Nasal NKp46⁺ cells are NK-lineage cells.

<p>a. Flow cytometry of CD45⁺ cells from nasal passage, spleen, lung, and Nasopharynx associated lymphoid tissue (NALT) of <i>Ncr1</i>^<i>GFP/+</i> 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 <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p

Nasal NK cells have a unique expression pattern of Ly49 family receptors.

<p>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; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (Mann-Whitney <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p

Visualization of nasal NKp46⁺ cells by using immunohistochemistry.

<p>Frozen sections of nasal tissue obtained from 8-week-old <i>Ncr1</i>^<i>GFP/+</i> 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.</p

Indispensable role of nasal NK cells in influenza virus infection.

<p>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). *, <i>P</i> < 0.05 **, <i>P</i> < 0.01 (Mann-Whitney <i>U</i> 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; *, <i>P</i> < 0.05 **, <i>P</i> < 0.01 (Mann-Whitney <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p

Identification and Analysis of Natural Killer Cells in Murine Nasal Passages

<div><p>Background</p><p>Natural killer (NK) cells in the upper respiratory airways are not well characterized. In the current study, we sought to characterize and functionally assess murine nasal NK cells.</p><p>Methods</p><p>Using immunohistochemistry and flow cytometry, we compared the nasal NK cells of <i>Ncr1</i>^<i>GFP/+</i> knock-in mice, whose NK cells produced green fluorescent protein, with their splenic and pulmonary counterparts. In addition, we functionally analyzed the nasal NK cells of these mice <i>in vitro</i>. To assess the <i>in vivo</i> functions of nasal NK cells, C57BL/6 mice depleted of NK cells after treatment with PK136 antibody were nasally infected with influenza virus PR8.</p><p>Results</p><p>Immunohistochemical analysis confirmed the presence of NK cells in the lamina propria of nasal mucosa, and flow cytometry showed that these cells were of NK cell lineage. The expression patterns of Ly49 receptor, CD11b/CD27, CD62L and CD69 revealed that nasal NK cells had an immature and activated phenotype compared with that of their splenic and pulmonary counterparts. Effector functions including degranulation and IFN(interferon)-γ production after <i>in vitro</i> stimulation with phorbol 12-myristate-13-acetate plus ionomycin or IL(interleukin)-12 plus IL-18 were dampened in nasal NK cells, and the depletion of NK cells led to an increased influenza virus titer in nasal passages.</p><p>Conclusions</p><p>The NK cells of the murine nasal passage belong to the conventional NK cell linage and characteristically demonstrate an immature and activated phenotype. Despite their hyporesponsiveness <i>in vitro</i>, nasal NK cells play important roles in the host defense against nasal influenza virus infection.</p></div

Unique maturation and activation status of nasal NK cells.

<p>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 CD27^highCD11b^low 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; *, <i>P</i> < 0.05**, <i>P</i> < 0.01 (Mann-Whitney <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p

Impaired effector function of nasal NK cells.

<p>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; *, <i>P</i> < 0.05 **, <i>P</i> < 0.01 (Mann-Whitney <i>U</i> test with Ryan’s multiple comparison method). Data are obtained from at least 3 independent experiments.</p

Schematic diagram of the genome structures of wild type YK304 and the relevant domains of the recombinant viruses used in this study.

<p>Line 1, YK304 genome carrying a bacmid (BAC) in the intergenic region between UL3 and UL4. Line 2, domains encoding the UL40 to UL42 open reading frames. Line 3, UL41 gene encoding vhs. Lines 4-7, recombinant viruses with mutations in the UL41 gene. Line 8, domains encoding the Us11 to Us12 open reading frames and the viral replication origin S (Ori-S). Line 9, recombinant virus with mutation in the Us12 gene encoding ICP47.</p

Effect of Us3 kinase activity on HSV-1-specific CTL induction in vivo.

<p>Six-week-old female C57BL/6J mice were mock-infected (n=21) or infected with 1 x 10⁶ PFU HSV-1(F) (n=21), YK511 (Us3-K220M) (n=21), YK513 (Us3-repair) (n=21) or YK591 (ΔICP47) (n=10)/footpad. At 4 d post-infection, spleen (A) and popliteal lymph node (B) cells were obtained and stained with MHC-I tetramers specific for the H-2K^b-restricted HSV-1 gB immunodominant epitope (SSIEFARL). Cells were then stained with anti-CD8α and anti-CD3ε antibodies and analyzed by flow cytometry. The percentage of CD8⁺ and CD3⁺ cells from mock-infected mice that were positive for gB-specific MHC-I tetramer was subtracted from the percentage of CD8⁺ and CD3⁺ cells from mice infected with each virus that were also positive for gB-specific MHC-I tetramer. Each data point is the mean ± standard error.</p

Effect of depletion of CD8⁺ T cells or NK cells on YK511 (Us3K220M) replication in vivo.

<p>Six 6-week-old female C57BL/6J mice were mock-depleted or depleted of CD8⁺ T cells (A) or NK1.1⁺ cells (B) and infected with 1 x 10⁶ PFU YK511 (Us3-K220M) or YK513 (Us3-repair)/footpad. At 1 and 4 d post-infection, virus titers in the footpads from the infected mice were determined by standard plaque assays on Vero cells. Each data point is the mean ± standard error of the PFU/gram/footpad.</p