Interaction between DC-TNF and NK cell–TNFR2 mediates proliferation and increased cytotoxic activity of NK cells in DC–NK-cell crosstalk. (A) DC TNF, but not NK-cell TNF, is required for DC stimulation of NK-cell proliferation. (B) Neither NK-cell nor DC TNFR1 is required for DC induction of NK-cell proliferation. (C) NK-cell TNFR2, but not DC TNFR2, is required for DC induction of NK-cell proliferation. Wild-type (wt) or TNF^−/− (A), TNFR1^−/− (B), and TNFR2^−/− (C) fiDCs (irradiated with 30 Gy [3000 rad]) and fNK cells were cultured for 4 days in the presence of 200 ng/mL LPS and 300 IU/mL IL-2 either alone or in the following combinations: wild-type+wild-type, gene-deficient+gene-deficient, wild-type+gene-deficient, and gene-deficient+wild-type. Following this incubation, the cell cultures were supplemented with [³H]thymidine (1 μCi/200μL [0.037 MBq/200 μL]), incubated for additional 18 hours, harvested, and the DNA-incorporated radioactivity measured in a β-scintillation counter. Data are from a representative experiment of 2 to 5 similar experiments performed. They represent means ± SD of triplicates counts per minute of cocultured DC+NK cells following subtraction of counts per minute of DCs and NK cells cultured alone. Single asterisks indicate significant increases of counts per minute in cocultured DCs+NK cells in comparison with counts per minute of alone-cultured DCs or NK cells. Double asterisks indicate significant decreases of counts per minute in cocultured TNF^−/−DCs+TNF^−/−NK cells or TNF^−/−DCs+wtNK cells and TNFR2^−/−DCs+TNFR2^−/−NK cells or wtDCs+TNFR2^−/−NK cells in comparison with wtDCs+wtNK cells or wtDCs+TNF^−/−NK cells and wtDCs+wtNK cells or TNFR2^−/−DC+wtNK cells, respectively. Similar findings to those presented in panel C were obtained in 2 independent experiments with wild-type and TNFR2^−/− fiDC and fNK cells stimulated only by LPS (data not shown). (D-E) DC TNF, but not NK-cell TNF, is required for DC stimulation of NK-cell cytotoxic activity. Wild-type or TNF^−/− ciDCs and 24-hour IL-2–activated faNK cells were cultured for 24 hours in the absence of cytokines or microbial products either alone or in the following combinations and 1:2 ratio: wild-type+wild-type, TNF^−/−+TNF^−/−, wild-type+TNF^−/−, and TNF^−/−+wild-type. Following this incubation, the cells were assessed for their NK cytotoxicity using the standard 4-hour ⁵¹Cr-release assays against YAC-1 tumor-cell targets. Data are from a representative experiment of 3 similar experiments performed. They represent means ± SD of triplicates percentages of killing by alone-cultured NK cells and cocultured DCs+NK cells. Single asterisks indicate significant increases in cytotoxicity of DCs+NK cells in comparison with those of NK cells alone.