Peripheral T Cell Cytokine Responses for Diagnosis of Active Tuberculosis

BACKGROUND: A test for diagnosis of active Tuberculosis (TB) from peripheral blood could tremendously improve clinical management of patients. METHODS: Of 178 prospectively enrolled patients with possible TB, 60 patients were diagnosed with pulmonary and 27 patients with extrapulmonary TB. The frequencies of Mycobacterium tuberculosis (MTB) specific CD4(+) T cells and CD8(+) T cells producing cytokines were assessed using overnight stimulation with purified protein derivate (PPD) or early secretory antigenic target (ESAT)-6, respectively. RESULTS: Among patients with active TB, an increased type 1 cytokine profile consisting of mainly CD4(+) T cell derived interferon (IFN)-γ was detectable. Despite contributing to the cytokine profile as a whole, the independent diagnostic performance of one cytokine producing T cells as well as polyfunctional T cells was poor. IFN-γ/Interleukin(IL)-2 cytokine ratios discriminated best between active TB and other diseases. CONCLUSION: T cells producing one cytokine and polyfunctional T cells have a limited role in diagnosis of active TB. The significant shift from a "memory type" to an "effector type" cytokine profile may be useful for further development of a rapid immune-diagnostic tool for active TB

Cytokine ratios pulmonary TB/non-tuberculous diseases.

<p>Cytokine ratios pulmonary TB/non-tuberculous diseases.</p

Frequencies of ESAT-6 specific T cells.

<p>Frequencies of single cytokine producing T cells and multifunctional T cells of 60 patients with pulmonary TB (light grey), 27 extrapulmonary TB (dark grey) and 91 with non-tuberculous diseases (white) after overnight stimulation with ESAT-6 are depicted. Boxes and whiskers are shown; the black line marks the median. Differences between patient groups are marked with a bar. A significantly increased frequency of CD4⁺ T cells expressing IFN-γ (0; 0–0; 0–0.07 vs. 0; 0–0.02; 0–0.3 or 0; 0–0.03; 0–0.3 [median; 25%–75% percentile; min–max], respectively. p = 0.034) and a decreased frequency of CD4⁺ T cells expressing IL-2 (0; 0–0.02; 0–0.26 vs. 0; 0–0.01; 0–0.06 [median; 25%–75% percentile; min–max]. p = 0.037) were found in patients with pulmonary TB when compared to diseases other then TB. CD8⁺ T cells expressing IFN-γ and TNF-α⁺ (0; 0–0.02; 0–0.16 vs. 0.02 ; 0–0.05; 0–0.23 [median; 25%–75% percentile; min–max]. p = 0.041) were different when pulmonary and extrapulmonary TB were compared. An independent-samples t-test was used to test for significance.</p

Representative dot plots.

<p>Representative two-parameter dot plots of a patient with extrapulmonary TB (urogenital TB) indicating the frequency of PPD and ESAT-6-specific CD4⁺ T cells expressing IFN-γ and/or IL-2, respectively. PBMC were incubated with medium alone (control), PPD and ESAT-6, respectively.</p

Cytokine ratios extrapulmonary TB/non-tuberculous diseases.

<p>Cytokine ratios extrapulmonary TB/non-tuberculous diseases.</p

Frequencies of PPD specific T cells.

<p>Frequencies of single cytokine producing T cells and multifunctional T cells of 60 patients with pulmonary TB (light grey), 27 extrapulmonary TB (dark grey) and 91 with non-tuberculous diseases (white) after overnight stimulation with PPD are depicted. Boxes and whiskers are shown; the black line marks the median. CD4⁺IFN-γ⁺IL-10⁺ T cells (0.01 ; 0.02–0.33; 0–0.16 vs. 0; 0–0.01; 0–0.08 [median; 25%–75% percentile; min–max]. p = 0.007) were significantly different when extrapulmonary TB was compared to other diseases then TB. Comparing pulmonary and extrapulmonary TB, PPD specific CD8⁺ T cells expressing IL-2 (0.14 ; 0.09–0.365; 0–4.61 vs. 0.13; 0.06–0.33; 0–2.93 [median; 25%–75% percentile; min–max]. p = 0.011) were significantly different. Differences between patient groups are marked with a bar. An independent-samples t-test was used to test for significance.</p

Cytokine ratios pulmonary TB/extrapulmonary TB.

<p>Cytokine ratios pulmonary TB/extrapulmonary TB.</p

Cytokine ratios.

<p>Significant differences between different cytokine ratios are depicted, detected with a one way between-groups analysis of variance using the tukey test for post hoc analysis. Data from 60 patients with pulmonary TB (light grey), 27 extrapulmonary TB (dark grey) and 91 with non-tuberculous diseases (white) are depicted. Boxes and whiskers are displayed, the black line marks the median. Percentile ranges are additionally shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035290#pone-0035290-t003" target="_blank">Tables 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035290#pone-0035290-t004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035290#pone-0035290-t005" target="_blank">5</a>. (A) shows statistically significant differences between non-tuberculous diseases and pulmonary TB: PPD induced IFN-γ/IL-2 (p<0.001), ESAT-6 induced TNF-α/IFN-γ (p = 0.048), TNF-α/IL-2 (p = 0.03), IFN-γ/IL-2 (p = 0.005), all CD4⁺ T cell derived. (B) shows statistically significant differences between non-tuberculous diseases and extrapulmonary TB: PPD induced TNF-α/IL-2 (p<0.001), IFN-γ/IL-2 (p = 0.001), ESAT-6 induced TNF-α/IFN-γ (p = 0.026), TNF-α/IL-2 (p = 0.008), IFN-γ/IL-2 (p<0.001), CD4⁺ T cell derived. (C) shows statistically significant differences between pulmonary TB and extrapulmonary TB: PPD induced TNF-α/IL-2 (p = 0.001), CD4⁺ T cell derived.</p

Summary of receiver-operating-characteristic curves (ROC).

<p>Legends: AUC: area under the curve; S.E.: standard error; Sig.: significance level; CI: confidence interval.</p

ROC curves.

<p>Receiver-operating-characteristic curves (ROC) were calculated for every significantly different cytokine and cytokine ratio. AUCs are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035290#pone-0035290-t002" target="_blank">Table 2</a>.</p