Doxorubicin inhibits MDSC then enhances the killing effect of CTL in vitro
Under inverted microscope, the killing process of CTLs to SK-N-SH cells was clearly shown in each group respectively. From day 1 to day 10, we found that in SK-N-SH+CTL+MDSC groups, SK-N-SH cells still scattered in view with relative regular outline. However, in CTL+SK-N-SH group and CTL+SK-N-SH+MDSC+DOX groups, nearly all of SK-N-SH cells appeared apoptosis or necrosis, and CTLs also decreased significantly. During the whole period, in SK-N-SH group, tumor cells proliferated continuously and kept active state (Fig. 4A). So these results proved that doxorubicin inhibits MDSC then enhances the killing effect of CTL on SK-N-SH cells. The significant difference existed in the killing rate between the groups (F = 22.386, P < 0.001) except between CTL+SK-N-SH group and CTL+SK-N-SH+DOX group (P = 0.100 > 0.05) (Fig. 4B). IL-2 and IFN-γ in the supernatant were detected by ELISA. By repeated measurement analysis of variance, the results showed that there were significant differences in the secretion levels of IL-2 (F = 192.013, P = 0.000< 0.001) and IFN-γ (F = 519.274, P = 0.000< 0.001) among CTL+SK-N-SH group, CTL+MDSC+SK-N-SH group and CTL+MDSC+SK-N-SH+DOX group (Figs. 4C and 4D).