Project 5

In animal models of type-1 diabetes, systemic treatment with antibodies blocking CD40L has been shown to prevent disease. Previous work from this laboratory has demonstrated that a subset of cells generated during this treatment prevents type-1 diabetes when transferred to susceptible animals (1). These cells share properties of dendritic cells (DC) and natural killer (NK) cells and regulate in an antigen-specific fashion, but the mechanism by which anti-CD40L treatment generates this population and the means by which they suppress type-1 diabetes is currently unknown. The use of conditioned bitypic NK/DCs as a therapeutic tool to suppress type-1 diabetes in an antigen-specific manner is an exciting prospect and if successful, a therapy that could be applied to other immune-mediated diseases. Prerequisites to therapeutic use are, however, to elucidate the manner in which anti-CD40L treatment induces this subset of cells and to understand how the cells exert their suppressive effect. The aims for the following proposal are therefore twofold:

1. To dissect the mechanism(s) by which αCD40L-induced NK/DCs protect against diabetes Establishing the mechanism by which NK/DCs protect against diabetes could provide novel therapeutic opportunities by recreating their suppressive activity in vitro. To address this aim we will first compare the phenotype of “protective NK/DCs” from anti-CD40L treated mice with “non-protective NK/DC” from nontreated mice using flow cytometry and gene array methods. Subsequent studies will employ a combination of gene knockout or transgenic mice and stimulatory or inhibitory antibodies to investigate whether blocking or stimulating suspect molecules alters the diabetes protecting properties of NK/DCs. To date, knock out and systemic inhibition studies have identified three intriguing factors - perforin, interferon-γ and IDO.
2. To generate “protective NK/DCs” in vitro. The NK/DC subset isolated from mice represents only 3-5% of splenocytes and therefore several donor mice are generally treated with anti-CD40L to provide enough cells to protect recipient mice. For future use as a therapeutic intervention in humans, it will therefore be necessary to expand the NK/DC subset in vitro such that they retain the ability to suppress diabetes in vivo. As the CD11c+DX5+ subset is phenotypically similar to that of activated NK cells, methods previously used to expand and activate NK cells will be applied to the expansion of this population. These methods include culture with IL-15 and/or mature antigen presenting cells, together with in vitro blockade of CD40L. The phenotype and function of these cells will be compared to the phenotype of those isolated ex vivo from anti-CD40L treated mice and assayed for their ability to protect from diabetes in vivo.