Figure 5
TNF−/− DCs and NK cells are impaired in the ability to reciprocally stimulate IFN-γ secretion, and the impairment can be fully repaired by restoring TNF−/− DCs with tmTNF but not sTNF. (A) TNF−/− DCs and TNF−/− NK cells have a remarkably decreased ability to reciprocally stimulate IFN-γ secretion. Wild-type cmDCs and wild-type caNK cells or TNF−/− cmDCs and TNF−/− caNK cells were cultured either alone or together for 24 hours. cmDCs were generated by LPS stimulation of ciDCs. Following this culture, the cell-culture–conditioned media were assessed for IFN-γ using ELISA. Data are from a representative experiment of 7 similar experiments performed. They are means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (B) Transfer of wild-type Tnf restores cell-associated tmTNF in TNF−/− DCs. TNF−/− ciDCs were transduced with adenoviral vectors containing GFP or wild-type Tnf (wtTNF). Cell lysates of transfected cells were assessed using Western blot assays (inset) and ELISA (columns). Data are from an experiment. Western blot was assessed with anti-TNF antibody. ELISA data are means of triplicates TNF nanograms per 0.5 × 106 cells per milliliter (SD is 0.2 ng). (C) Transfer of wild-type Tnf into TNF−/− DCs restores DC ability to stimulate IFN-γ secretion by TNF−/− NK cells. Wild-type ciDCs were transduced with adenoviral vector containing GFP (wt+GFP), and TNF−/− ciDCs were transduced with adenoviral vectors containing either GFP (TNF−/−+GFP) or wild-type Tnf (TNF−/−+wtTNF). The transduced wild-type or TNF−/− DCs and respective wild-type or TNF−/− untreated caNK cells were cultured either alone or together, in the presence of LPS, for 24 hours. Following this culture, the cell-culture–conditioned media were assessed by IFN-γ ELISA. Presented data are from one experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (D) Transfer of noncleavable Tnf restores cell-surface expression of tmTNF in TNF−/− DCs. TNF−/− ciDCs were transduced with the plasmid DNA containing either wtTnf or nclTnf. Transduced DCs were compared with mock-transduced TNF−/− ciDCs for the expression of tmTNF on cell surface using flow cytometry and Western blot (insert). Flow cytometry presents log10 fluorescence intensity of TNF−/− ciDCs either mock transduced or transduced with nclTnf and stained with isotype-matched control antibody (thin-line open histogram) or anti-TNF antibody (thick-line open histogram, mock transduced; filled histogram, nclTnf transduced). Numbers in parentheses are MFI. Western blot presents TNF forms in lysates of TNF−/− DCs either mock transduced (0) or transduced with wtTNF or nclTnf. Presented data are from one experiment. (E) Transfer of nclTnf fully restores the ability of TNF−/− DCs to stimulate IFN-γ secretion by TNF−/− NK cells. TNF−/− cmDCs mock transduced (TNF−/−) or transduced with nclTnf (TNF−/−+nclTNF) and nontransduced TNF−/− caNK cells were cultured alone or together for 24 hours. For comparison, the ability of nontransduced LPS-stimulated wild-type cmDCs to stimulate wild-type caNK cells was assessed (wt). Following this culture, the cell-culture–conditioned media were assessed by IFN-γ ELISA. Presented data are from an experiment and are means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (F) sTNF is a poor stimulator of NK-cell IFN-γ secretion. Wild-type caNK cells were incubated for 24 hours in the absence or presence of grading concentrations of mouse recombinant sTNF. Following the incubation, the cell-culture–conditioned media were collected and assessed by IFN-γ ELISA. Presented data are from one experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. In panels A, C, and E, single asterisks indicate significant increases of IFN-γ in wild-type cmDC+wild-type caNK-cell cocultures in comparison with wild-type cmDC or caNK-cell cultures alone. In panel B, the single asterisk indicates a significant increase of cell-associated TNF in TNF−/− ciDCs transduced with wtTnf compared with TNF−/− ciDCs transduced with GFP. In panel F, single asterisks indicate significant increases of IFN-γ in sTNF-stimulated versus unstimulated caNK-cell cultures. Double asterisks indicate significant decreases of IFN-γ in cocultures of untreated, GFP-transduced, or mock-transduced TNF−/− cmDCs with TNF−/− untreated caNK cells in comparison with cocultures of untreated or GFP-transduced wild-type cmDCs with untreated wild-type caNK cells. Triple asterisks indicate significant increases of IFN-γ in cocultures of wtTnf- or nclTnf-transduced TNF−/− cmDCs with untreated TNF−/− caNK cells in comparison with cocultures of mock-transduced or GFP-transduced TNF−/− cmDCs with untreated TNF−/− caNK cells.

TNF−/− DCs and NK cells are impaired in the ability to reciprocally stimulate IFN-γ secretion, and the impairment can be fully repaired by restoring TNF−/− DCs with tmTNF but not sTNF. (A) TNF−/− DCs and TNF−/− NK cells have a remarkably decreased ability to reciprocally stimulate IFN-γ secretion. Wild-type cmDCs and wild-type caNK cells or TNF−/− cmDCs and TNF−/− caNK cells were cultured either alone or together for 24 hours. cmDCs were generated by LPS stimulation of ciDCs. Following this culture, the cell-culture–conditioned media were assessed for IFN-γ using ELISA. Data are from a representative experiment of 7 similar experiments performed. They are means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (B) Transfer of wild-type Tnf restores cell-associated tmTNF in TNF−/− DCs. TNF−/− ciDCs were transduced with adenoviral vectors containing GFP or wild-type Tnf (wtTNF). Cell lysates of transfected cells were assessed using Western blot assays (inset) and ELISA (columns). Data are from an experiment. Western blot was assessed with anti-TNF antibody. ELISA data are means of triplicates TNF nanograms per 0.5 × 106 cells per milliliter (SD is 0.2 ng). (C) Transfer of wild-type Tnf into TNF−/− DCs restores DC ability to stimulate IFN-γ secretion by TNF−/− NK cells. Wild-type ciDCs were transduced with adenoviral vector containing GFP (wt+GFP), and TNF−/− ciDCs were transduced with adenoviral vectors containing either GFP (TNF−/−+GFP) or wild-type Tnf (TNF−/−+wtTNF). The transduced wild-type or TNF−/− DCs and respective wild-type or TNF−/− untreated caNK cells were cultured either alone or together, in the presence of LPS, for 24 hours. Following this culture, the cell-culture–conditioned media were assessed by IFN-γ ELISA. Presented data are from one experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (D) Transfer of noncleavable Tnf restores cell-surface expression of tmTNF in TNF−/− DCs. TNF−/− ciDCs were transduced with the plasmid DNA containing either wtTnf or nclTnf. Transduced DCs were compared with mock-transduced TNF−/− ciDCs for the expression of tmTNF on cell surface using flow cytometry and Western blot (insert). Flow cytometry presents log10 fluorescence intensity of TNF−/− ciDCs either mock transduced or transduced with nclTnf and stained with isotype-matched control antibody (thin-line open histogram) or anti-TNF antibody (thick-line open histogram, mock transduced; filled histogram, nclTnf transduced). Numbers in parentheses are MFI. Western blot presents TNF forms in lysates of TNF−/− DCs either mock transduced (0) or transduced with wtTNF or nclTnf. Presented data are from one experiment. (E) Transfer of nclTnf fully restores the ability of TNF−/− DCs to stimulate IFN-γ secretion by TNF−/− NK cells. TNF−/− cmDCs mock transduced (TNF−/−) or transduced with nclTnf (TNF−/−+nclTNF) and nontransduced TNF−/− caNK cells were cultured alone or together for 24 hours. For comparison, the ability of nontransduced LPS-stimulated wild-type cmDCs to stimulate wild-type caNK cells was assessed (wt). Following this culture, the cell-culture–conditioned media were assessed by IFN-γ ELISA. Presented data are from an experiment and are means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. (F) sTNF is a poor stimulator of NK-cell IFN-γ secretion. Wild-type caNK cells were incubated for 24 hours in the absence or presence of grading concentrations of mouse recombinant sTNF. Following the incubation, the cell-culture–conditioned media were collected and assessed by IFN-γ ELISA. Presented data are from one experiment and represent means ± SD of triplicates IFN-γ nanograms per 0.5 × 106 cells per milliliter. In panels A, C, and E, single asterisks indicate significant increases of IFN-γ in wild-type cmDC+wild-type caNK-cell cocultures in comparison with wild-type cmDC or caNK-cell cultures alone. In panel B, the single asterisk indicates a significant increase of cell-associated TNF in TNF−/− ciDCs transduced with wtTnf compared with TNF−/− ciDCs transduced with GFP. In panel F, single asterisks indicate significant increases of IFN-γ in sTNF-stimulated versus unstimulated caNK-cell cultures. Double asterisks indicate significant decreases of IFN-γ in cocultures of untreated, GFP-transduced, or mock-transduced TNF−/− cmDCs with TNF−/− untreated caNK cells in comparison with cocultures of untreated or GFP-transduced wild-type cmDCs with untreated wild-type caNK cells. Triple asterisks indicate significant increases of IFN-γ in cocultures of wtTnf- or nclTnf-transduced TNF−/− cmDCs with untreated TNF−/− caNK cells in comparison with cocultures of mock-transduced or GFP-transduced TNF−/− cmDCs with untreated TNF−/− caNK cells.

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