Structure-Function Relationship of XCL1 Used for in vivo Targeting of Antigen Into XCR1+ Dendritic Cells.

XCL1 is the ligand for XCR1, a chemokine receptor uniquely expressed on cross-presenting dendritic cells (DC) in mouse and man. We are interested in establishing therapeutic vaccines based on XCL1-mediated targeting of peptides or proteins into these DC. Therefore, we have functionally analyzed various XCL1 domains in highly relevant settings in vitro and in vivo. Murine XCL1 fused to ovalbumin (XCL1-OVA) was compared to an N-terminal deletion variant lacking the first seven N-terminal amino acids and to several C-terminal (deletion) variants. Binding studies with primary XCR1+ DC revealed that the N-terminal region stabilizes the binding of XCL1 to its receptor, as is known for other chemokines. Deviating from the established paradigm for chemokines, the N-terminus does not contain critical elements for inducing chemotaxis. On the contrary, this region appears to limit the chemotactic action of XCL1 at higher concentrations. A participation of the XCL1 C-terminus in receptor binding or chemotaxis could be excluded in a series of experiments. Binding studies with apoptotic and necrotic XCR1-negative cells suggested a second function for XCL1: marking of stressed cells for uptake into cross-presenting DC. In vivo studies using CD8+ T cell proliferation and cytotoxicity as readouts confirmed the critical role of the N-terminus for antigen targeting, and excluded any involvement of the C-terminus in the uptake, processing, and presentation of the fused OVA antigen. Together, these studies provide basic data on the function of the various XCL1 domains as well as relevant information on XCL1 as an antigen carrier in therapeutic vaccines.

Induction of potent CD8 T cell cytotoxicity by specific targeting of antigen to cross-presenting dendritic cells in vivo via murine or human XCR1.

Current subunit vaccines are incapable of inducing Ag-specific CD8(+) T cell cytotoxicity needed for the defense of certain infections and for therapy of neoplastic diseases. In experimental vaccines, cytotoxic responses can be elicited by targeting of Ag into cross-presenting dendritic cells (DC), but almost all available systems use target molecules also expressed on other cells and thus lack the desired specificity. In the present work, we induced CD8(+) T cell cytotoxicity by targeting of Ag to XCR1, a chemokine receptor exclusively expressed on murine and human cross-presenting DC. Targeting of Ag with a mAb or the chemokine ligand XCL1 was highly specific, as determined with XCR1-deficient mice. When applied together with an adjuvant, both vector systems induced a potent cytotoxic response preventing the outgrowth of an inoculated aggressive tumor. By generating a transgenic mouse only expressing the human XCR1 on its cross-presenting DC, we could demonstrate that targeting of Ag using human XCL1 as vector is fully effective in vivo. The specificity and efficiency of XCR1-mediated Ag targeting to cross-presenting DC, combined with its lack of adverse effects, make this system a prime candidate for the development of therapeutic cytotoxic vaccines in humans.

Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.