Figure 8.

Host CD4⁺ T cells are indispensible for potent effector function and antitumor activity of IL-12/15/18–preactivated NK cells. (a and b) IL-12/15/18–pretreated NK cells (CD45.1⁺) were transferred into tumor-bearing, irradiated mice (CD45.2⁺) as described in Fig. 2 b. Anti-CD4 or anti-CD8 mAb was applied as described in Materials and methods. 11 d later, splenocytes were restimulated by RMA-S cells and stained for IFN-γ (a) or granzyme B (b). Dot plots and histograms shown are gated on CD3ε⁻NK1.1⁺ cells. Numbers indicate percentages among the host or transferred NK cells. One representative staining from each group is shown. Percentages of IFN-γ and levels of granzyme B produced by host (CD3ε⁻NK1.1⁺CD45.1⁻) and transferred (CD3ε⁻NK1.1⁺CD45.1⁺) NK cells are depicted in the bottom panels. Graphs indicate mean + SD (n = 3). Data shown are representative of two independent experiments. (c) NK cells were preactivated with IL-12/15/18 for 16 h and transferred into tumor-bearing, irradiated mice as described in Fig. 1 b. Anti-CD4 mAb was applied as described in Materials and methods. Tumor growth was monitored. Graphs display mean + SEM (n = 4–8). *, P < 0.05 compared with the group with RT treatment. Data shown are representative of two independent experiments. (d) Representative staining (n = 3) of frozen tumor sections obtained from mice 4 d after RT and transfer of IL-12/15/18–preactivated NK cells is depicted. Transferred NK cells and CD4⁺ and CD8⁺ T cells were stained with anti-CD45.1, anti-CD8, and anti-CD4 mAbs. Data shown are representative of two independent experiments. Bars, 25 µm.