Figure 1.
DCs Activate NK Cells Most Efficiently after Maturation with the dsRNA Analog polyI:C
Differently matured DCs were compared to immature DCs for their ability to induce peripheral blood NK cell proliferation (A) and IFN-γ secretion (B). The tested maturation stimuli included (i) the standard proinflammatory cytokine cocktail consisting of IL-1β, IL-6, TNF-α, and prostaglandin E2 (cyt DC); (ii) the TLR3 and mda-5 agonist polyI:C (polyI:C DC); (iii) a cocktail of inflammatory cytokines (IL-1β, TNF-α IFN-α, IFN-γ) and polyI:C (DC1); (iv) the TLR4 agonist LPS (lps DC). These differently matured DCs were compared to immature DCs (iDC) and stimulation with IL-2 alone.
(A) CFSE-labeled NK cells and DCs were cultured for 6d at a ratio of 5:1, and proliferation was analyzed by gating on CD3−CD56+ cells. Percentages of CFSE dilute CD16+ and CD16− NK cells are indicated.
(B) NK cells and DCs were cultured for 20 h at a ratio of 2:1 and BFA was added for the last 8 h. IFN-γ production of CD3−CD56+ cells was analyzed. Percentages of IFN-γ positive NK cells are indicated. Similar results were obtained in three independent experiments.
Table 1.
Secretion of IL-12, IL-15, and IL-18 by DCs
Figure 2.
NK Cells from Blood and Tonsil Activated by PolyI:C-matured DCs Limit EBV-mediated B Cell Transformation
(A) B cells were infected with EBV and cultured for 12 d alone, with DCs or NK cells and with DCs plus NK cells. The indicated percentage of B cell transformation was evaluated by determining the percentage of transformed CD19+CD23+B cells within gated CD19+CD21+ B cells.
(B and C) Total numbers of transformed B cells were determined from live cell numbers and the percentage of transformed CD19+CD23+B cells in the different cultures. Total transformed B cell numbers for one representative experiment (B), and differences in total transformed B cell numbers for all experiments (percentage restriction of B cell transformation) (C), were analyzed with or without peripheral blood NK cell addition for the indicated DC maturation conditions. Data represent results from eight independent experiments (mean ± standard error of the mean).
(D and E) Mononuclear cells from tonsils were depleted of CD3+ T cells or CD3+ T and CD56+ NK cells. Total numbers of transformed B cells were determined from live cell numbers and the percentage of transformed CD19+CD23+ B cells in the different cultures. Total transformed B cell numbers for one representative experiment (D), and differences in total transformed B cell numbers for all experiments (percentage restriction of B cell transformation) (E), were analyzed with and without tonsilar NK cells for the indicated DC maturation conditions. Data represent results from six independent experiments (mean ± standard error of the mean).
Figure 3.
CD56brightCD16− Cells from Blood, Tonsil, and Spleen Limit EBV-mediated B Cell Transformation after Activation by DCs
(A and B) B cells were infected with EBV and cultured for 12 d with peripheral blood NK cells and DC1s at the indicated NK cell to B cell ratios. Total NK cells were compared to sorted CD56brightCD16− and CD56dimCD16+ NK cells in their ability to limit EBV-mediated B cell transformation. Total transformed B cell numbers for one representative experiment (A) and restriction of B cell transformation for all experiments (B) were compared after addition of unseparated bulk NK cells (total NK), CD56dimCD16+ NK cells (90% of the bulk NK cell number) or CD56brightCD16− NK cells (10% of the bulk NK cell number, to respect the NK subset distribution in peripheral blood) (*, p < 0.01).
(C and D) Tonsilar B cells were infected with EBV and cultured for 12 d alone or with autologous tonsilar NK cell subsets at 2 ratios (NK, 5,000 and 5-fold NK, 25,000) in the absence or presence of allogeneic iDCs or DC1s (*, p < 0.01). Total transformed B cell numbers for one representative experiment (C), and restriction of B cell transformation for all experiments are shown (D).
(E and F) Splenic B cells were infected with EBV and cultured for 12 d alone or with autologous splenic NK cell subsets in the absence or presence of autologous splenic DCs matured with polyI:C, TNF-α, IL-1β, IFN-α and IFN-γ (*, p < 0.01). Total transformed B cell numbers for one representative experiment (E), and restriction of B cell transformation for all experiments are shown (F). Data in (A–F) represent results from three independent experiments (mean ± standard error of the mean).
Figure 4.
IFN-γ Secreted by NK Cells Restricts EBV-mediated B Cell Transformation
(A) Sorted NK cell subsets from blood, spleen, tonsil, and lymph node were cultured alone or with DC1s, and IFN-γ levels were quantified by ELISA after 20 h.
(B) IFN-γ secreted by tonsilar NK cells after activation by differently matured DCs was detected in supernatants of B cell transformation assays by ELISA. (Tonsil–T, tonsilar cultures depleted of T cells; Tonsil–T-NK, tonsilar cultures depleted of T and NK cells.)
(C) IFN-γ secreted by peripheral blood NK cells and NK cell subsets was detected in supernatants of B cell transformation assays by ELISA.
(D) IFN-γ secreted by purified tonsilar NK cell subsets was detected in supernatants of B cell transformation assays by ELISA.
(E) Peripheral blood B cells were infected with EBV and increasing concentrations of IFN-γ were added. Restriction of B cell transformation was analyzed after 12 d by comparing numbers of transformed B cells with and without IFN-γ.
(F) Blocking antibodies against IFN-γ were added to B cell transformation assays with B cells, NK cells, and DC1s from peripheral blood. Where indicated, NK cells were separated from B cells and DCs by transwell membranes. Results from at least three independent experiments were summarized (mean ± standard deviation) (*, p < 0.03).
Figure 5.
Myeloid DCs Can Sense EBV and Subsequently Activate NK Cells via IL-12
(A) CD11c+ myeloid DCs (1 × 105) were purified to 99.8% purity by flow cytometric sorting and exposed to polyI:C (25 μg/ml), EBV (MOI of 1, 5 × 105 RIU/ml), and LPS in the absence and presence of polymyxin B (25 μg/ml), an inhibitor of LPS-mediated TLR4 activation. IL-12p70 was detected by ELISA 24 h later.
(B) Purified DCs were exposed to polyI:C (25 μg/ml) or EBV (MOI of 1, 5 × 105 RIU/m), and upregulation of the DC maturation marker CD83 was detected by flow cytometry 24 h later.
(C) Flow-sorted CD11c+ DCs and peripheral blood NK cells were cultured together or separately in the presence of polyI:C (25 μg/ml), or EBV (MOI of 1, 5 × 105 RIU/ml). IFN-γ was detected by ELISA 24 h later. Where indicated, IL-12 was blocked in selected experiments with a specific antibody (n.d., not determined). Data represent results from at least three independent experiments performed in duplicates.
Figure 6.
IFN-γ Impairs Transformation of B Cells by EBV
(A) Cell numbers were compared at different time points between control and IFN-γ-treated B cells with and without EBV infection.
(B) CFSE-labeled B cells were infected with EBV, and proliferation was compared between controls and IFN-γ-treated samples at the indicated time points.
(C) Expression of EBV-encoded genes was quantified by RT-PCR at different time points and compared between controls and IFN-γ-treated cells. M, 100 bp ladder (D) Expression of the oncogene LMP-1 was quantified by real time-PCR and normalized to GAPDH expression.
(E) Peripheral blood B cells were infected with EBV, and 10,000 pg/ml IFN-γ was added at the indicated time points. Restriction of B cell transformation was analyzed after 12 d by comparing numbers of transformed B cells with and without IFN-γ. Results represent data from at least three independent experiments.