Figure 1. Proportion of peripheral blood CD8+CD57+ T cells. PBMC from TB-patients and healthy controls were incubated with monoclonal antibodies against CD8 and CD57 and analyzed by flow cytometry. The percentage of CD8+CD57+ T cells is shown. Results are expressed as median and interquartile range using the Wilcoxon rank-sum (Mann–Whitney); the p value of the difference between TB-patients and healthy controls is indicated; n = 20.

Figure 2. Expression of phenotypic markers on CD8+CD57+ and CD8+CD57− T cells from TB-patients and healthy controls. Immunomagnetic beads purified CD8+ cells were analyzed for cell surface markers by two-color immunofluorescence. (A) Cells were stained with mAbs to either CD28, CD44, CD45RA, CD62L, or CD57. Results are expressed as median and interquartile range using the Wilcoxon rank-sum (Mann–Whitney), n = 6. (B) Cells stained with anti-CD57 monoclonal antibody and either anti-CD161 mAbs or α-GalCer-loaded CD1d tetramers; the circle within each dot plot indicates the gate used to determine the percentage of double positive cells. Data are representative of three independent experiments. *p < 0.03, **p < 0.009, ***p < 0.006.

Figure 3. Differential expression of CD69 in CD8+CD57 positive and negative T cells. Freshly obtained PBMC from TB-patients were analyzed for CD8, CD57, and CD69 expression by three-color immunofluorescence. CD69 was evaluated in CD57 positive and negative fluorescent CD8+ cells. Fifty percent of CD8+CD57+ and 19% of CD8+CD57− cells expressed CD69 (the arrows indicate the respective histogram). Activated NK cells are situated exactly under CD8dim+CD57+ T cells. Data are representative of three independent experiments.

Figure 4. In vitro cytotoxic activity of purified CD8+ T cell subsets. Freshly isolated CD8+CD57+ and CD57− T cell subsets (effector cells) from healthy controls or TB-patients were co-cultured with autologous monocytes (target cells) in the absence (A) or presence (B) of RvCFP for 4 h at 37°C. The activity of released lactate dehydrogenase enzyme from damaged cells was determined by a colorimetric assay calculating the percentage of cytolysis by an equation (see Materials and methods). Results are expressed as median and interquartile range using the Wilcoxon rank-sum (Mann–Whitney). All assays were performed in triplicate, n = 6. *p < 0.01, **p < 0.0005, ***p < 0.006.

Figure 5. Expression of perforin and granzyme-A on purified CD8+ T cell subsets. Freshly isolated CD8+CD57+ and CD57− T cell subsets from healthy controls and TB-patients were analyzed for the intracellular expression of granzyme-A and perforin. (A) Percentage of cells positive to perforin and granzyme, n = 6. Results are expressed as median and interquartile range using the Wilcoxon rank-sum (Mann–Whitney) *p < 0.05. (B) Representative staining of individual perforin and granzyme-A expression in purified CD8+CD57+ and CD8+CD57− T cells from a TB-patient. Tin lines indicate the staining pattern of isotype-matched control mAbs, whereas the thick lines denote the fluorescence intensity of the specific antibody.

Percentage of IFN-γ and TNF-α expression on CD8+ T cell subsets

Percentage of CD8+CD57+ and CD8+CD57− T cells positive to IFN-γ and TNF-α. PBMC from TB-patients or healthy controls were stimulated with phorbol-12-myristate-13-acetate (50 ng/ml) plus ionomycin (500 nM) for 4 h at 37°C, before staining the cells with specific IFN-γ and TNF-α monoclonal antibodies. The cells were analyzed by flow cytometry considering a three-color immunofluorescence. Results represent the mean ± standard deviation, n = 5. The p value is indicated; ns = not statistically significant.