MDSC separation, extraction, identification and
neuroblastoma Ag-specific CTL preparation, cytokine release test
By percoll density gradient centrifugation, the 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 (Fig. 1A), and
the rate of Gr-1+CD11b+MDSC was
22.6% (Fig. 1B). Furthermore, MDSC suspension was sorted by CD11b
magnetic bead and purification of
Gr-1+CD11b+MDSC was shown 84.6% by
flow cytometry (Fig. 1C). By flow cytometry, the expressive rates of
CD11c, CD86 and MHC-II on dendritic cells (DCs) wituout antigen-loaded
were 10.9%, 3.8% and 27.9% respectively which suggested their weaker
antigen presenting ability (Fig. 1D). Most of DCs can be seen adherent
growth with different size, star or spindle shape and stretching tubers.
At the 7th day, DCs were 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 which indicated mature DCs with efficient presenting
antigen ability (fig. 1E). CD3+T cells, extracted from
spleen lymphocytes by MACS, reached 87.3% by flow cytometry. The living
cells rate was 96.38% by Trypan blue test. After 3 to 4 days’
cultivation with antigen-loaded DCs, antigen-specific
CD3+T cells were prepared and gathered into many small
colonies. The levels of interleukin-2 (IL-2) (1.092 +/- 0.010 ng/l) and
interferon-γ (0.855 +/- 0.038 ng/l) in the supernatant of antigen-loaded
CD3+T cells were significantly higher than IL-2 (0.962
+/- 0.007 ng/l) and IFN-γ (0.765 +/- 0.010 ng/l) in the supernatant of
CD3+T cells without antigen-loaded
(P <0.05).