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

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

Cytokine profile in response to <i>Mtb</i>-specific antigens in active TB.

<p>Polyfunctional cytokine production analysis of Mtb-specific CD4⁺ T-cells by FACS. PB and BALC from active TB patients were stimulated overnight with Rv2628- and RD1-antigens. T- cells were classified as single IFN-γ-producing, single IL-2-producing or double IFN-γ/IL-2–producing cells in response to Rv2628- (A) and RD1-antigens (B). The results are reported as relative median in PB compared to BALC samples (A–B). Black triangles: PB; open triangles: BALC. <b>Footnote:</b> PB: peripheral blood; BALC: bronchoalveolar lavage cells; IFN: interferon; IL: interleukin.</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

Magnitude of RD1-response is significantly higher than Rv2628-response in PBMC and BALC, evaluated by ELISPOT.

<p>ELISPOT evaluation of IFN-γ-producing CD4⁺ T-cells in PBMC and BALC in response to Rv2628- and RD1-antigens in LTBI and active TB subjects. Response to Rv2628- and RD1-antigens in PBMC of active TB and LTBI subjects (A), response to Rv2628- and RD1-antigens in BALC of LTBI and active TB subjects (B) <b>Footnote:</b> PBMC: peripheral blood mononuclear cells; BALC: bronchoalveolar lavage cells; SFC: spot-forming cells; IFN: interferon; TB: tuberculosis; LTBI: latent TB infection.</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