Fig. 1.

γδ T-cells within BCG-, M. vaccae- and M. obuense-treated PBMCs produce granzyme B and T_H1 cytokines. PBMCs were cultured with heat-killed BCG, M. vaccae (Mv) and M. obuense (Mo) and responses measured within gated γδ T-cells. Untreated (un) cells were used as a negative control. a Gating strategy used to identify γδ T-cells within PBMCs. Lymphocytes were gated according to size (forward scatter; FSC) and granularity (side scatter; SSC). Within lymphocytes, γδ T-cells were gated as CD3⁺TCRγδ⁺. Mean percentages of γδ T-cells expressing CD69 (at 24 h; n = 5), CD25 and HLA-DR (both at 48 h; n = 3) are shown. b Mean percentages of Vδ1⁺ and Vδ2⁺ cells expressing CD69 (at 24 h; n = 3). c CFSE⁺ PBMCs were cultured for 6 days with mycobacteria and proliferation measured within the γδ T-cell compartment. Mean percentages of γδ T-cells proliferating are shown (n = 6). d Mean fluorescent intensities (MFI) of granzyme B expression within γδ T-cells (at 24 h; n = 3). e Percentages of γδ T-cells expressing IFN-γ, TNF-α and IL-10 were measured after 24 h of stimulation. Representative flow cytometric dot plots from one donor and mean values for n = 3 are shown. Error bars represent SD. * and ** indicates P values of <0.05 and <0.001, respectively, for statistical comparisons between treated and untreated cells