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. 2017 Oct 26;66(4):554–563. doi: 10.1093/cid/cix834

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© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Bacille Calmette-Guérin (BCG)-specific CD4 cytokine responses. Fresh whole blood was stimulated with BCG for 12 hours prior to intracellular cytokine staining and flow cytometry analysis. A, Longitudinal changes of BCG-specific CD4+ T cells expressing any combination of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin (IL) 2, IL17, and/or IL22 are indicated by arrows (red for MVA85A arm and black for control arm). Medians and 95% confidence intervals (CIs; for the medians) are shown. Unadjusted P values were calculated by mixed effects models. B, Cytokine coexpression patterns of BCG-specific CD4 responses at week 16 (left) and week 52 (right) in the MVA85A (top) and control (bottom) arms by permutation test. Pies represent total BCG-specific CD4+ T cells expressing any cytokine; slices show the relative proportion of cells coexpressing 1 (blue), 2 (green), 3 (yellow), or 4 (orange) cytokines, identified by the external arcs: IFNγ (red), IL2 (black), IL17 (orange), IL22 (purple), and TNFα (dark green). C, Differentiation profiles were defined based on expression patterns of CD45RA and CCR7 as follows: naive-like (T_NL, CD45RA+ CCR7+), central memory (T_CM, CD45RA- CCR7+), effector (T_E, CD45RA- CCR7-), and terminal effector (T_TE, CD45RA+ CCR7-). Representative flow cytometry plot of BCG-specific cytokine+ CD4+ T cells (pink) overlaid on total CD4+ T cells (black). D, Longitudinal changes of BCG-specific cytokine+ CD4+ T cells expressing T_NL (maroon), T_CM (blue), T_E (orange), or T_TE (green) phenotype in MVA85A (left) or control (right) recipients. Medians and 95% CIs (for the medians) are shown. The number of participants meeting cutoff criteria for this analysis (see methods) is shown for each visit. Frequencies of all subsets significantly increased (P < .025) upon BCG vaccination (week 8 vs week 16) and decreased (P < .025) between week 16 and 52, with the exception of T_CM in the MVA85A arm. Unadjusted P values were calculated by Wilcoxon matched-pairs test; week 4 and 8 were not compared due to low numbers of paired samples (less than 10). No significant differences were observed when comparing frequencies of each subset at each visit between study arms. Abbreviations: BCG, bacille Calmette-Guérin; IFNγ, interferon gamma; IL, interleukin; TNFα, tumor necrosis factor alpha.