Figure 4.

Flow cytometry expression of NK receptors and cytotoxic molecules in BCG-activated γδ T-cells.

PBMC from healthy donors were incubated with the sub-strains of BCG iBCG or Oncotice at a 6:1 ratio (total bacteria to PBMC). After 7 days in culture, cells in suspension were recovered and analyzed by flow cytometry. The percentage of cells expressing each marker and standard deviation are plotted in black. Gray bars represent the average percentage of the CD3⁺TCRɣδ⁺ subset in the lymphocyte gate. a. NK Receptors in γδ T-cells. The graphs represent the percentage of γδ T-cells, positive for the indicated marker (black), within the γδ TCR gate of total lymphocytes. In certain cases, the relative fluorescence intensity (RFI) is shown to analyze the different levels of expression, since the whole population was positive for these markers. An RFI >1 means above the Ig control (RFI = MFI sample/MFI IgG, where MFI is mean fluorescence intensity). b. Granzyme A and perforin in γδ T-cells. N = 6 healthy donors in each case, except for CD27 (seven donors). c. Purified γδ T-cells. γδ T-cells were negatively selected from 20 · 10⁶ PBMC. Either purified γδ T-cells or the remaining PBMC (γδ T-cell-depleted, PBMC [-γδ cells]) were co-cultured with iBCG for 7 days and analyzed by flow cytometry. The γδ T-cell number before (D0) and after iBCG culture (D7) are depicted, together with average and standard deviation. For comparison, the activation obtained for each donor after co-culture of PBMC with iBCG was also analyzed. Data from four healthy donors, each one depicted with a different symbol, are shown (purity after γδ T-cell selection > 95%). d. γδ T-cells grown with supernatant from iBCG-stimulated cultures. Supernatants from the experiment in C were recovered, centrifuged at 10,000x g to eliminate any remaining bacteria, and used to feed a second culture of autologous PBMC. After 1 week in culture, cells were characterized by flow cytometry. The graphs show the percentage of γδ T-cells expanded in each condition. For comparison, a second co-culture of PBMC with iBCG was also set for each donor.