<i>Ex vivo</i> cytolytic potential and cytokine secretion are lower in pancreatic and liver NK cells.

<p>A) NK cells pooled from five 8-week-old NOD mice were stimulated for four hours with plate-bound anti-Ly49D in the presence of an antibody to CD107a to mark cytolytic granule release. B) Summary of results from 3 experiments. * p<0.05 versus all other groups (unpaired T test). C) NK cells pooled from 8-week-old NOD mice were stimulated with IL-12+IL-18 in the presence brefeldin A for four hours and then stained for intracellular IFNγ. Results are pooled from 4 independent experiments. * The frequency of NK cells producing IFNγ in blood was significantly higher than NK cells in pancreatic (Pan) or inguinal (Ing) lymph nodes (<i>p</i><0.005) and in the liver and pancreas (<i>p</i><0.0001), **% of NK cells expressing IFNγ in both nodes was higher than in the pancreas (<i>p</i><0.001), # % of NK cells expressing IFNγ in the spleen was significantly higher than in pancreas (<i>p</i> = 0.04). NS, there was not a significant difference between the percentage of NK cells expressing IFNγ in the liver and pancreas according to an unpaired T test.</p

NK cells within the pancreas proliferate at a higher rate than NK cells in the periphery.

<p>Plots shown are gated on NKp46⁺ TCRβ⁻ lymphocytes day 3 after daily injections of 200 µg BrdU. A) NK cells from NOD mice incorporate BrdU at a higher rate than T cells in the same tissue. B) A higher frequency of NK cells in the pancreas are proliferating compared to NK cells in the spleen of NOD mice, relative to the same comparison of T cells in the pancreas and spleen. Bar graph of three NOD mice in one experiment C) The higher proliferation of pancreatic NK cells in NOD mice is dependent on the presence of adaptive immunity as pancreatic NK cells in <i>Rag2^−/−</i> NOD mice did not incorporate more BrdU than splenic or liver NK cells. D) Bar graph of three <i>Rag2^−/−</i> NOD mice. In some cases mice were pooled to obtain sufficient numbers of NK cells. One representative experiment of three independent experiments is shown.</p

<i>In vivo</i> activation of pancreatic NK cells is intact.

<p>A) CD69 expression on NK cells from NOD mice injected with 200 µg poly I:C (solid line) versus control NOD mice injected with PBS (filled histogram). Colored numbers matching the respective histograms indicate median fluorescence intensity. One representative experiment of three independent experiments is shown. B) NK cells harvested from the mice in (A) were immediately stained <i>ex vivo</i> (without any <i>in vitro</i> stimulation) for intracellular IFNγ. Results were combined from 3 independent experiments.</p

NK cells reside in the pancreas.

<p>A) Flow cytometric analysis of leukocytes isolated from enzyme-digested tissues of NOD mice. Plots are of CD45⁺ cells and numbers are percentages of CD45⁺ cells. Example of one of six similar experiments. B) Using immunohistochemistry, NK cells (red) were found in the surrounding peri-islet infiltrates (left panel 40×) and in infiltrated islets (right panel 20×). Insulin was stained green to define islets. Example of sections of pancreas from three different NOD mice, evaluating a minimum of 10 slides per mouse. C) NK cells were more numerous in NOD pancreas compared to B6.g7 pancreas– dots were enlarged to allow visualization of NK cells due to low number of events obtained from B6.g7 mice. D) NOD.NK1.1 pancreata contain similar frequencies of NK cells as NOD mice. E) <i>Rag2^−/−</i> NOD mice have low numbers of NK cells in their pancreas, whereas <i>Rag1^−/−</i> B6.g7 mice have very few, almost undetectable numbers of NK cells in the pancreas. F) Absolute numbers of NK cells recovered from the pancreas of individual mice from noted strains. Example from one of six similar experiments, each with a minimum of three mice per strain is shown.</p

NK cells are not required for the progression to diabetes.

<p>A) The left panel shows the rate of spontaneous diabetes in NOD.NK1.1 littermates that received either anti-NK1.1 or an isotype-matched control IgG from 3 through 10 weeks of age (10 litters). In the middle panel, purified CD4⁺ T cells from diabetic NOD mice were transferred into <i>Rag2^−/−</i> NOD.NK1.1 mice and treated with anti-NK1.1 or an isotype-matched control IgG. Both groups in the adoptive transfer experiments also received anti-CD8 to ensure exclusively CD4⁺ T cell-mediated disease. The far right panel shows the incidence of disease in unmanipulated NOD mice and NOD.NK1.1 mice in our colony. P values were calculated using the Mann-Whitney U test. B) Five hundred micrograms of BrdU was in injected intravenously and mice were sacrificed one hour later. Flow cytometric plots of cells isolated from the pancreas from one representative experiment are shown. Graphs are cumulative of 3 independent experiments involving littermates. C) NOD.NK1.1 mice were sacrificed after 10 weeks after receiving noted treatment regimens. Isolated leukocytes from the pancreatic lymph node or pancreas with stimulation with PMA and ionomycin for 3 hours in the presence of GolgiStop and assayed for intracellular IFNγ or IL-17 production by CD4⁺ T cells or CD8⁺ T cells, as indicated. Graphs are cumulative of 3 independent experiments involving littermates. No significant difference between groups was demonstrated in any parameter assayed.</p

Pancreatic NK cells have an activated phenotype.

<p>A) NK cells pooled from five 8-week-old NOD mice were assayed for maturational status using CD11b and CD27. Both the pancreas and liver had more CD11b^loCD27^lo NK cells than in the spleen. B) Histograms of CD11b and CD27 (gated on CD3-, NKp46⁺ cells) demonstrate a lower mean fluorescence intensity of these markers on pancreatic NK cells relative to splenic NK cells. CD11c, CD69, and KLRG1 were increased on pancreatic NK cells. Lower expression of CD49b, CD43, and NKp46 on pancreatic NK cells was also indicative of activation. Example from one of five similar experiments is shown.</p

NK cells do not influence intra-islet DC maturation in NOD mice.

<p>A) NK cells were not detected in islets from NSG mice (first panel on left) or NOD.NK1.1 mice treated with anti-NK1.1 (far right panel), but were found in islets from <i>Rag2^−/−</i> NOD mice and NOD.NK1.1 mice treated for 8 weeks with isotype-matched control IgG. Plots are of CD45⁺ cells isolated from islets of 10 week-old mice and numbers represent percentages of total CD45⁺ cells. B) DCs were detected in islets of NSG and <i>Rag2^−/−</i> NOD mice, and in the islets of NOD.NK1.1 mice treated with isotype-matched control IgG or anti-NK1.1 (α-NK1.1). Plots are of CD45⁺ cells isolated from islets of 10 week-old mice and numbers represent percentages of total CD45⁺ cells. C) NK cells in <i>Rag2^−/−</i> NOD mice do not promote DC maturation relative to the NK cell-deficient NSG mice (top row) as there was no difference in maturation marker expression on DC in <i>Rag2^−/−</i> NOD and NSG mice. NOD.NK1.1 mice treated with anti-NK1.1 or an isotype-matched control IgG have DCs of similar maturation status (bottom row). One representative experiment of three independent experiments is shown.</p