zed work indicating that the protozoan parasite Toxoplasma gondii contains a major agonist for IL-12 production by CD8alpha+ dendritic cells (DC) that signals through Toll-like receptor(TLR)TLR11. In work performed this year we demonstrated that this TLR ligand, T.gondii profilin, is also an immunodominant antigen (Ag) in the CD4+ T cell response to a crude extract of the parasite (STAg). We hypothesized that the latter property stems from the selective processing and presentation of profilin by DC because of its dual function as Toll ligand and protein Ag Indeed, CD8 alpha + but not CD8 alpha- DC from STAg injected mice potently stimulated proliferation of profilin specific CD4+ T cells in an IL-12 independent fashion. Importantly this response was severely impaired when DC from either TLR11-/- or MyD88-/- mice were substitituted for WT cells in the assay. The requirement for TLR11/MyD88 was not due merely to the role of TLR signaling in DC maturation since it could not be overcome by co-injection of LPS. Furthermore, in mixed WT/MyD88-/-chimera experiments impaired antigen presentation was associated with the MyD88 deficient DC population arguing that the defect is DC intrinsic. Finally, using flourescent tagged profilin, we showed that the molecule is selectively taken up by TLR11+, CD8+ DC in vivo and that these labeled cells preferentially activate profilin specific CD4+ T cells in vitro. These findings support a major influence of TLR recognition in antigen selection by DC in vivo and establish a mechanism by which TLR ligand association regulates the immunogenicity of microbial antigens.? ? In a related collaborative project with David Sacks's group we studied the role of DC in the activation of CD8+ T cells by T.gondii comparing the requirements for antigen processing and presentation with those for Leishmania major. In carrying out these studies we used strains of T.gondii and L. major genetically engineered to express the foreign Ag ovalbumin (OVA) and measured activation of OVA specific transgenic CD8+ T cells. In the case of T.gondii we found that effective T cell activation occured only when OVA was expressed in secreted form and that active infection of DC by the parasite is required. Importantly, Ag presentation by T. gondii infected DC was found to be dependent both on the peptide transporter TAP and on proteosomal processing in direct contrast to Ag presentation by L. major which was both TAP and proteosome independent. Finally, studies in TAP deficient mice confirmed the requirement for the transporter in CD8+ dependent immunity to T. gondii but not L.major. Together these experiments established that although dwelling in a parasitophorous vacuole T.gondii utilizes conventional cytosolic pathways for antigen processing and loading of MHC class I molecules and established a major dichotomy with a second vacuole dwelling protozoan, L. major, which employs a distinct mechanism currently under investigation in the Sacks laboratory.
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