Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma - PubMed (original) (raw)

Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma

Till Strowig et al. PLoS Pathog. 2008.

Abstract

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56(bright)CD16(-) NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-gamma (IFN-gamma) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-gamma concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

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.

Figure 2

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

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

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

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

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.

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