Fig. 5.

Assessing effector cytokine production of Ag85A-specific CD4⁺ T cells expressing CXCR3⁺ KLRG1^- in the lung parenchyma of naïve and vaccinated mice. Mice were immunised as shown in Fig. 3a and sacrificed 4 weeks post vaccination. Fluorochrome-conjugated CD45.2 was injected intravenously before sacrificing. Lungs cells were stimulated ex-vivo with Ag85A (85A) peptide pool. The cells were stained and analysed using flow cytometry (Appendix 3). (a) Representative flow plot shows lung parenchymal CD4⁺ T cells producing Ag85A-specific IFN-γ and/or TNF-α in the lungs of B-C-Mid vaccinated mice. (b) The cytokine producing cells were then gated on the expression of CXCR3⁺ KLRG1^-. (c) Total cell number of Ag85A-specific IFN-γ and/or TNF-α producing lung parenchymal CXCR3⁺ KLRG1^- CD4⁺ T cells. (d) Representative flow plot shows lung parenchymal CD8⁺ T cells producing Ag85A-specific IFN-γ and/or TNF-α of B-C-Mid group. (e) The cytokine producing cells were then selected on the expression of CXCR3⁺ KLRG1^-. (f) Total cell number of Ag85A-specific IFN-γ and/or TNF-α producing lung parenchymal CXCR3⁺ KLRG1^- CD8⁺ T cells. Each symbol represents one animal and the line is the median of each group (n = 4–6 mice). Data are representative of three independent experiments. Statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.