NK Cells Are Not Required for Spontaneous Autoimmune Diabetes in NOD Mice

NK cells have been shown to either promote or protect from autoimmune diseases. Several studies have examined the role of receptors preferentially expressed by NK cells in the spontaneous disease of NOD mice or the direct role of NK cells in acute induced disease models of diabetes. Yet, the role of NK cells in spontaneous diabetes has not been directly addressed. Here, we used the NOD.NK1.1 congenic mouse model to examine the role of NK cells in spontaneous diabetes. Significant numbers of NK cells were only seen in the pancreas of mice with disease. Pancreatic NK cells displayed an activated surface phenotype and proliferated more than NK cells from other tissues in the diseased mice. Nonetheless, depletion of NK cells had no effect on dendritic cell maturation or T cell proliferation. In spontaneous disease, the deletion of NK cells had no significant impact on disease onset. NK cells were also not required to promote disease induced by adoptively transferred pathogenic CD4+ T cells. Thus, NK cells are not required for spontaneous autoimmune diabetes in NOD mice

Effects of NKG2D ligand expression on host immune responses

Natural Killer (NK) cells are a component of the innate immune system. They play an important role in the defense against pathogens and tumors, and use a diverse pool of receptors to eliminate their targets. NK cell-mediated cytotoxicity is determined by a balance of signals emanating from the recognition of "missing-self" and "induced-self" on target cells. NK cells are best know for their detection of "missing-self", which involves the activation of NK cells when they encounter cells with low or absent expression of major histocompatibility complex class I molecules. However, NK cells can also mediate the recognition of "induced-self", or self-proteins upregulated in infected or transformed cells. Although many NK cell activating receptors have been proposed to mediate "induced-self" recognition, the exact ligands recognized remain in many cases unknown.An exception to this is NKG2D, an invariant receptor shared by NK cells and T cells that can potently induce killing. NKG2D recognizes a family of "induced-self" ligands that has been extensively studied and remains a field of active investigation. This dissertation describes my efforts at gaining a better understanding of the effect of "induced-self" recognition by the NKG2D receptor on host immunity. First, using a transgenic mouse model, I discuss the effect of constitutive NKG2D ligand expression on global NK cell function. I show that NKG2D-independent functions are unaltered when NK cells are exposed to NKG2D ligands constitutively. Second, I describe a novel model created to selectively express the NKG2D ligand Rae-1 on targeted cell subsets and, in particular, on dendritic cells (DCs). I use this novel tool to investigate the role of NKG2D in the crosstalk between DCs and NK cells and CD8+ T cells. My studies suggest that the NKG2D pathway is dispensable for NK and CD8+ T cell activation by dendritic cells. Finally, I discuss a novel model developed to determine the role of soluble NKG2D ligands on NKG2D function. My studies suggest that soluble NKG2D ligands can potently impair NK cell function in the absence of any additional accelerating factors

Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function

To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand-expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk

Chronic In Vivo Interaction of Dendritic Cells Expressing the Ligand Rae-1ε with NK Cells Impacts NKG2D Expression and Function.

To investigate how dendritic cells (DCs) interact with NK cells in vivo, we developed a novel mouse model in which Rae-1ε, a ligand of the NKG2D receptor, is expressed in cells with high levels of CD11c. In these CD11c-Rae1 mice, expression of Rae-1 was confirmed on all subsets of DCs and a small subset of B and T cells, but not on NK cells. DC numbers and activation status were unchanged, and NK cells in these CD11c-Rae1 mice presented the same Ly49 repertoire and maturation levels as their littermate wildtype controls. Early NK cell activation after mouse CMV infection was slightly lower than in wildtype mice, but NK cell expansion and viral control were comparable. Notably, we demonstrate that chronic interaction of NK cells with NKG2D ligand-expressing DCs leads to a reversible NKG2D down-modulation, as well as impaired NKG2D-dependent NK cell functions, including tumor rejection. In addition to generating a useful mouse model, our studies reveal in vivo the functional importance of the NK cell and DC cross-talk

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>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

<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

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