Fig 1.
Visualization of nasal NKp46+ cells by using immunohistochemistry.
Frozen sections of nasal tissue obtained from 8-week-old Ncr1GFP/+ mouse were stained with 4′,6-diamidino-2-phenylindole (nucleus), anti-CD45, and anti-GFP antibodies and examined under a fluorescence microscope. Arrows indicate CD45+GFP(NKp46)+ cells. Bar, 50 μm. Data are representative of at least 3 independent experiments. a. Nasal septum. b. Nasal concha.
Fig 2.
Nasal NKp46+ cells are NK-lineage cells.
a. Flow cytometry of CD45+ cells from nasal passage, spleen, lung, and Nasopharynx associated lymphoid tissue (NALT) of Ncr1GFP/+ mice stained with CD3. Numbers in quadrants indicate the percentages of cells in each. Dot plot below shows percentage of GFP (NKp46)+ cells in CD45+ cells from nasal passage, spleen, lung, and NALT. b. Flow cytometry of CD3−NKp46+ cells from spleen, lung, and nasal passages stained with CD122, NK1.1, 2B4, CD49b, and CD127. Continuous lines, specific antibodies; Dashed lines, isotype-matched control antibodies. Dot plot below shows the percentage of positively stained cells. Bar, mean; n.s.; not significant (Mann-Whitney U test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.
Fig 3.
Nasal NK cells have a unique expression pattern of Ly49 family receptors.
a, b, c. Flow cytometry of CD3−NKp46+ cells from nasal passage, spleen, and lung stained with (a) Ly49A, (b) Ly49C/F/H/I, and (c) Ly49D. Numbers in histograms indicate the percentages of positive cells. Continuous lines, specific antibodies; Dashed lines, isotype-matched control antibodies. Dot plots below shows the percentage of positively stained cells. Bar, mean; n.s., not significant; *, P < 0.05; **, P < 0.01 (Mann-Whitney U test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.
Fig 4.
Unique maturation and activation status of nasal NK cells.
a. Flow cytometry of CD3−NKp46+ cells from nasal passage, spleen, and lung, and double-stained with CD11b and CD27. Numbers in quadrants indicate the percentages of cells in each. Dot plots below shows percentage of CD27highCD11blow cells from nasal passage, spleen, and lung. b. Flow cytometry of CD3−NKp46+ cells from spleen, lung, and nasal passages with CD62L, CD69, and CD69/CD103. The numbers in the histograms indicate the percentage of positive cells. Solid line, specific antibody; dashed line, isotype-matched control antibody. Dot plots below shows the percentage of positively stained cells. Bar, mean; n.s.; not significant; *, P < 0.05**, P < 0.01 (Mann-Whitney U test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.
Fig 5.
Impaired effector function of nasal NK cells.
a. Intracellular expression of granzyme B by CD3−NKp46+ cells. Dashed line, isotype-matched control antibody; solid line, specific antibody. Dot plots below shows the mean fluorescence intensity (MFI). b, c. Intracellular staining of (b) CD107a and (c) IFN-γ production by lymphocytes isolated from nasal passage, spleen, and lung and stimulated for 4 h with various stimuli. Isolated lymphocytes were stimulated and then stained for surface antigen followed by intracellular staining. Signal assessed by gating on CD3−NKp46+ cells. Black line, nasal passage; red line, spleen; blue line, lung. d, e. Dot plots showing the percentage of positively stained cells after stimulation. Control, isotype matched control; PMA/iono, phorbol-12-myristate-13-acetate and ionomycin. Bar, mean; n.s.; not significant; *, P < 0.05 **, P < 0.01 (Mann-Whitney U test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.
Fig 6.
Indispensable role of nasal NK cells in influenza virus infection.
a. Change of nasal NK cells after infection with PR8 1×103 pfu/mouse intranasally. Horizontal axis, day after infection; vertical axis, percentage of nasal NK cells in CD45+ lymphocytes (left) or absolute count of nasal NK cells (right). *, P < 0.05 **, P < 0.01 (Mann-Whitney U test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments. b. CD69 expression of nasal NK cells after intranasal infection with influenza virus PR8 (1×103 pfu/mouse). Histogram (left). Dashed line, naïve; solid line, day 2 after infection; bold line, day 5 after infection. Dot plot of MFI (right). Data are representative of 2 independent experiments with 4 mice. c. Nasal virus titer of mice intranasally infected with influenza virus PR8 (1×103 pfu/mouse). Mice were injected intraperitoneally with 100 mg PK136 antibody or an isotype-matched control on days –2, 0, 2 after infection. Bar, mean; horizontal axis, day after infection; vertical axis, virus titer (pfu). Data are representative of 3 independent experiments with 4 to 6 mice in each group. n.s., not significant; *, P < 0.05 **, P < 0.01 (Mann-Whitney U test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.