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Both conventional and interferon killer dendritic cells have antigen-presenting capacity during influenza virus infection.

GeurtsvanKessel CH, Bergen IM, Muskens F, Boon L, Hoogsteden HC, Osterhaus AD, Rimmelzwaan GF, Lambrecht BN - PLoS ONE (2009)

Bottom Line: Here, using a model of influenza infection, we found recruitment of both conventional B220(-) NK cells and IKDCs to the lung.In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung.In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Pulmonary Medicine, Erasmus University Medical Centre, Rotterdam, The Netherlands.

ABSTRACT
Natural killer cells are innate effector cells known for their potential to produce interferon-gamma and kill tumour and virus-infected cells. Recently, B220(+)CD11c(int)NK1.1(+) NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220(-) NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.

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Depletion of NK1.1+ cells during in vivo influenza virus infection affects infection parameters.(A) 2 days after injection of PK136 (NK1.1 depleting antibody), mice were infected with X31 virus. Two days later, depletion of NK subsets and cDCs was checked in lung tissue by using the gating strategy in Figure 1. (B) IFN-γ was measured in BALf of infected mice versus mock-infected mice with or without NK1.1+ cell depletion. Bars represent mean values of IFN-γ +/− SEM. At least 5 mice per group were used, * p<0,05. (C) Viral titers were measured in lung tissue of at least 5 mice per group and expressed as mean TCID50 +/− SEM. * p<0,05. (D) Absolute numbers of TM specific CD8+ T cells were measured in spleen and lung tissue at 8 dpi and represented as mean values +/− SEM, of at least 5 mice/group. (E) Hemagluttinin specific antibodies in serum were measured at 8 dpi and depicted as geometric mean titer +/− SE. * p<0,05. (F) Weight loss of mice during infection depicted as % of initial weight. Values indicate mean of at least 5 mice per group +/− SEM.*p<0,05. The demonstrated data are representative of 4 independently performed experiments.
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pone-0007187-g005: Depletion of NK1.1+ cells during in vivo influenza virus infection affects infection parameters.(A) 2 days after injection of PK136 (NK1.1 depleting antibody), mice were infected with X31 virus. Two days later, depletion of NK subsets and cDCs was checked in lung tissue by using the gating strategy in Figure 1. (B) IFN-γ was measured in BALf of infected mice versus mock-infected mice with or without NK1.1+ cell depletion. Bars represent mean values of IFN-γ +/− SEM. At least 5 mice per group were used, * p<0,05. (C) Viral titers were measured in lung tissue of at least 5 mice per group and expressed as mean TCID50 +/− SEM. * p<0,05. (D) Absolute numbers of TM specific CD8+ T cells were measured in spleen and lung tissue at 8 dpi and represented as mean values +/− SEM, of at least 5 mice/group. (E) Hemagluttinin specific antibodies in serum were measured at 8 dpi and depicted as geometric mean titer +/− SE. * p<0,05. (F) Weight loss of mice during infection depicted as % of initial weight. Values indicate mean of at least 5 mice per group +/− SEM.*p<0,05. The demonstrated data are representative of 4 independently performed experiments.

Mentions: The described experiments suggested that IKDCs and B220− NK cells had APC potential for CD8 cells ex vivo. To study the role of NK1.1+ cells during infection in vivo we injected the NK1.1-specific depleting antibody PK136 before and throughout influenza infection or mock infection [30]. Both in mock infected and infected mice, intra-peritoneal (i.p.) injection of the antibody efficiently depleted NKG2D+ B220−NK cells and IKDCs from the lung tissue (Figure 5A) when compared to injection with isotype Ig diluted in PBS, although depletion was more absolute in mock infected mice. On the contrast, cDC numbers were unaffected by treatment with NK1.1. Previously an enhancing effect of NK cells on the state of maturation and antigen-presenting capacity of cDCs has been suggested [31], [32], [33]. To exclude any confounding effects of NK1.1 depletion on cDC functions, we additionally compared the antigen presenting capacity of sorted cDCs after infection with influenza virus containing the OVA MHCI epitope, in mice treated or not with PK136. At 4 dpi cDCs were sorted from both groups and expressed similar levels of co-stimulatory molecules (CD86) and MHCII and induced the same amount of T cell proliferation (Figure S1). We next addressed the impact of NK1.1 depletion on induction of antiviral innate and adaptive immunity to a mild X-31 pulmonary influenza infection. During the early innate response to influenza, IFN-γ is found in high levels at the site of infection (lung tissue and BAL fluid) [13] and NK cells are the most prominent source of this cytokine [6], [11]. Not surprisingly, we found a decrease of IFN-γ at 4 dpi in mice depleted of NK1.1 cells. At 8 dpi this mild influenza X-31 infection has been cleared from the lungs and therefore IFN-γ levels start decreasing at this time point (Figure 5B). Viral titers in mice lacking NK1.1 cells were increased 5 fold at 4 dpi compared with mice treated with isotype control antibody. Nevertheless virus was efficiently cleared at 8 dpi (Figure 5C). Most importantly, to address the contribution of NK1.1 positive cells on induction of adaptive CTL immunity, we measured the percentage of virus-specific CD8 T cells at day 8 post infection by using a PE labeled H-2Db tetramer with the NP366–374 epitope ASNENMETM [13]. At day 8 we found decreased numbers of tetramer (TM) positive cells in both spleen and lung tissue (Figure 5D). In addition to this we determined the hemagluttinin specific antibodies in serum at 8 dpi (Figure 5E) and found increased levels following NK1.1 depletion, supporting the higher viral titers. Both at earlier and later time points, similar conclusions on tetramer positive cell induction and humoral immune responses were reached (Figure S2). Surprisingly, NK1.1 depleted mice did not appear to have much more severe systemic morbidity as their weight loss did not exceed 10% of initial weight, in contrast with isotype treated mice, which lost significantly more weight during the peak of infection (Figure 5F). As a final support for an antigen presenting capacity of NK cells in vivo, we also found colocalization of naïve OVA-specific CD8 T cells with NK cells in the draining nodes 48 h after infection with influenza-OVA virus but to a much lower extent with wild type influenza virus (Figure S3).


Both conventional and interferon killer dendritic cells have antigen-presenting capacity during influenza virus infection.

GeurtsvanKessel CH, Bergen IM, Muskens F, Boon L, Hoogsteden HC, Osterhaus AD, Rimmelzwaan GF, Lambrecht BN - PLoS ONE (2009)

Depletion of NK1.1+ cells during in vivo influenza virus infection affects infection parameters.(A) 2 days after injection of PK136 (NK1.1 depleting antibody), mice were infected with X31 virus. Two days later, depletion of NK subsets and cDCs was checked in lung tissue by using the gating strategy in Figure 1. (B) IFN-γ was measured in BALf of infected mice versus mock-infected mice with or without NK1.1+ cell depletion. Bars represent mean values of IFN-γ +/− SEM. At least 5 mice per group were used, * p<0,05. (C) Viral titers were measured in lung tissue of at least 5 mice per group and expressed as mean TCID50 +/− SEM. * p<0,05. (D) Absolute numbers of TM specific CD8+ T cells were measured in spleen and lung tissue at 8 dpi and represented as mean values +/− SEM, of at least 5 mice/group. (E) Hemagluttinin specific antibodies in serum were measured at 8 dpi and depicted as geometric mean titer +/− SE. * p<0,05. (F) Weight loss of mice during infection depicted as % of initial weight. Values indicate mean of at least 5 mice per group +/− SEM.*p<0,05. The demonstrated data are representative of 4 independently performed experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747012&req=5

pone-0007187-g005: Depletion of NK1.1+ cells during in vivo influenza virus infection affects infection parameters.(A) 2 days after injection of PK136 (NK1.1 depleting antibody), mice were infected with X31 virus. Two days later, depletion of NK subsets and cDCs was checked in lung tissue by using the gating strategy in Figure 1. (B) IFN-γ was measured in BALf of infected mice versus mock-infected mice with or without NK1.1+ cell depletion. Bars represent mean values of IFN-γ +/− SEM. At least 5 mice per group were used, * p<0,05. (C) Viral titers were measured in lung tissue of at least 5 mice per group and expressed as mean TCID50 +/− SEM. * p<0,05. (D) Absolute numbers of TM specific CD8+ T cells were measured in spleen and lung tissue at 8 dpi and represented as mean values +/− SEM, of at least 5 mice/group. (E) Hemagluttinin specific antibodies in serum were measured at 8 dpi and depicted as geometric mean titer +/− SE. * p<0,05. (F) Weight loss of mice during infection depicted as % of initial weight. Values indicate mean of at least 5 mice per group +/− SEM.*p<0,05. The demonstrated data are representative of 4 independently performed experiments.
Mentions: The described experiments suggested that IKDCs and B220− NK cells had APC potential for CD8 cells ex vivo. To study the role of NK1.1+ cells during infection in vivo we injected the NK1.1-specific depleting antibody PK136 before and throughout influenza infection or mock infection [30]. Both in mock infected and infected mice, intra-peritoneal (i.p.) injection of the antibody efficiently depleted NKG2D+ B220−NK cells and IKDCs from the lung tissue (Figure 5A) when compared to injection with isotype Ig diluted in PBS, although depletion was more absolute in mock infected mice. On the contrast, cDC numbers were unaffected by treatment with NK1.1. Previously an enhancing effect of NK cells on the state of maturation and antigen-presenting capacity of cDCs has been suggested [31], [32], [33]. To exclude any confounding effects of NK1.1 depletion on cDC functions, we additionally compared the antigen presenting capacity of sorted cDCs after infection with influenza virus containing the OVA MHCI epitope, in mice treated or not with PK136. At 4 dpi cDCs were sorted from both groups and expressed similar levels of co-stimulatory molecules (CD86) and MHCII and induced the same amount of T cell proliferation (Figure S1). We next addressed the impact of NK1.1 depletion on induction of antiviral innate and adaptive immunity to a mild X-31 pulmonary influenza infection. During the early innate response to influenza, IFN-γ is found in high levels at the site of infection (lung tissue and BAL fluid) [13] and NK cells are the most prominent source of this cytokine [6], [11]. Not surprisingly, we found a decrease of IFN-γ at 4 dpi in mice depleted of NK1.1 cells. At 8 dpi this mild influenza X-31 infection has been cleared from the lungs and therefore IFN-γ levels start decreasing at this time point (Figure 5B). Viral titers in mice lacking NK1.1 cells were increased 5 fold at 4 dpi compared with mice treated with isotype control antibody. Nevertheless virus was efficiently cleared at 8 dpi (Figure 5C). Most importantly, to address the contribution of NK1.1 positive cells on induction of adaptive CTL immunity, we measured the percentage of virus-specific CD8 T cells at day 8 post infection by using a PE labeled H-2Db tetramer with the NP366–374 epitope ASNENMETM [13]. At day 8 we found decreased numbers of tetramer (TM) positive cells in both spleen and lung tissue (Figure 5D). In addition to this we determined the hemagluttinin specific antibodies in serum at 8 dpi (Figure 5E) and found increased levels following NK1.1 depletion, supporting the higher viral titers. Both at earlier and later time points, similar conclusions on tetramer positive cell induction and humoral immune responses were reached (Figure S2). Surprisingly, NK1.1 depleted mice did not appear to have much more severe systemic morbidity as their weight loss did not exceed 10% of initial weight, in contrast with isotype treated mice, which lost significantly more weight during the peak of infection (Figure 5F). As a final support for an antigen presenting capacity of NK cells in vivo, we also found colocalization of naïve OVA-specific CD8 T cells with NK cells in the draining nodes 48 h after infection with influenza-OVA virus but to a much lower extent with wild type influenza virus (Figure S3).

Bottom Line: Here, using a model of influenza infection, we found recruitment of both conventional B220(-) NK cells and IKDCs to the lung.In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung.In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Pulmonary Medicine, Erasmus University Medical Centre, Rotterdam, The Netherlands.

ABSTRACT
Natural killer cells are innate effector cells known for their potential to produce interferon-gamma and kill tumour and virus-infected cells. Recently, B220(+)CD11c(int)NK1.1(+) NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220(-) NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.

Show MeSH
Related in: MedlinePlus