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. Author manuscript; available in PMC: 2020 Oct 15.
Published in final edited form as: J Immunol. 2019 Sep 20;203(8):2194–2209. doi: 10.4049/jimmunol.1900230

Figure 7.

Figure 7.

Mtb-responsive resident airway CD4+ T-cells of LTBI individuals produce multiple cytokines. Supernatants of Mtb-infected BAL cells from cultures used in our gene expression studies were evaluated for production of IFNγ, TNFα, IL-2 and IL-17. As illustrated in Figure 7A, levels of IFNγ, IL-2, and IL-17 were all significantly reduced in cultures of BAL cells from which CD4+ T cells were depleted as compared to those of unsorted BAL cells (based on paired t-tests); reduction in TNFα was observed as well but was not statistically significant. In contrast, depletion of CD8+ T cells had no significant impact on production on any of these cytokines by BAL cell of LTBI subjects (not shown). In all figures, results are represented as cytokine levels for Mtb-infected cultures subtracted from cultures of uninfected, unsorted BAL cells from the same individual. Cytokine levels are reported as pg/mL of culture supernatants, with the exception of TNFα, which is reported as ng/mL. Based on these findings, we also examined polyfunctionality of BAL CD4+ TEM from LTBI subjects using intracellular cytokine staining for these same four cytokines in response in vitro stimulation with PPD. Figure 7B shows the percentage of effector-memory (CD45RA-/CCR7-) CD4+ T cells (TEM) in each subject’s samples that produced each of the possible 15 cytokine combinations in response to PPD. As illustrated, the most commonly observed PPD-induced cytokine profiles were single production of TNFα and dual production of TNFα and IFNγ, with polyfunctional production of TNFα, IFNγ and IL-2 being the next most common. These findings are represented in pie chart form in Figure 7C. The very low number of CD4+ TEM producing all 4 cytokines are indicated by the thin black slice pointing straight upward; from there, the remaining combinations of three, two and single cytokines proceed clockwise in the same order displayed in 7B. The colored arcs indicate production of each of the measured cytokines. As indicated, TNFα (dark blue arc) is the most frequently observed cytokine produced by PPD-responsive BAL CD4+ TEM, followed by IFNγ (red arc), IL-2 (green arc), and IL-17 (light blue arc).