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, 83 (16), 8108-21

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

Affiliations

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

Mostafa Jarahian et al. J Virol.

Abstract

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3zeta lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.

Figures

FIG. 1.
FIG. 1.
Soluble NKp44 and NKp46 receptors bind to various NDV-infected tumor cell lines. (A) PANC-1 pancreatic carcinoma, HeLa cervical carcinoma, A549 lung carcinoma, and Ma-Mel-8a melanoma cells were infected for 20 h with the nonlytic NDV strain Ulster (100 HU/106 cells) (black lines) or left uninfected (gray lines). Cells were stained with NKp30-, NKp44-, and NKp46-IgG1 Fc fusion proteins in complexes with goat anti-hIgG-PE secondary antibodies as indicated. Results for Ma-Mel-8a cells stained with a secondary antibody alone are represented by filled curves. To monitor infection efficiencies, tumor cells were stained with anti-NDV MAb HN.B, recognizing HN (top panels, black lines). For a control, uninfected cells were stained with HN.B (top panels, gray lines). (B) HeLa cells were infected for 18 h with the NDV strain Ulster (black lines) or left uninfected (gray lines). Cells were stained with the NKG2D-Fc fusion protein or with MAbs recognizing NDV HN, to control for infection, or the NKG2D ligands MICA, MICB, and ULBP1 to ULBP4, as indicated. Results for secondary antibody controls are represented by filled curves.
FIG. 2.
FIG. 2.
Anti-NDV HN antibody blocks NKp44 and NKp46 binding. (A) Staining of Ma-Mel-8a, PANC-1, and HeLa cells infected for 20 h with NDV Ulster (100 HU/106 cells), as well as Ma-Mel-8a cells infected for 20 h with NDV MTH-68 (25 HU/106 cells), with NKp30-Fc, NKp44-Fc, or NKp46-Fc fusion proteins without preincubation (black lines) or after preincubation with MAb HN.B (gray lines). Results for secondary antibody controls are represented by filled curves. To monitor infection efficiencies, tumor cells were stained with anti-HN MAb HN.B (top panels, black lines) and secondary antibodies for control (top panels, filled curves). (B) HeLa cells were stably transfected with either NDV HN or F0 cDNA individually or cotransfected with both cDNAs. Panel 1 shows HN expression (black line) and F0 expression (gray line) in HeLa/HN+ F0 cells as detected using MAbs HN.B and Icii, respectively. Panel 2 shows HN expression in HeLa/HN (black line) and F0 expression in HeLa/F0 (gray line) single transfectants. Results for the secondary antibody controls are shown with filled curves. For panels 3 to 5, HeLa/HN+F0 (black lines), HeLa/HN (gray lines), and HeLa/F0 (broken lines) transfectants and HeLa cells carrying the pcDNA3.1(+) vector control (filled curves) were stained with NKp30-Fc, NKp44-Fc, and NKp46-Fc as indicated. (C) HeLa cells infected for 20 h with NDV Ulster (200 HU/106 cells) were stained with NKp44-Fc or NKp46-Fc desialylated with immobilized neuraminidase (gray lines) or untreated NCR fusion proteins as controls (black lines). Filled curves depict results for secondary antibody controls. The infection efficiency was monitored with MAb HN.B (top panel, black line) and a secondary antibody control (top panel, filled curve). +, with; −, without.
FIG. 3.
FIG. 3.
NKp44-Fc and NKp46-Fc bind to NDV particle-coated plates. (A) Standard ELISA plates were coated with purified NDV (strain Ulster and MTH-68) particles, and the particles were allowed to react with NKp44-Fc and NKp46-Fc fusion proteins, followed by peroxidase-labeled anti-hIgG secondary antibody (anti-hIgG-POX). The assay results were developed using the peroxidase substrate o-phenylenediamine dihydrochloride and evaluated in an ELISA reader as the absorption (Abs.) at 450 nm. The binding of soluble NCR can be blocked by preincubation of the virus-coated plate with the anti-HN MAb HN.B but not the anti-F MAb Icii. Plate coating was checked by using anti-HN or anti-F0 MAb, followed by peroxidase-labeled anti-mouse IgG secondary antibody (anti-mIgG-POX). Wells without virus show low levels of nonspecific binding of NCR-Fc, anti-HN, anti-F, and secondary antibodies. Means and SEM for triplicate samples are shown. (B to F) Activation of NKp44-CD3ζ and NKp46-CD3ζ reporter cells by NDV-infected cells. (B, left) Schematic diagram showing NCR-CD3ζ reporter proteins. The ectodomains of NKp46, NKp44, and NKp30 were fused to the transmembrane domain (TMD) and cytoplasmic domain (cyt) of mouse CD3ζ (mCD3ζ) by using cDNA constructs. Residues of the respective proteins contained in the chimeras are indicated. (Right) BWZ.36 mouse thymoma cells harboring the lacZ gene under the control of the NFAT promoter were transfected with the cDNA constructs. Stainings with MAbs specific for NKp46 (αNKp46), NKp44 (αNKp44), and NKp30 (αNKp30) demonstrated surface expression of the indicated NCR-CD3ζ fusion proteins. (C) The indicated NCR-CD3ζ reporter lines were cocultured with uninfected HeLa cells or NDV Ulster-infected HeLa cells for 18 h, after which the lacZ response was measured with the colorigenic substrate CPRG in an ELISA reader. Absorption at 595 nm, expressed as means of triplicate values ± SEM, is shown. ***, P < 0.001; n.s., not significant. (D) Untransfected HeLa cells as well as HeLa cells transfected with HN cDNA were analyzed as described in the legend to panel C. Reporter cell stimulation by NDV-infected cells transfected with HN cDNA (HeLa/HN) and that by untreated HeLa cells were compared by Student's t test. **, P < 0.01; n.s., not significant. (E) The indicated NCR-CD3ζ reporter cells were cultured for 20 h in wells of an ELISA plate treated with coating buffer (no virus) for a control or coated with purified NDV Ulster particles. Other wells were coated with NKp30, NKp44, and NKp46 antibodies (1 μg/well) as indicated to monitor reporter cell activation following antibody-mediated NCR cross-linking.
FIG. 4.
FIG. 4.
NK cells produce enhanced amounts of IFN-γ and TNF-α upon coincubation with NDV-infected cells. HeLa or T98G cells were infected with NDV Ulster or MTH-68 and cultured with primary NK cells in the presence of IL-2. For comparison, uninfected HeLa and T98G cells were cocultured with NK cells, or NK cells and infected (Inf.) tumor cells were cultured alone. IFN-γ and TNF-α secreted into the supernatants were quantified using respective lymphokine immunoassays. Means of triplicate values ± SEM are shown. Lymphokine production elicited by NDV-infected cells was statistically compared with lymphokine secretion in the presence of uninfected cells by Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG. 5.
FIG. 5.
(A to D) NDV-infected tumor cells show enhanced susceptibility to NK cell lysis. HeLa, PANC-1, Ma-Mel-8a, and Ma-Mel-8b tumor cell lines were infected with NDV Ulster (100 HU/106 cells; 20 h) or left uninfected and were then subjected to a 4-h chromium release assay using primary, IL-2-activated NK cells from one to three different donors as effectors. The mean percentages of specific lysis ± SEM for triplicate samples at different effector/target cell ratios are indicated. (E) T98G cells were infected with NDV Ulster (100 HU/106 cells; 20 h) or MTH-68 (25 HU/106 cells; 12 h) or left uninfected and were then subjected to a 4-h chromium release assay using primary IL-2-activated NK cells. The anti-F protein MAb Icii was present during infection with MTH-68 to prevent syncytium formation. (F) Results from cytofluorometric staining of uninfected HeLa cells and NDV Ulster- and NDV MTH-68-infected HeLa cells with the pan-HLA-A-, HLA-B-, HLA-C-reactive antibody W6/32.
FIG. 6.
FIG. 6.
Tumor cells transfected with HN cDNA show enhanced susceptibility to NK cell lysis. (A) Ma-Mel-8a cells transiently transfected with NDV HN or F0 cDNA or an empty vector were used in a chromium release assay with the NK cell line NK-92 as effectors. (B) HeLa cells were stably transfected with pcDNA3.1(+)/HN, doubly transfected with pcDNA3.1(+)/HN and pcDNA3.1(+)/F0, or transfected with an empty vector for a control. Transfectants were subjected to a 4-h chromium release assay using NK-92 cells as effectors. (C) Results of a chromium release assay using HeLa target cells together with NK-92 effector cells. NK-92 cells were either preincubated (pre-inc.) with UV-inactivated NDV particles or left untreated. P < 0.02 by paired Student's t test. (D) Results of a chromium release assay using HeLa or T98G target cells and primary, IL-2-prestimulated NK cells (pNK) as effectors. Primary NK cells were either preincubated with UV-inactivated NDV particles or left untreated. P < 0.02 by paired Student's t test for both HeLa and T98G targets.
FIG. 7.
FIG. 7.
(A, B, D, and E) NDV-induced enhancement of lysis is blocked by anti-NCR and anti-NDV antibodies. Ma-Mel-8a, T98G, and HeLa cells were either infected with NDV Ulster (100 HU/106 cells; 20 h) or left uninfected (uninf.). (C) Alternatively, HeLa cells were infected with NDV MTH-68 (25 HU/106 cells; 16 h) in the presence of anti-F protein MAb Icii to prevent syncytium formation or left uninfected. Fc receptor-deficient NK-92 effector cells were used throughout. For panel A, antibodies against NKp44 or NKp46 or the control antibody MOPC21 was included in the assay mixture as indicated. For panels B and C, polyclonal rabbit anti-NDV antibodies were present in a standard chromium release assay mixture with NDV-infected cells. As controls, rabbit anti-vaccinia virus polyclonal antibodies were used or no antibody was added. For panels D and E, the anti-HN MAb HN.B, HN.B plus the anti-F antibody Icii, or a combination of soluble NKp44-Fc and NKp46-Fc receptors was included in the assay mixture with infected or uninfected targets as indicated.
FIG. 8.
FIG. 8.
The neuraminidase inhibitor Neu5Ac2en blocks NDV-mediated enhancement of NK lysis. T98G and HeLa target cells were infected with NDV Ulster (100 HU/106 cells; 20 h) or left uninfected and were then subjected to a 4-h chromium release assay using primary IL-2-activated NK cells (pNK) or NK-92 effectors. Target cells were preincubated with 100 μM neuraminidase inhibitor Neu5Ac2en during the 1-h chromium labeling period as indicated.

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