The goal of this project is a thorough analysis of the role of the human homologue of the drosophila Toll (dToll) protein in innate and adaptive immunity. This homologue, which we call hToll, was recently cloned by us from a human spleen cDNA library. We observed that hToll had the predicted properties for a pattern recognition receptor (PRR), namely induction of co-stimulatory molecules and inflammatory cytokines upon transfection of cells with a dominantly active hToll construct. We have evidence from northern blots of its distribution in tissues and cells, but to completely analyze its expression we need to develop antibodies against the native protein for use in cell sorter analyses and in immunohistochemical staining. We will use the ectodomain fused to the Ig heavy chain to probe for ligands for the hToll protein. When a ligand(s) is identified, we will prepare such ligands as pure proteins for use in analyses of the function of the intact protein. The upstream components are likely to be more readily defined in Drosophila, where we have an interesting lead, but the downstream elements are now known to include the Toll-like protein MyD88, a general adapter for receptors with the Toll signalling domain. We will prepare mice that are deficient in the expression of mToll and of mMyD88, and analyze their phenotype if they are viable. If they are not viable, several alternatives are available to us to prepare conditional or lineage specific gene knock outs. Finally, we have identified several other Toll family members by screening a human spleen cDNA library, and we will analyze the functions of these proteins in the later years of the grant.
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