A detailed understanding of the trafficking behavior of the endosomal TLRs, as exemplified by that of TLR9, should aid in the design of strategies that improve delivery of antigen to processing compartments in professional antigen presenting cells, concomitant with appropriate activation of the antigen presenting cell. Consequently, this knowledge is expected to contribute to an understanding of the efficacy of vaccines currently in use, and how to improve on them. B cells represent a special target in this regard, as they not only capture antigen via a signaling receptor, the B cell receptor for antigen, which then activates the B cell, but they also possess TLRs whose engagement contributes to B cell activation per se. Other antigen presenting cells that require engagement of TLRs for full activation, production of cytokines and display of costimulatory molecules capture antigen by other means, including phagocytosis and receptor mediated endocytosis, using signaling apparatus distinct form that available to B cells. Cell-autonomous and cell type-specific factors that control endosomal TLR trafficking and function are therefore likely to be important. To address these questions, new animal models have been generated. These include a TLR9-GFP transgenic mouse, to be complemented by a TLR7-GFP mouse, and a transnuclear (TN) mouse model made by somatic cell nuclear transfer, using the nucleus of an ovalbumin-specific B cell as nucleus donor;the resulting mice possess B cells that produce ovalbumin-specific IgG1. Having established an important role for Unc93B1 in escorting functional endosomal TLRs to their site of action, much of the biochemical and cell biological details that underlie Unc93B1s function remain to be determined. The differential requirements displayed by TLR7 and TLR9 for distinct structural elements within Unc93B1 require a molecular explanation. Combined, the results of the proposed experiments, performed with altogether new mouse models, should illuminate the cell biology and function of a class of TLRs of known importance to host defense and implicated in autoimmunity. A combination of live cell imaging, biochemistry and functional readouts (cytokine production, expression of activation markers, antigen presentation) will be applied to the following aims:
Aim 1 : The TLR-GFP animal models will be applied to study endosomal Toll-like receptor 7 and 9 (TLR7/9) ligand binding, trafficking and proteolytic cleavage, to identify novel TLR7/9 cofactors required for trafficking, signaling and/or cleavage and to identify new players in TLR7/9 signaling.
Aim 2 : The TLR-GFP mice will be combined with the ovalbumin-specific TN mice to analyze the synergy between TLR and BCR signaling in B cell activation in response to a combination of BCR and TLR ligands.
Aim 3 : The molecular determinants of UNC93B1 required for trafficking, interaction and activation of TLRs will be examined, making use of new chemoenzymatic tools to study its topology and interacting partners.
. New animal models are proposed to study the trafficking properties and interacting proteins of the endosomal TLRs (TLR7 and TLR9) and their essential partner, Unc93B1, through construction of animals transgenic for GFP-tagged TLRs. These models will be combined with transnuclear mice, created by somatic cell nuclear transfer to yield animals with ovalbumin-specific B cells, to study the interplay between TLR- and BCR derived signals for proper B cell activation, using live cell imaging and biochemistry.
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