Figure Legends
Figure1 Extraction, identification, purification of myeloid-derived suppressor cell (MDSC) and cultivation and tumor antigen loading of dendritic cell (DC) A, Cells were extracted from bone marrow of BALB/c mice and stained by monoclonal antibodies. By flow cytometry, the expressive rate of Gr-1+MDSC, CD11b+MDSC, CD11c+MDSC, CD80+MDSC, F4/80+MDSC and MHC-II+MDSC were 70.4%, 3.5%, 4.8%, 1.2%, 0.3%, 2.1% respectively. B, The expressive rate of Gr-1+CD11b+MDSC was 22.6%.C, After Magnetic-activated cell sorting (MACS) by CD11b magnetic bead, purification of Gr-1+CD11b+MDSC reached 84.6%.D, Most of dendritic cells wituout antigen-loaded can be seen adherent growth with different size, star or spindle shape and stretching tubers, but part of the cells seemed half adherent state with rough surface. The expressive rates of CD11c, CD86 and MHC-II on DCs were 10.9%, 3.8% and 27.9% respectively by flow cytometry.E, At the 7th day, DCs were stimulated and activated by tumor antigen. DCs in half adherent state increased obviously with radial spike and bigger shape. The expressive rates of CD11c, CD86 and MHC-II were 74.8%, 50.3% and 49.8% respectively by flow cytometry.
Figure2 Myeloid-derived suppressor cell (MDSC) inhibiting proliferation of neuroblastoma antigen-specific cytotoxic T lymphocyte (CTL) Neuroblastoma antigen-specific CTLs in the two groups were stained by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE).A, Under fluorescence microscopy, the same number of CTLs was seen in CTL group and CTL+MDSC group before cultivation. B,After having been cultivated for 4 days, cell proliferated obviously but fluorescent intensity weakened in CTL group. However, in CTL+MDSC group, cell fluorescent intensity remain strong and cell number scarcely increased. C, By flow cytometry, the results showed the consistent results with the view in microscopy. After cultivation, In CTL+MDSC group, the rate of CTLs with strong fluorescence was 87.6%. However, only 44.1% of CTLs with strong fluorescence were found in CTL group.
Figure3 Expressions of cluster of differentiation 3ζ (CD3ζ) and L-selectin (CD62L) in CTL in different groups The levels of CD3ζ and CD62L in CTL were detected and compared by reai-time PCR and western-blot analysis respectively. A, In reai-time PCR test, significant difference of CD3ζ (F = 20.315, P =0.002 < 0.05) and CD62L (F = 13.858, P =0.006 < 0.05) occurred between CTL group, CTL+MDSC group and CTL+MDSC+DOX group. B, Western-blot analysis showed that significant difference of CD3ζ (F = 28.241, P =0.001 < 0.05) and CD62L (F = 41.142, P < 0.001) were seen among the three groups. *P ﹤0.05; **P ﹤0.01.
Figure4 Immunosuppressive action of MDSC on neuroblastoma antigen-specific CTL and 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. A , As the control group, In SK-N-SH group, tumor cells proliferated continuously and kept active state. At d1, CTLs and SK-N-SH cells were mixed and no obvious difference was seen between the groups. At d2, in CTL+SK-N-SH group and CTL+SK-N-SH+MDSC+DOX group, CTLs and SK-N-SH cells gathered and began to interact. But CTLs and SK-N-SH cells scattered in view in CTL+SK-N-SH+MDSC group. At d4, in CTL+SK-N-SH+MDSC group, CTLs and SK-N-SH cells all proliferated slowly. However, in the other two groups, CTL proliferated but SK-N-SH cells began to deform. At d7, in CTL+SK-N-SH group and CTL+SK-N-SH+MDSC+DOX group, SK-N-SH cells furtherly deformed and lost their cellular shape meanwhile CTLs began to decrease. However, SK-N-SH cells kept regular shape in CTL+SK-N-SH+MDSC group. At d10, in SK-N-SH+CTL+MDSC groups, SK-N-SH cells still scattered in view with relative regular outline. However, in the other two groups, nearly all of SK-N-SH cells appeared apoptosis or necrosis, and CTLs also decreased significantly.B, 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). C and D, Interleukin-2 (IL-2) and interferon-γ (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. *P ﹤0.05.