Fig. 3.

Secondary effectors among memory mice differ in cytokine multifunctionality with responses in H56 memory mice dominated by IL-2- and IL-17A-producing CD4 T cell subsets.

Ten weeks into Mtb challenge, lung cells from memory mice were i.v. stained with anti-CD45 prior to euthanization and subsequently stimulated ex vivo with ESAT-6_1-15 and stained by ICS to determine the frequency of antigen-specific CD4 T cells expressing IFN-g, TNF-a, IL-2 or IL-17A in any combination based on combinatorial Boolean gating analysis. Upper panels show lung vascular associated responses (IV +); lower panels responses in the lung parenchyma (IV −). Bar charts show mean frequencies ± s.d. (n = 3), white bars: IL-17A-; grey bars: IL-17A co-producers. Pies embedded into bar charts denote the proportion of each cytokine-producing subset of the responding cells from each lung compartment (unperfused lungs). Cells gated on Singlets > Lymphocytes > CD4 + > Combinatorial/Boolean gating on IV +, IFN-g +, TNF +, IL-2 +, IL-17A +. Repeated once with similar results.