Volume 66, Issue 6, Pages 475-486 (June 2013) IFNγ/TNFα specific-cells and effector memory phenotype associate with active tuberculosis  Elisa Petruccioli, Linda Petrone, Valentina Vanini, Alessandro Sampaolesi, Gina Gualano, Enrico Girardi, Fabrizio Palmieri, Delia Goletti  Journal of Infection  Volume 66, Issue 6, Pages 475-486 (June 2013) DOI: 10.1016/j.jinf.2013.02.004 Copyright © 2013 The British Infection Association Terms and Conditions

Figure 1 Increased frequency of cytokine response to “RD1” proteins in CD4+ T-cells in active TB. Flow cytometry evaluation of CD4+ T-cell response to “RD1” recombinant proteins (ESAT-6 and CFP-10). A response was scored as positive considering the production of any cytokines (IFNγ and/or IL2 and/or TNFα) with a detection limit of 0.03% corresponding to at least 30 events analyzed. The horizontal lines represent the median; the dotted line indicates the limit of detection (0.03%) to score a response as positive; statistical analysis was performed using the Mann–Whitney test with Bonferroni correction and p value was considered significant if ≤0.016. Footnotes: TB: tuberculosis; LTBI: latent TB infection. Journal of Infection 2013 66, 475-486DOI: (10.1016/j.jinf.2013.02.004) Copyright © 2013 The British Infection Association Terms and Conditions

Figure 2 Representative flow cytometry panel of an LTBI subject. Whole blood cells were stimulated overnight with or without “RD1” recombinant proteins (ESAT-6 and CFP-10) and analyzed by flow cytometry for intracellular production of IFNγ IL2 TNFα. Cells were gated according to forward and side scatter plots and the frequency of single, double and triple cytokines producing CD4+ T-cells was evaluated using boolean combination gates. Footnotes: IFN: interferon; IL: interleukin; TNF: tumor necrosis factor. Journal of Infection 2013 66, 475-486DOI: (10.1016/j.jinf.2013.02.004) Copyright © 2013 The British Infection Association Terms and Conditions

Figure 3 Bifunctional IFNγ+ TNFα+ CD4+ T-cells are associated with active TB disease. Whole blood cells were stimulated overnight with “RD1” recombinant proteins (ESAT-6 and CFP-10) or CMV and analyzed by flow cytometry for intracellular production of IFNγ IL2 TNFα. The graphs show the proportion of cytokine-producing subsets of the antigen-specific response in the different groups. The horizontal lines represent the median; statistical analysis was performed using Mann–Whitney test with Bonferroni correction and p value was considered significant if ≤0.016. Pie charts represent the proportion of cytokine-producing cell subsets in response to each stimulus. A) Functional cytokine profile of “RD1”-specific CD4+ T-cells. B) Functional cytokine profile of CMV-specific CD4+ T-cells. Footnotes: TB: tuberculosis; LTBI: latent TB infection; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; *: p ≤ 0.016. Journal of Infection 2013 66, 475-486DOI: (10.1016/j.jinf.2013.02.004) Copyright © 2013 The British Infection Association Terms and Conditions

Figure 4 Central memory phenotype associates with M. tuberculosis control and effector memory phenotype associates with active TB. Memory status of CD4+ T-cell response was evaluated by flow cytometry according to the surface expression of CD45RA and CD27. We defined naïve (N) as CD45RA+ CD27+, central memory (CM) as CD4RA− CD27+, effector memory (EM) as CD45RA− CD27−, terminally differentiated effector memory T-cells (E) as CD4RA+ CD27−. The graphs show the proportion of N, CM, EM and E CD4+ T-cells in the different groups in response to overnight stimulation with the “RD1” recombinant proteins (ESAT-6 and CFP-10) or CMV. The horizontal lines represent the median; statistical analysis was performed using the Mann–Whitney test with Bonferroni correction and p value was considered significant if ≤0.016. A, B, C, D, E) Phenotype profile of “RD1”-specific CD4+ T-cells. F, G, H, I, L) Phenotype profile of CMV-specific CD4+ T-cells. A, F) Cells gated on total IFNγ+ CD4+ T-cells. B, G) Cells gated on total TNFα+ CD4+ T-cells. C, H) Cells gated on total IL2+ CD4+ T-cells. D, I) Cells gated on IFNγ+ TNFα+ CD4+ T-cells. E, L) Cells gated on IFNγ+ TNFα+ IL2+ CD4+ T-cells. Footnotes: TB: tuberculosis; LTBI: latent TB infection; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; N: naïve; CM: central memory; EM effector memory; E: terminally differentiated effector memory; *: p ≤ 0.016; **: p ≤ 0.001. Journal of Infection 2013 66, 475-486DOI: (10.1016/j.jinf.2013.02.004) Copyright © 2013 The British Infection Association Terms and Conditions

Figure 5 Among the subjects at different TB stages, the memory status of the specific CD4+ T-cells in response to “RD1” (ESAT-6 and CFP-10) differs from the response to an unrelated antigen such as CMV. Memory status of CD4+ T-cell response was evaluated by flow cytometry according to the surface expression of CD45RA and CD27. We defined naïve (N) as CD45RA+ CD27+, central memory (CM) as CD4RA− CD27+, effector memory (EM) as CD45RA− CD27−, terminally differentiated effector memory T-cells (E) as CD4RA+ CD27−. Pie charts represent the proportion of N, CM, EM and E CD4+ T-cells in the different groups in response to overnight stimulation with “RD1” recombinant proteins (ESAT-6 and CFP-10) or CMV. A, F) Cells gated on total IFNγ+ CD4+ T-cells. B, G) Cells gated on total TNFα+ CD4+ T-cells. C, H) Cells gated on total IL2+ CD4+ T-cells. D, I) Cells gated on IFNγ+ TNFα+ CD4+ T-cells. E, L) Cells gated on IFNγ+ TNFα+ IL2+ CD4+ T-cells. Footnotes: TB: tuberculosis; LTBI: latent TB infection; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; N: naïve; CM: central memory; EM effector memory; E: terminally differentiated effector memory. Journal of Infection 2013 66, 475-486DOI: (10.1016/j.jinf.2013.02.004) Copyright © 2013 The British Infection Association Terms and Conditions

Figure 6 Comparison of the memory status found after “RD1” and CMV stimulation in the subjects concomitantly responding to both antigens in the active TB, cured TB and LTBI groups. Memory status of CD4+ T-cell response was evaluated by flow cytometry according to the surface expression of CD45RA and CD27. We defined naïve (N) as CD45RA+ CD27+, central memory (CM) as CD4RA− CD27+, effector memory (EM) as CD45RA− CD27−, terminally differentiated effector memory T-cells (E) as CD4RA+ CD27−. Pie charts represent the proportion of N, CM, EM and E CD4+ T-cells in the different groups in response to overnight stimulation with “RD1” recombinant proteins (ESAT-6 and CFP-10) or CMV. Cells were gated on total IFNγ+ CD4+ T-cells. Each pie represents the response found in a single subject. Footnotes: TB: tuberculosis; LTBI: latent TB infection; IFN: interferon; N: naïve; CM: central memory; EM effector memory; E: terminally differentiated effector memory. Journal of Infection 2013 66, 475-486DOI: (10.1016/j.jinf.2013.02.004) Copyright © 2013 The British Infection Association Terms and Conditions