Figure 5. This tetramer-enriched approach detected BCG-elicited resting memory P65-specific CD4 T cells at a frequency of 2-3/10,000 in PBL.

(a) Left bar graph shows that the frequency of ≤0.03% tetramer-bound CD4⁺ T-cells detected by tetramer direct staining in the BCG-vaccinated Mamu-DRB*W201⁺ macaques was difficult to distinguish from background staining (p>0.05). The bar graph on right shows that intracellular IFN-γ staining could not detect the epitope-specific IFN-γ-producing memory CD4 T cells. (b) The flow cytometry histograms on left show the total numbers of tetramer-bound epitope specific CD4 T cells detected by the tetramer-based enrichment approach in 10⁷ PBL from each macaque. The enriched tetramer⁺ CD4⁺ T-cell population is counted by flow cytometry and displayed in the contour plot in the upper right of the CD4 quadruple, with the total numbers shown in the upper left CD4 quadruple. The bar graph on right shows that this tetramer enriched approach can detect significantly greater numbers of P65-specific CD4 T cells in BCG-vaccinated Mamu-DRB*W201⁺ macaques than those in the vaccinated Mamu-DRB*W201^- animals. (c) The bar graph shows that the tetramer staining after P65 stimulation detects about ten-fold greater numbers of the BCG-elicited tetramer-bound CD4 T memory cells than without stimulation. The numbers of tetramer-bound cells in PBL from Mamu-DRB*W201⁺ macaques were significantly greater than those in naïve or BCG-vaccinated Mamu-DRB*W201^- animals (**, p<0.01).