Fig 4. Clofazimine enhances the capacity of BCG revaccination to increase the frequency of T_SM cells.

Spleens were harvested and single cell suspensions were made. Cells were cultured overnight with M.tb lysate (CSA) and stained with, anti-CD4, -CD62L, -CD45RA, -CD95 and -CD122 antibodies. (a) Representative gating strategy for isolation of CD4⁺CD45RA⁺CD62L⁺CD95⁺CD122⁺ T_SM cells from the lymphocyte population and percentage of T_SM cells in different experimental groups. (b) Percentage of IL-6- and TGFβ-producing peritoneal macrophages cultured in the presence or absence of clofazimine. (c) Peritoneal macrophages were primed with M.tb antigen in the presence or absence of clofazimine for 4 hrs. Then, the primed macrophages were cocultured with T cells from OT-II transgenic mice in the presence of ovalbumin overnight. Finally, the percentage of IL17-producing CD4⁺ cells was analysed and (d) expression of RORγt was determined by qPCR analysis. All data are representative of 3 independent experiments and each group included at least 5 mice in each experiment. For in vitro experiments at least 3 biological replica and 3 technical replica were performed. All values are represented as Mean±SD. Statistical analyses were performed by ANOVA with Tukey’s post hoc test. * denotes P ≤0.05. CF, clofazimine.