Regulation of NK cell activity : studies of DAP12-associated receptors in immune synapse formation and in responses to cytomegalovirus infection

Abstract

Natural killer (NK) cell effector functions are important for innate resistance against tumor cells and viral infections. NK cells display a broad range of inhibitory and activating receptors on the cell surface, which ensure specificity. Several activating NK cell receptors are co-expressed with and function through the immunoreceptor tyrosinebased activation motif (ITAM)-bearing molecule DAP12. The general aim of this thesis was to address the role of a specific signaling pathway for activating NK cell receptors, the DAP12 pathway, in complex situations such as cellular or hostpathogen interactions.

The redistribution of inhibiting and activating receptors and their ligands to the NK cell immune synapse have recently been investigated. To determine the role of DAP12 signaling in activating NK cell immune synapse formation, we established a system based on in vitro co-incubation with mouse NK cells and ligand-expressing target cells, using NK cells from mice bearing DAP12 molecules with non-functional ITAMs. We showed that the recruitment of the DAP12-associated activating NK cell receptor Ly49D to the NK cell immune synapse upon ligand-interaction was independent of DAP12 signaling. Signaling was however crucial for ligand induced down-modulation of Ly49D, similar to TCR-downmodulation.

To address specific activating pathways in the regulation of NK cells in the host-pathogen interaction, we studied the role of DAP12 in the early response to murine cytomegalovirus (MCMV). In DAP12 mutant mice bearing a non-functional ITAM, we found a considerable increase in viral titers in the spleen (30-40 fold) and in the liver (2-5 fold). The difference compared to wild type mice could be attributed to NK cells. Moreover, the percentage of hepatic NK cells producing IFN-γ was strongly reduced in the absence of a functional DAP12. This was the first study showing a crucial role for a particular activating signaling pathway in the NK cell-mediated resistance to an infection in vivo. Our results were in line with three concurrent reports demonstrating that innate resistance to MCMV requires the presence of NK cells expressing the activating receptor Ly49H, known to associate with DAP12. The DAP12 signaling pathway was critical also for the specific expansion of Ly49H+ NK cells upon M~ infection, most likely by enhancement of cytokine-driven NK cell proliferation, indicating an adaptive component in the NK cell response.

Upon MCWV infection, NK cell stimulating cytokines such as IFN-α/β, and to some extent IL-12, are produced by plasmacytoid dendritic cells (pDCs). Murine pDCs deficient for the signaling molecule DAP12 produced increased amounts of IFN-α/β and IL-12 in response to cytomegalovirus (MONT) infection or to CpG challenge in vivo. In the case of CpG challenge, this was regulated by endogenous DAP12 signaling. However, during MCNW infection, endogenous DAP12 signaling in pDCs limited IL-12 production but did not significantly modulate IFN-α/β induction. It is possible that DAP12 signaling influences some but not all of the pathways for induction of IFN-α/β. The DAP12 mediated regulation of pDC functions may be important to allow viral defense but limit immunopathology and avoid autoimmunity. NK cells have multiple functions and our results show that these can be dissected and explored also in complex situations. DAIP12 mediated signaling regulates NK cell receptor downmodulation effector functions as well as proliferation.