Figure 7.
In vitro inflammatory cytokine production profiles of splenocytes from mice treated with granulocyte macrophage colony‐stimulating factor (GM‐CSF)‐transduced RENCA vaccine cells. (a,b) Interferon (IFN)‐γ and interleukin (IL)‐4 production by splenocytes from mice immunized with irRC/AdV/GM or irRC/SeV/GM cells were evaluated using mouse (a) IFN‐γ and (b) IL‐4 ELISPOT assays. Ten thousand splenocytes, as responder cells (R), from RENCA‐bearing mice treated with the indicated tumor vaccines were incubated for 20 h with or without stimulator cells (S) or PMA at the indicated R : S ratios. Bound cytokines were visualized by incubation with biotinylated anti‐IFN‐γ and anti‐IL‐4 monoclonal antibodies, followed by streptavidin–horseradish peroxidase, and the premixed peroxidase substrate 3‐amino‐9‐ethylcarbazole (AEC). Results are expressed as the mean number of spot‐forming cells + SD from quadruplicate determinations per 1 × 105 splenocytes. (c–h) Splenocytes were harvested from mice 5 days after the last inoculation of the indicated tumor vaccines and then cocultured with or without irradiated RENCA stimulator cells. Twenty hours after the mixed lymphocyte and tumor incubation, the concentrations of mouse (c) tumor necrosis factor (TNF)‐α, (d) IFN‐γ, (e) IL‐2, (f) IL‐4, (g) IL‐5, and (h) IL‐6 in the culture supernatants were measured by (c–g) cytometric bead array and (h) enzyme‐linked immunosorbent assays. *P < 0.05 represents significant difference compared with indicated group.