The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gammaRIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gammaRIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.

The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gammaRIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gammaRIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.

L'échinococcose alvéolaire (EA) est une maladie parasitaire en relation non seulement avec la destruction hépatique quiaccompagne le développement du stade larvaire (métacestode) d'Echinococcus multilocularis, mais aussi avecl'importante réponse immunitaire granulomateuse qui l'entoure. La plupart des travaux antérieurs visant à caractériser le;relations hôte-parasite ont porté sur les cellules de l'immunité dans des sites éloignés de l'habituelle localisationparasitaire, dans le foie. Ce travail de thèse rapporte les mécanismes impliqués dans les modifications de l'homéostasiehépatique aux différents stades de l'infection, mais aussi l'analyse détaillée des profils de cytokines et chimiokinesprésents dans l'infiltrât cellulaire périparasitaire hépatique, de la présence du transforming growth factor-beta et desautres acteurs de sa voie d'activation, et de l'implication possible du Fibrinogen-like protein-2 (FGL2), une moléculeeffectrice des lymphocytes T-régulateurs récemment identifiée. Les résultats indiquent que la réaction inflammatoire quientoure le métacestode dans le foie contribue significativement à la sécrétion de cytokines et de chimiokines et auxmécanismes fonctionnels immunitaires de l'interaction hôte-parasite ; ils révèlent aussi pour la première foisl'intervention cruciale de FGL2 dans la tolérance vis-à-vis d'£. multilocularis. Ces résultats contribuent à identifier denouvelles cibles pour une thérapeutique immunologique qui permettrait de pallier les conséquences pathologiques del'infection par E. multilocularis et de complémenter l'action seulement parasitostatique des benzimidazoles, seuls agentsthérapeutiques connus actuellement. / Alveolar echinococcosis (AE) is a parasitic disease predominantly caused not only by thé direct hepatic damage whichfollows thé continuous tumor-like prolifération of thé larval stage (métacestode) of Echinococcus multilocularis, but alsoindirectly by thé intense local granulomatous immune response which surrounds thé parasitic tissue. Most of previousstudies which aimed at characterizing thé host-parasite relationship hâve been performed on cells of thé immune responsiin peripheral sites, far from thé usual location of E. multilocularis larvae, i.e. thé liver. This PhD thesis reports on thémechanisms involved in thé changes in hepatic homeostatis at thé various stages of infection, and also on a detailedanalysis of thé cytokine and chemokine profiles in thé hepatic periparasitic cell infiltrate, of thé présence of transforminggrowth factor-beta and other actors of its metabolic pathway, and of thé possible involvement of Fibrinogen-like protein-:(FGL2), a recently identified effector molécule of T-regulatory lymphocytes. Results indicate that thé inflammatoryreaction which surrounds thé métacestode in thé liver significantly contributes to cytokine and chemokine sécrétion and tthé functional immune mechanisms of thé host-parasite interactions; they also reveal for thé first time thé crucialintervention of FGL2 in thé tolérance towards E. multilocularis. Thèse results contribute to identify new targets fortherapeutic immune modulation in order to alleviate thé pathological conséquences of E. multilocularis and tocomplément thé parasitostatic-only action of benzimidazoles, thé currently available chemotherapy of thé disease