Figure 3
Expressions of tmTNF by DCs and tmTNFRs by NK cells correlate with DC and NK-cell ability to cross-stimulate IFN-γ secretion. (A) LPS rapidly induces increases in cell-surface expression of TNF on fiDCs. fiDCs were either untreated (fiDC) or 6-hour LPS stimulated (fmDC). (B) LPS rapidly induces increases in cell-surface expression of TNF on ciDCs. ciDCs were either untreated (ciDC) or 6-hour LPS induced (cmDC). (C) IL-2 rapidly stimulates increases in cell-surface expression of TNFR2 on fNK cells. fNK cells were either untreated (fNK) or 6-hour IL-2 activated (faNK). Panels A-C present flow cytometry analyses of the cell-surface molecules (thin-line open histograms, isotype control mAb; thick-line open histograms, nonstimulated cells stained with specific mAb; filled histograms, stimulated cells stained with specific mAb). The numbers in parentheses are mean fluorescence intensity (MFI). (D) LPS-stimulated fiDCs and IL-2–stimulated fNK cells rapidly increase the expression of cell-associated TNF and TNFR2, respectively, and enhance their abilities to cross-stimulate IFN-γ secretion. fiDCs or fNK cells were cultured for the indicated periods of time either unstimulated (data not shown, no changes observed) or stimulated with LPS or IL-2, respectively. Following these cultures, the cell-associated TNF was measured in the cell lysates of DCs (DC (TNF)), and cell-associated TNFR2 was measured in cell lysates of NK cells (NK (TNFR2)) using ELISA. In parallel, fiDCs and fNK cells were incubated alone (NK-cell data are shown [NK (IFN-γ)]; DC data are negative, not shown) or together (DC+NK (IFN-γ)) in the presence of LPS and IL-2 for the indicated periods of time. The cell-culture–conditioned media were assessed by IFN-γ ELISA. Data are from an experiment. The results are means ± SD of triplicates TNF, TNFR2, and IFN-γ nanograms per 0.5 × 106 cells per milliliter. (E) LPS-stimulated ciDCs rapidly increase the expression of cell-associated TNF and acquire increased ability to stimulate caNK-cell IFN-γ secretion. ciDCs were cultured for the indicated periods of time either unstimulated (data not shown, no changes observed) or stimulated with LPS. Following these cultures, cell-associated TNF was measured in the cell lysates using ELISA (DC (TNF)). In parallel, ciDC and caNK cells were incubated alone (negative data, not shown) or together (DC+NK (IFN-γ)) in the presence of LPS for the indicated periods of time. The cell-culture–conditioned media were assessed by IFN-γ ELISA. Data are from a representative experiment of 3 similar experiments performed. The results are means ± SD of triplicates TNF and IFN-γ nanograms per 0.5 × 106 cells per milliliter. Asterisks in panels D-E indicate significant increases in the cell-associated proteins in stimulated (1 to 48 hours) versus unstimulated (0 hours) NK cells and/or DCs (NK (TNFR2); DC (TNF)) or in IFN-γ secretion in DC+NK-cell cocultures (DC+NK (IFN-γ)) versus NK-cell cultures alone.

Expressions of tmTNF by DCs and tmTNFRs by NK cells correlate with DC and NK-cell ability to cross-stimulate IFN-γ secretion. (A) LPS rapidly induces increases in cell-surface expression of TNF on fiDCs. fiDCs were either untreated (fiDC) or 6-hour LPS stimulated (fmDC). (B) LPS rapidly induces increases in cell-surface expression of TNF on ciDCs. ciDCs were either untreated (ciDC) or 6-hour LPS induced (cmDC). (C) IL-2 rapidly stimulates increases in cell-surface expression of TNFR2 on fNK cells. fNK cells were either untreated (fNK) or 6-hour IL-2 activated (faNK). Panels A-C present flow cytometry analyses of the cell-surface molecules (thin-line open histograms, isotype control mAb; thick-line open histograms, nonstimulated cells stained with specific mAb; filled histograms, stimulated cells stained with specific mAb). The numbers in parentheses are mean fluorescence intensity (MFI). (D) LPS-stimulated fiDCs and IL-2–stimulated fNK cells rapidly increase the expression of cell-associated TNF and TNFR2, respectively, and enhance their abilities to cross-stimulate IFN-γ secretion. fiDCs or fNK cells were cultured for the indicated periods of time either unstimulated (data not shown, no changes observed) or stimulated with LPS or IL-2, respectively. Following these cultures, the cell-associated TNF was measured in the cell lysates of DCs (DC (TNF)), and cell-associated TNFR2 was measured in cell lysates of NK cells (NK (TNFR2)) using ELISA. In parallel, fiDCs and fNK cells were incubated alone (NK-cell data are shown [NK (IFN-γ)]; DC data are negative, not shown) or together (DC+NK (IFN-γ)) in the presence of LPS and IL-2 for the indicated periods of time. The cell-culture–conditioned media were assessed by IFN-γ ELISA. Data are from an experiment. The results are means ± SD of triplicates TNF, TNFR2, and IFN-γ nanograms per 0.5 × 106 cells per milliliter. (E) LPS-stimulated ciDCs rapidly increase the expression of cell-associated TNF and acquire increased ability to stimulate caNK-cell IFN-γ secretion. ciDCs were cultured for the indicated periods of time either unstimulated (data not shown, no changes observed) or stimulated with LPS. Following these cultures, cell-associated TNF was measured in the cell lysates using ELISA (DC (TNF)). In parallel, ciDC and caNK cells were incubated alone (negative data, not shown) or together (DC+NK (IFN-γ)) in the presence of LPS for the indicated periods of time. The cell-culture–conditioned media were assessed by IFN-γ ELISA. Data are from a representative experiment of 3 similar experiments performed. The results are means ± SD of triplicates TNF and IFN-γ nanograms per 0.5 × 106 cells per milliliter. Asterisks in panels D-E indicate significant increases in the cell-associated proteins in stimulated (1 to 48 hours) versus unstimulated (0 hours) NK cells and/or DCs (NK (TNFR2); DC (TNF)) or in IFN-γ secretion in DC+NK-cell cocultures (DC+NK (IFN-γ)) versus NK-cell cultures alone.

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