M. hyopneumoniae-specific T cell responses following vaccination and challenge infection. Pigs were prime-boost vaccinated on D0 and D14 with three different experimental M. hyopneumoniae bacterins (Lipo_DDA:TDB, PLGA_TLR, SWE_TLR), challenge infected on D28–29 and euthanized on D56. M. hyopneumoniae-specific T cells were determined by in vitro restimulation of PBMC followed by intracellular cytokine staining and multicolour flow cytometry. Following doublet exclusion, live cells were gated and the percentage of TNF+ CD4+, IFNγ+TNF+ double positive CD4+ and CD8β+ T cells, and IL-17A+ CD4+ T cells was determined. The mean values obtained from triplicate cultures for individual animals are shown. Positive animals are marked in red (defined as being above the mean + 3*SD of all control animals on D28 and being above the mean + 3*SD of the NCG on D56). The horizontal line in each group represents the group mean. For each time point, significance between groups was calculated using one-way ANOVA followed by Tukey–Kramer’s test. The NCG was not included in the statistical analyses. Groups that have no superscript in common are significantly different from each other (P ≤ 0.05). NCG, non-challenge control group (PBS-injected, non-challenge infected); PCG, PBS-injected control group (PBS-injected, challenge infected); PBMC, peripheral blood mononuclear cells.