Fig. 4
Adenosine generated by CeCa-MSCs strongly inhibits the proliferation of CD8+ T-cells. A total of 5 × 105 CD8+ T-cells obtained by negative selection were cultured with beads containing anti-CD2/CD3/CD28 antibodies in a 2:1 ratio and in the presence of various concentrations of synthetic adenosine (a). Some cells were cultured with supernatants (Sup) obtained from NCx-MSC or CeCa-MSC cultured in the presence of the AMP (bars with diagonal lines), ADP (grey bars) or ATP (black bars), or in the presence of adenine nucleotides plus 300 μM caffeine (CAF), a nonselective antagonist of Ado receptors; 1 µM ZM241385 (ZM), a selective antagonist of A2A receptor, or CAF:ZM (300 μM:1 µM), as indicated (b). CD8+ T-cells cultured in the presence of 500 μM synthetic Ado were used as positive controls of inhibition. After 96 h of culture, CD8+ T-cell proliferation was determined by a colorimetric method described in “Methods” section. Significant differences *(P < 0.05) and **(P < 0.01) were obtained compared with CD8+ T-cells cultured with beads in the absence of Ado (negative control of inhibition). Data are representative of three independent experiments, and the mean ± SEM are shown