Differential activation of NK cells by influenza A pseudotype H5N1 and 1918 and 2009 pandemic H1N1 viruses

Abstract

Natural killer (NK) cells are the effectors of innate immunity and are recruited into the lung 48 h after influenza virus infection. Functional NK cell activation can be triggered by the interaction between viral hemagglutinin (HA) and natural cytotoxicity receptors NKp46 and NKp44 on the cell surface. Recently, novel subtypes of influenza viruses, such as H5N1 and 2009 pandemic H1N1, transmitted directly to the human population, with unusual mortality and morbidity rates. Here, the human NK cell responses to these viruses were studied. Differential activation of heterogeneous NK cells (upregulation of CD69 and CD107a and gamma interferon [IFN-gamma] production as well as downregulation of NKp46) was observed following interactions with H5N1, 1918 H1N1, and 2009 H1N1 pseudotyped particles (pps), respectively, and the responses of the CD56(dim) subset predominated. Much stronger NK activation was triggered by H5N1 and 1918 H1N1 pps than by 2009 H1N1 pps. The interaction of pps with NK cells and subsequent internalization were mediated by NKp46 partially. The NK cell activation by pps showed a dosage-dependent manner, while an increasing viral HA titer attenuated NK activation phenotypes, cytotoxicity, and IFN-gamma production. The various host innate immune responses to different influenza virus subtypes or HA titers may be associated with disease severity.

FIG. 1.

Expression of the early activation marker CD69 on NK cells in response to influenza virus pps. Freshly purified human NK cells from 18 different donors were cocultured overnight with H1N1-2009pps, H1N1-1918pps, or H5N1pps at doses ranging from 50 to 1,000 HAU/ml. (A) CD69 expression on mock-treated NK cells and on NK cells stimulated with pps at a dosage of 1,000 HAU/ml from one representative donor. FACS plots are gated on CD56⁺ CD3⁻ lymphocytes. Percentages indicate the proportions of CD56^bright or CD56^dim cells that were positive for CD69. Data are based on the collection of 20,000 total events. (B) CD69 expression on CD56^bright NK cells. The percentage of CD69⁺ cells (left) and the fold increase in CD69 median fluorescence intensity (MFI) (right) are shown. (C) CD69 expression on CD56^dim NK cells. The percentage of CD69⁺ cells (left) and the fold increase in CD69 MFI (right) are shown. *, P < 0.05; **, P < 0.01.

FIG. 2.

Expression of CD107a on NK cells in response to influenza virus pps and the cytotoxicity of NK cells toward K562 cells after stimulation by pps. Freshly purified human NK cells from 18 different donors were cocultured overnight with H1N1-2009pps, H1N1-1918pps, or H5N1pps at doses ranging from 50 to 1,000 HAU/ml. CD107a expression was assessed by intracellular staining followed by flow cytometric analysis. For the cytotoxicity assay, K562 cells were prestained with calcein AM, cocultured with pp-stimulated NK cells or mock-treated NK cells for 3 h, and then stained with ethidium homodimer-1. Cytotoxicity was analyzed by flow cytometry and calculated as the percentage of cells stained with calcein AM plus ethidium homodimer-1 cells among the total cells stained with calcein AM. (A) CD107a expression on mock-treated NK cells and on NK cells stimulated with pps at a dose of 1,000 HAU/ml from one representative donor. FACS plots are gated on CD56⁺ CD3⁻ lymphocytes. Percentages indicate the proportions of CD56^bright or CD56^dim cells that were positive for CD107a. Data are based on the collection of 50,000 total events. (B) CD107a expression on CD56^bright NK cells. The percentage of CD107a⁺ cells (left panel) and the fold increase in CD107a MFI (right panel) are shown. (C) CD107a expression on CD56^dim NK cells. The percentage of CD107a⁺ cells (left) and the fold increase in CD107a MFI (right) are shown. (D) Cytotoxic activity of mock-treated and pp-stimulated NK cells from 6 individuals toward K562 cells at an E/T ratio of 20:1. (E) Cytotoxic activity of mock-treated NK cells and NK cells from 6 individuals stimulated by pps at a dose of 200 HAU/ml toward K562 cells at different E/T ratios. E/T ratio, ratio of effector to target cells; *, P < 0.05; **, P < 0.01.

FIG. 3.

Influenza virus pp-induced IFN-γ in NK cells. Freshly purified human NK cells from 18 different donors were cocultured overnight with H1N1-2009pps, H1N1-1918pps, or H5N1pps at doses ranging from 50 to 1,000 HAU/ml. IFN-γ was determined with cell-free supernatants using human IFN-γ Flex Set reagents (BD Biosciences) according to the manufacturer's protocol. Concentrations are shown in pg/ml. **, P < 0.01.

FIG. 4.

Expression of NKp46 on NK cells in response to influenza virus pps. Freshly purified human NK cells from 18 different donors were cocultured overnight with H1N1-2009pps, H1N1-1918pps, or H5N1pps at doses ranging from 50 to 1,000 HAU/ml. NKp46 expression was analyzed by surface staining and flow cytometric analysis. (A) NKp46 expression on mock-treated NK cells and on NK cells stimulated with pps at a dose of 1,000 HAU/ml from one representative donor. FACS plots are gated on CD56⁺ CD3⁻ lymphocytes. Percentages indicate the proportions of CD56^bright or CD56^dim cells that were positive for NKp46. Data are based on the collection of 20,000 total events. (B) NKp46 expression on CD56^bright NK cells. The percentage of NKp46⁺ cells (left) and the decrease percentage in NKp46 MFI (right) are shown. (C) NKp46 expression on CD56^dim NK cells. The percentage of NKp46⁺ cells (left) and the decrease percentage in NKp46 MFI (right) are shown.

FIG. 5.

Enhanced NK cell activation partially induced by NKp46 downregulation. Freshly purified human NK cells from 6 different donors were first incubated for 1 h with or without unlabeled anti-NKp46 MAb at a concentration of 10 μg/ml and then cocultured overnight with H1N1-2009pps, H1N1-1918pps, or H5N1pps at a dose of 200 HAU/ml. Corresponding isotype antibody was added as control. (A) CD69 expression on mock-treated NK cells and on NK cells stimulated with pps from one representative donor. FACS plots are gated on CD56⁺ CD3⁻ lymphocytes. Percentages indicate the proportions of CD56^bright or CD56^dim cells that were positive for CD69. Data are based on the collection of 50,000 total events. (B) CD69 expression on CD56^bright NK cells. The percentage of CD69⁺ cells (left) and the fold increase in CD69 MFI (right) are shown. (C) CD69 expression on CD56^dim NK cells. The percentage of CD69⁺ cells (left) and the fold increase in CD69 MFI (right) are shown. *, P < 0.05; **, P < 0.01.