Figure 4
Increases in expression of tmTNF induced by TNF gene transfer augment the ability of DCs to induce IFN-γ secretion by NK cells. (A) Transfer of Tnf results in increases in cell-surface expression of TNF on DCs. ciDCs were transduced with adenoviral vectors containing GFP (GFP) or wild-type Tnf (TNF) and stimulated with LPS for 6 hours, stained with isotype control mAb (isotype) and anti-TNF mAb (αTNF), and examined by flow cytometry. The numbers adjacent to histograms are MFI. (B) Transfer of Tnf results in increases in expression of tmTNF in ciDCs. Lysates of the GFP- or Tnf-transduced ciDCs were assessed for TNF using Western blot (inset) and ELISA (columns). Data are from one experiment. Western blot shows the 26 kDa tmTNF. The 17 kDa sTNF was 15-fold less expressed than tmTNF (data not shown). ELISA data are means of triplicates TNF nanograms per 0.5 × 106 cells per milliliter. (C) Transfer of Tnf leads to high increases in the ability of ciDCs to induce IFN-γ secretion by caNK cells. GFP- or Tnf-transduced ciDCs (cmDC) and untreated caNK cells (caNK) were cultured either alone or together (cmDC+caNK) in the presence of LPS for 24 hours. After this incubation, the cell-culture–conditioned media were assessed using IFN-γ ELISA. Data are from one experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (D) Tnf- transduced wild-type ciDCs are only capable of efficiently inducing secretion of IFN-γ in wild-type caNK cells in cell-cell contact but not physically separated. GFP- or Tnf-transduced ciDCs (cmDC) and untreated caNK cells (caNK) were cultured in the presence of 200 ng/mL LPS for 24 hours either alone or together in standard wells (cmDC+caNK) or physically separated in Transwells (cmDC/caNK Transwell). After this incubation, the cell-culture–conditioned media were assessed using IFN-γ ELISA. Data are from an experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. In panel B, the single asterisk indicates a significant increase of cell-associated TNF in ciDCs transduced with Tnf compared with ciDCs transduced with GFP. In panels C and D, single asterisks indicate a significant increase in IFN-γ in cocultures of GFP-transduced cmDCs+caNK cells versus cultures of cmDCs or caNK cells alone. Double asterisks indicate a significant enhancement of IFN-γ in cocultures of TNF-transduced cmDCs+caNK cells versus cocultures of GFP-transduced cmDCs+caNK cells. Triple asterisks indicate significant decreases of IFN-γ in cocultures of cmDC/caNK cells separated by Transwell membrane versus cmDC+caNK-cell cocultures without separation.

Increases in expression of tmTNF induced by TNF gene transfer augment the ability of DCs to induce IFN-γ secretion by NK cells. (A) Transfer of Tnf results in increases in cell-surface expression of TNF on DCs. ciDCs were transduced with adenoviral vectors containing GFP (GFP) or wild-type Tnf (TNF) and stimulated with LPS for 6 hours, stained with isotype control mAb (isotype) and anti-TNF mAb (αTNF), and examined by flow cytometry. The numbers adjacent to histograms are MFI. (B) Transfer of Tnf results in increases in expression of tmTNF in ciDCs. Lysates of the GFP- or Tnf-transduced ciDCs were assessed for TNF using Western blot (inset) and ELISA (columns). Data are from one experiment. Western blot shows the 26 kDa tmTNF. The 17 kDa sTNF was 15-fold less expressed than tmTNF (data not shown). ELISA data are means of triplicates TNF nanograms per 0.5 × 106 cells per milliliter. (C) Transfer of Tnf leads to high increases in the ability of ciDCs to induce IFN-γ secretion by caNK cells. GFP- or Tnf-transduced ciDCs (cmDC) and untreated caNK cells (caNK) were cultured either alone or together (cmDC+caNK) in the presence of LPS for 24 hours. After this incubation, the cell-culture–conditioned media were assessed using IFN-γ ELISA. Data are from one experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (D) Tnf- transduced wild-type ciDCs are only capable of efficiently inducing secretion of IFN-γ in wild-type caNK cells in cell-cell contact but not physically separated. GFP- or Tnf-transduced ciDCs (cmDC) and untreated caNK cells (caNK) were cultured in the presence of 200 ng/mL LPS for 24 hours either alone or together in standard wells (cmDC+caNK) or physically separated in Transwells (cmDC/caNK Transwell). After this incubation, the cell-culture–conditioned media were assessed using IFN-γ ELISA. Data are from an experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. In panel B, the single asterisk indicates a significant increase of cell-associated TNF in ciDCs transduced with Tnf compared with ciDCs transduced with GFP. In panels C and D, single asterisks indicate a significant increase in IFN-γ in cocultures of GFP-transduced cmDCs+caNK cells versus cultures of cmDCs or caNK cells alone. Double asterisks indicate a significant enhancement of IFN-γ in cocultures of TNF-transduced cmDCs+caNK cells versus cocultures of GFP-transduced cmDCs+caNK cells. Triple asterisks indicate significant decreases of IFN-γ in cocultures of cmDC/caNK cells separated by Transwell membrane versus cmDC+caNK-cell cocultures without separation.

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