L-arginine metabolism is correlated with the inhibition activity of MDSCs.

<p>Levels of (A) L-arginine, (B) Ornithine and (C) Nitrate in the serum of healthy donors (HD, N.15) and active TB patients (TB, N.30). Results are expressed as the median ± IQR. *P<0.05 **P<0.02.</p

Successful TB treatment reduces the concentration of MDSCs in peripheral blood cells.

<p>(A) Analysis of MDSC frequency in patients with active TB (N.30) and cured TB (N.10). (B) MDSC frequency in cured TB at the end of therapy (N.5) and 1–3 years after the end of therapy (past TB, N.5). Results are expressed as the median ± IQR. *P<0.05.</p

Concentration-dependent IFN-γ response after <i>in vitro</i> short-term whole blood stimulation with rHBHAms in subjects with LTBI.

<p>Whole blood from 10 subjects with LTBI was stimulated with or without rHBHAms at different concentrations (between 25 and 1 µg/ml). IFN-γ response was evaluated after a short-term stimulation (1 day post-<i>in vitro</i> stimulation). A significant difference was found for the IFN-γ response obtained between 25 and 5 µg/ml and that obtained at a concentration of 1 µg/ml.</p

IFN-γ response to rHBHAms in short- and long-term-“<i>in vitro</i>” stimulation and to QFT-IT: ROC analysis.

<p>A ROC analysis for the response to rHBHAms obtained in whole blood by short-term stimulation (<b>A</b>), long- term stimulation (<b>B</b>) and QFT-IT (<b>C</b>) was performed using the active TB patients and the subjects without active TB as comparator groups.</p

CD4⁺ effector memory T lymphocytes produce IFN-γ in response to rHBHAms.

<p>The phenotypic characteristics of the cells responding to the rHBHAms in the HBHA-responders were evaluated. As shown in a representative subject, a significant IFN-γ response to the rHBHAms was observed for CD4⁺ T-cells (<b>D</b>) over the negative control (<b>B</b>), whereas no response was detected for CD8⁺ T-cells (<b>C</b>) over the negative control (<b>A</b>). To characterize this immune response, naive and memory phenotypes were studied. Most of the CD4⁺ T-cells IFN-γ responding to the rHBHAms presented an effector memory phenotype (84%) defined as CD45R0⁺CD62L (<b>E</b>).</p

Demographic and clinical characteristics of the subjects enrolled in the study.

<p> <b>Footnotes: TB: tuberculosis; IQR: interquartile range; BCG: Bacillus Calmètte et Guerin; QFT-IT:QuantiFERON TB Gold In Tube; NA: not available.</b></p

Response to rHBHAms is significantly impaired in patients with active TB after short- or long- term stimulation.

<p>A significant difference was found for the IFN-γ response to rHBHA between the subjects with and without active TB (p = 0.001) evaluated by short- or long- term stimulation.</p

Memory responses to rHBHAms.

<p>Memory response (long-term stimulation) to rHBHAms was evaluated in the subjects who scored negative on the short- test. A significant difference was found between the IFN-γ value obtained in the short-term stimulation compared to the long-term stimulation among those without active TB (p = 0.0003). Differently, no significant difference was found between the short- and long- term stimulation among those with active TB (p = 0.4). The data are shown after the subtraction of the results obtained in the unstimulated samples. Dotted lines indicate the cut-off obtained for the short- and long-term tests.</p

Response to rHBHAms is significantly impaired in patients with active TB.

<p>Whole blood was stimulated with or without QFT-IT (<b>A</b>), rHBHAms (<b>B</b>) and CMV lysate (<b>C</b>). IFN-γ response was evaluated after a short-term stimulation (1 day post-<i>in vitro</i> stimulation). The data are shown after the subtraction of the results obtained in the unstimulated sample. A significant difference in terms of IFN-γ release to QFT-IT (<b>A</b>) and CMV lysate (<b>C</b>) was found between those with active TB and recent infection (p = 0.01 and p = 0.002 respectively). A significant lower response to rHBHAms (<b>B</b>) was found in patients with active TB compared to those with remote LTBI (p = 0.001) and past TB (p = 0.02).</p

Increased frequency of Rv2628- and RD1-response in BALC than PBMC in active TB, evaluated by ELISPOT.

<p>ELISPOT evaluation of IFN-γ-producing CD4⁺ T-cells in circulating and BAL lymphocytes in response to Rv2628- and RD1-antigens in active TB and LTBI subjects. Response to Rv2628- (A) and RD1-antigens (B) in active TB patients; response to Rv2628- (C) and RD1-antigens (D) in LTBI subjects. <b>Footnote:</b> PBMC: peripheral blood mononuclear cells; BALC: bronchoalveolar lavage cells; SFC: spot-forming cells; IFN: interferon; TB: tuberculosis; LTBI: latent TB infection. Dotted lines link the results obtained for circulating and local lymphocytes for the same patient.</p