mtb infection activity was evaluated based on radiological data of lung tissue infiltration, destruction, dissemination or calcification, and condensation. The rate of positive QFT results and the levels of antigen-driven IFN-γ secretion increased in a row: patients with residual TB lesions < patients with low TB activity < patients with high TB activity. Thus, antigen-driven IFN-γ secretion and QFT results did not associate with TB severity but associated with the infection activity. The results suggest that quantitative parameters of IFN-γ secretion play a minor role in determining the course of TB disease but mirror the activity of the infectious process

<em>Mtb</em>-Specific CD27^low CD4 T Cells as Markers of Lung Tissue Destruction during Pulmonary Tuberculosis in Humans

<div><h3>Background</h3><p>Effector CD4 T cells represent a key component of the host’s anti-tuberculosis immune defense. Successful differentiation and functioning of effector lymphocytes protects the host against severe <em>M. tuberculosis</em> (<em>Mtb</em>) infection. On the other hand, effector T cell differentiation depends on disease severity/activity, as T cell responses are driven by antigenic and inflammatory stimuli released during infection. Thus, tuberculosis (TB) progression and the degree of effector CD4 T cell differentiation are interrelated, but the relationships are complex and not well understood. We have analyzed an association between the degree of <em>Mtb</em>-specific CD4 T cell differentiation and severity/activity of pulmonary TB infection.</p> <h3>Methodology/Principal Findings</h3><p>The degree of CD4 T cell differentiation was assessed by measuring the percentages of highly differentiated CD27^low cells within a population of <em>Mtb</em>- specific CD4 T lymphocytes (“CD27^lowIFN-γ⁺” cells). The percentages of CD27^lowIFN-γ+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p<0.0005). Within the group of patients, the percentages of CD27^lowIFN-γ⁺ cells were uniformly high in the lungs (>76%), but varied in blood (12–92%). The major correlate for the accumulation of CD27^lowIFN-γ⁺ cells in blood was lung destruction (r = 0.65, p = 2.7×10⁻⁷⁾. A cutoff of 47% of CD27^lowIFN-γ⁺ cells discriminated patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27^lowIFN-γ⁺cells following TB therapy correlated with repair and/or reduction of lung destruction (p<0.01).</p> <h3>Conclusions</h3><p>Highly differentiated CD27^low Mtb-specific (CD27^lowIFN-γ⁺) CD4 T cells accumulate in the lungs and circulate in the blood of patients with active pulmonary TB. Accumulation of CD27^lowIFN-γ⁺ cells in the blood is associated with lung destruction. The findings indicate that there is no deficiency in CD4 T cell differentiation during TB; evaluation of CD27^lowIFN-γ⁺ cells provides a valuable means to assess TB activity, lung destruction, and tissue repair following TB therapy.</p> </div

Correlation between TB manifestations and percentages of CD27^lowIFN-γ⁺ CD4 T cells in the blood of TB patients.

1<p>Analysis was initially performed in 50 patients. Subsequently, 12 patients from validation cohort were added (n = 62), mainly to check the consistency of the results. The results obtained in both cohorts are shown.</p>2<p>Simple correlation analysis selects five major predictors for the accumulation of IFN-γ⁺CD27^low cells in the blood of TB patients (highlighted in bold). For TB duration and <i>Mtb</i> multi-drug resistance p-values were >0.007 (insignificant for multiple (seven) parameter testing); these factors were not included in multiple linear regression analysis. rho, Spearman coefficient, p, significance value of the test.</p>3<p>Multiple linear regression identified lung tissue destruction and clinical TB severity as the main correlates for the accumulation of CD27^lowIFN-γ⁺ cells in the blood of TB patients (highlighted in bold).</p>4<p>NA, not included in multiple linear regression analysis.</p

Characterization of groups included in the analysis.

1<p>Indicated are numbers (%).</p>2<p>NA, not applicable.</p

TB patients have increased percentages of CD27^lowIFN-γ⁺ CD4 T cells in their blood.

<p>A–C, Strategies for determining percentages of CD27^low (A), IFN-γ⁺ (B) and CD27^lowIFN-γ⁺ (C) CD4 T cells. A, CD27^low cells were gated within the total population of CD4⁺ T cells. B, To identify IFN-γ⁺ CD4 T cells, an aliquot of blood was stimulated with <i>Mtb</i> sonicate; another aliquote was left un-stimulated. During the analysis, the gates for IFN-γ⁺ cells in <i>Mtb</i>-stimulated samples were plotted based on <i>Mtb</i> un-stimulated samples (Fig. B, dotted line). To identify CD27^lowIFN-γ⁺ cells, the expression of CD27 was first analyzed in IFN-γ⁻ population. Because this population was always numerous, CD27^low and CD27^hi cells could be easily separated. The gates for CD27^low cells were then applied to IFN-γ⁺ population (C, dotted line). D–F, Percentages of CD27^low (D), IFN-γ⁺ (E), and CD27^lowIFN-γ⁺ (F) cells in TB patients (n = 50), TB contacts (n = 21) and <i>Mtb</i>-unexposed individuals (n = 15). G, Lack of correlation between the percentages of IFN-γ⁺ and CD27^lowIFN-γ⁺ cells in TB patients, TB contacts and <i>Mtb</i>-unexposed individuals (n = 86). H, ROC-curve of CD27^lowIFN- _γ⁺ cell percentages for discriminating TB patients from healthy individuals (TB contacts and <i>Mtb</i>-unexposed). I, Percentages of CD27^lowIFN-γ⁺ cells in TB contacts with positive and negative results of QFT assay *p<0.0005 compared to TB patients.</p

Selection of minimal model to explain variability in the percentages of CD27^lowIFN-γ⁺ cells between TB patients.

1<p>Best minimal models are those that differ insignificantly from the full model (highlighted in bold).</p>2<p>Analysis was initially performed in 50 patients. Subsequently, 12 patients from validation cohort were added (n = 62), mainly to check the consistency of the results. In both cohorts, lung destruction and clinical disease severity predicted best the accumulation of CD27^lowIFN-γ⁺ cells.</p>3<p>In Akaike Information Criterion, this combination was the best minimal model to predict the accumulation of CD27^lowIFN-γ⁺ cells in the blood ( Δ_n = 50 = 4.7; Δ_n = 62 = 4.8).</p

Evaluation of lung tissue destruction.

<p>Examples of X-ray computer tomograpy with different degrees of tissue destruction are shown. Lung destruction was evaluated based on the number and size of destructive (lucent) foci (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043733#s2" target="_blank">Methods</a> for the details). A, score 0, no destruction; B, score 1, one small (<2 cm) destruction; C, score 2, one large (>2 cm) destruction; D, score 3, multiple lung destructions. Arrows, lung destructions. Asterisk, infiltrative focus without destruction.</p

Discrimination of TB patients from different groups of healthy participants by the percentages of CD27^lowIFN-γ⁺ cells¹.

1<p>The percentages of CD27^lowIFN-γ⁺ cells were compared in TB patients and indicated groups of healthy participants. AUC, area under curve; CI, confidence interval; LR, likelihood ratio; OR, odds ratio.</p