Figure 4.

SAHA treated AM enhanced BCG-primed CD4 Th cell responses to Mtb infection. Human AM were infected with H37Ra (MOI 1-10) in the presence of SAHA or DMSO. After 24 h, AM were washed and co-cultured with CFSE-labelled PBMC from a BCG-vaccinated healthy donor (IGRA negative) who responds to PPD antigens in vitro. Uninfected AM co-cultured with PBMC and Mtb-infected AM not co-cultured with PBMC were assayed in parallel as controls. (A) The concentrations of IFN-γ present in the supernatants on the indicated days were analysed by ELISA (n = 3). On day 10 post co-culture, PBMC were removed, stimulated with PMA/ionomycin in the presence of brefeldin A, or left unstimulated. (B,C) Cells were stained with fluorochrome-conjugated antibodies specific for CD3, CD4, CD8, IFN-γ, TNF and GM-CSF, and analysed by flow cytometry. Cells were gated on the basis of forward and side scatter, doublets were then excluded and the population of proliferating CFSE^lo cells were gated. The CD4 and CD8 cell subpopulations were gated within proliferating cells, then cytokines were examined within these populations. Graphs illustrated collated data (n = 4) from unstimulated samples for the frequencies of proliferating cells expressing CD4 (B; left) or CD8 (C; left) and cytokine production from stimulated samples within the population of proliferating (CFSE^lo) CD3⁺ CD8⁻ (CD4⁺) T helper cells (B) and CD3⁺ CD8⁺ cytotoxic T cells (C). (D) Representative dot plots illustrate co-staining of IFN-γ and GM-CSF within the CFSE^lo CD3⁺ CD8⁻ Th cell gate. (E) Collated data shows the frequencies of IFN-γ⁺ GM-CSF⁺ double-positive and IFN-γ⁺ GM-CSF⁺ TNF⁺ triple-positive cells in the CD4 Th cell population that are proliferating. Statistically significant differences between DSMO and SAHA treated groups were determined by two-way ANOVA with Sidak's multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.