DCs stimulate NK-cell IFN-γ secretion via transmembrane but not secreted molecules, and DCs and NK cells express on cell-surface transmembrane TNF and TNFRs. (A) Secretion-deficient DCs mediate NK-cell IFN-γ secretion. Viable or paraformaldehyde-fixed ciDCs (ciDC and FxciDC, respectively) and 6-hour LPS-prestimulated viable or formaldehyde-fixed ciDCs (cmDC and FxcmDC, respectively) and viable caNK cells (caNK) were plated into 96-well plates either alone or together in a 1:1 ratio and incubated for 24 hours. Following the culture, cell-culture–conditioned media were assessed by IFN-γ ELISA. Data are from a representative experiment of 2 similar experiments performed. The results are means ± SD of triplicates IFN-γ nanograms per 0.5 × 10⁶ cells per milliliter. Asterisks indicate significant increases in IFN-γ secretion in DC+NK-cell cocultures versus DC or NK-cell cultures alone. (B) Transmembrane but not soluble TNF, TNFR1, and TNFR2 are expressed in fiDCs, fNK cells, ciDCs, and caNK cells. Western blot analyses of the DC and NK-cell lysates were performed using 120 μg cell proteins per lane. Relative quantities of the proteins in the Western blotting bands were determined by densitometry. Protein bands are as follows: 26 kDa, tmTNF; 17 kDa, sTNF; 55 kDa, tmTNFR1; 28 kDa, sTNFR1; 75 kDa, tmTNFR2; 40 kDa, sTNFR2; and 42 kDa, β-actin. (C) TNF, TNFR1, and TNFR2 are expressed on cell surface of ciDCs and caNK cells. Flow cytometry analyses of viable ciDCs and caNK cells were performed. The results are log₁₀ fluorescence intensity obtained with isotype-matched nonreactive control antibodies (open histograms) and specific antibodies (filled histograms).