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

Strowig T, Brilot F, Arrey F, Bougras G, Thomas D, Muller WA, Münz C - PLoS Pathog. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Immunobiology, The Rockefeller University, New York, New York, United States of America.

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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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.
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ppat-0040027-g005: 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.

Mentions: In order to extend our findings from monocyte-derived DCs to human blood DCs and from polyI:C to maturation by EBV, we exposed sorted human CD11c+ myeloid DCs to EBV particles directly (DC:EBV MOI = 1:1). We observed 189 ± 20 pg/ml IL-12 secretion and upregulation of the maturation marker CD83 upon coculture of myeloid DCs with EBV (Figure 5A and 5B). Both infectious and heat-inactivated EBV elicited this DC maturation (Figure 5A). DC maturation by EBV was not due to endotoxin contamination of the EBV virus preparations, since we detected less than in 0.1 ng endotoxin in 1 × 105 EBV RIU, a concentration insufficient for human DC maturation (Figure 5A). Furthermore polymyxin B, which inhibits TLR4 stimulation by LPS [36], had no effect on EBV-mediated DC maturation, but significantly inhibited DC maturation by LPS (Figure 5A). While EBV induced IL-12 levels were lower than IL-12 concentrations in response to polyI:C and to high levels of LPS (1286 ± 188 pg/ml and 763 ± 87 pg/ml, respectively; Figure 5A), EBV-matured DCs stimulated purified autologous NK cells to secrete IFN-γ in excess of 4000 pg/ml via IL-12 (Figure 5C). These IFN-γ concentrations are high enough to inhibit B cell transformation by EBV in vitro (Figure 4E). These data suggest that human myeloid DCs can be matured by EBV and then activate NK cells to produce protective amounts of IFN-γ.


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

Strowig T, Brilot F, Arrey F, Bougras G, Thomas D, Muller WA, Münz C - PLoS Pathog. (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233668&req=5

ppat-0040027-g005: 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.
Mentions: In order to extend our findings from monocyte-derived DCs to human blood DCs and from polyI:C to maturation by EBV, we exposed sorted human CD11c+ myeloid DCs to EBV particles directly (DC:EBV MOI = 1:1). We observed 189 ± 20 pg/ml IL-12 secretion and upregulation of the maturation marker CD83 upon coculture of myeloid DCs with EBV (Figure 5A and 5B). Both infectious and heat-inactivated EBV elicited this DC maturation (Figure 5A). DC maturation by EBV was not due to endotoxin contamination of the EBV virus preparations, since we detected less than in 0.1 ng endotoxin in 1 × 105 EBV RIU, a concentration insufficient for human DC maturation (Figure 5A). Furthermore polymyxin B, which inhibits TLR4 stimulation by LPS [36], had no effect on EBV-mediated DC maturation, but significantly inhibited DC maturation by LPS (Figure 5A). While EBV induced IL-12 levels were lower than IL-12 concentrations in response to polyI:C and to high levels of LPS (1286 ± 188 pg/ml and 763 ± 87 pg/ml, respectively; Figure 5A), EBV-matured DCs stimulated purified autologous NK cells to secrete IFN-γ in excess of 4000 pg/ml via IL-12 (Figure 5C). These IFN-γ concentrations are high enough to inhibit B cell transformation by EBV in vitro (Figure 4E). These data suggest that human myeloid DCs can be matured by EBV and then activate NK cells to produce protective amounts of IFN-γ.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Immunobiology, The Rockefeller University, New York, New York, United States of America.

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.

Show MeSH
Related in: MedlinePlus