Dual staining of porcine peripheral blood lymphocytes with Porcine CD335 detected with Goat anti Mouse IgG (H/L):FITC and Porcine wCD8a:RPE

Porcine CD335, used for the identification of NK cells in influenza infected porcine by flow cytometry. NK cell numbers in the blood of influenza infected porcine. Blood was taken from influenza A virus infected (n = 12) and control porcine (n = 9) on day 0-3 pi in a second experiment. (A) Isolated PBMCs were analysed by flow cytometry and live CD3- lymphocytes were gated as NKp46- or NKp46+ NK cells according to CD8a and NKp46 expression. Plots are taken from a representative control animal. Absolute numbers of (B) NKp46+ NK cells in PBMC were analysed by flow cytometry in control (left) and infected animals (right). (C) Results for the NKp46+ cells in the two groups were compared on each sampling day. (D) NKp46- NK cells in PBMC in control (left) and infected animals (right). (E) NKp46- NK cells were compared for the two groups. Each line in (B) and (D) represents one animal. **p=0.01. Early Responses of Natural Killer Cells in Porcine Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.

Porcine CD335, used for the identification of NK cells in influenza infected porcine by flow cytometry. Percentages of NK cells and expression of NKp46 and CD25 in lung tissue. Mononuclear cells were isolated from lung tissue of pigs infected with influenza A virus (n = 12) and control animals (n = 6) during the first 3 days pi and analysed by flow cytometry. (A) Live CD3- lymphocytes were gated as described in Fig 2. NK cells were gated according to CD8a and NKp46 expression and defined as NKp46-, NKp46int or NKp46high cells. Plot shown is from a representative control animal. (B) Proportions of NKp46- (green), NKp46int (blue) and NKp46high (purple) NK cells in individual animals, shown as percentages of gated cells in lymphocytes. (C) Percentages of NKp46- (left), NKp46int (middle) and NKp46high (right) NK cells among lymphocytes. (D) Median fluorescence intensity (MFI) in the NKp46- gate (left), the NKp46+ gate (middle) and in the NKp46high gate (right) are shown. (E) CD25+ cells were gated in the NKp46- and NKp46int NK cells (left) and in the NKp46high NK cells (right). Plots shown are from a representative control animal. (F) The percentages of CD25+ cells in each gate were calculated in control animals (green), infected animals from day 1 (purple), day 2 (blue) and day 3 (pink) pi. *p=0,05, **p=0.01. Early Responses of Natural Killer Cells in Porcine Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.

Porcine CD335, used for the identification of NK cells in influenza infected porcine by immunofluorescence. NKp46+ cells in the lungs of influenza virus infected porcine. Lung tissue sections from porcine infected with influenza A virus and control porcine were stained with immunofluorescence markers for cytokeratin (blue), NKp46 (green) and influenza A virus NP (red). NKp46+ cells were counted in areas were influenza A virus NP was (A) detected and (B) not detected. Representative pictures taken from the same animal on day 1 pi are shown. Arrows point at NKp46+ cells. Immunofluorescence staining, 200x. (C) Plot shows number of NKp46+ cells per 0,1 mm2 in sections (n = 24 per animal) from control animals (n = 6) and in areas with and without virus in infected animals (n = 4 per day) calculated as described in Material and Methods. Groups with different letters differ significantly (p=0.05). (D) NKp46+ cells in the lumen of a bronchus (BL). Arrows point at the epithelial lining. Representative picture of luminal exudate, taken from an infected animal on day 2 pi. Insert shows NKp46+ and influenza A virus NP+ cell in the lung tissue of an infected animal on day 1 pi. Immunofluorescence staining, 400x. Early Responses of Natural Killer Cells in Porcine Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus.