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).