The effort during 2007 has been mostly concentrated in setting up the new laboratory and recruiting the new members of the group. Progress has been made in several of the aims of the project, in particular on the role of TLR signaling in regulating proliferation and survival of fibroblasts and dendritic cells, on the regulation of cytokine production from human DC, on the role of plasmacytoid DC in regulating oral tolerance, and on the effect of NK cells on Th1 differentiation in Toxoplasma Gondii infection. Cell proliferation and survival induced by TLRs is antagonized by type I Interferons (collaboration with Dr. Uzma Hasan, Lyon, France; publiseh in PNAS 2007) TRIF is an adaptor protein associated with the signalling by TLR3 and TLR4 for the induction of type-I IFNs. We demonstrated a novel mechanism by which TLR signalling controls cell proliferation and survival. We showed that TLR3 and TLR4 can induce cell cycle entry via TRIF, which targets the cell cycle inhibitor p27kip1 for re-localisation, phosphorylation by cyclin/cdk complexes, and proteasome degradation. These events are antagonized by type-I IFN induced by the TRIF pathway. Furthermore, in human dendritic cells treated with TLR3, TLR4 or TLR5 ligands, we demonstrated that interferon signalling modulates p27kip1 degradation and apoptosis, identifying a novel immunoregulatory switching function of type-I IFNs. These findings revealed a new function of TLR signalling in cell proliferation and survival. Differential regulation of interleukin-12 and interleukin-23 production in human dendritic cells (collaboration with Dr. Franca Gerosa, Verona, Italy; submitted) We analyzed Interleukin (IL)-12 and IL-23 production by human antigen-presenting cells. The two cytokines were effectively produced by peripheral blood (DCs) or monocyte-derived DCs (mono-DCs). Heat-killed Mycobacterium tuberculosis H37Rv and zymosan were studied as two stimuli that preferentially induced IL-23. IL-23 was efficiently induced by the combination of NOD-2 and TLR-2 ligands that mimicked activation by M. tuberculosis or with the human Dectin-1 ligand -glucan alone or in combination with TLR-2 ligands, conditions mimicking zymosan. Although in those conditions IL-12 was either not induced or very poorly induced, strong induction of IL-12 was observed by NOD2 or beta-glucan in cooperation with the TLR7/8 ligand R848. IFN-gamma-priming of mono-DCs decreased the ability of mono-DCs to produce IL-23 in response to beta-glucan whereas it was required for optimal IL-12 production. TLR-2 ligands inhibited IL-12 and increased IL-23 production in mono-DC stimulated by zymosan or beta-glucan associated with R848. The differential regulation of IL-12 and IL-23 was found to reflect the accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not those of the IL-12/23p40. The differential expression of the two cytokines is likely important for the regulation of the immune response to different pathogens or at different times during infection. Hepatic plasmacytoid dendritic cells contribute to orally induced CD8+ T cell tolerance (collaboration with Dominique Kaserlian, Lyon, France; Submitted)The liver is thought to contribute to systemic T cell tolerance to orally absorbed antigens, although the precise mechanism of its tolerogenic effect is unclear. Here we show that the liver is a site of oral antigen presentation and that hepatic dendritic dells (DC) can tolerize mice to subsequent CD8+ T cell priming, in a model of hapten-specific contact sensitivity (CHS). The tolerogenic potential of liver DC is confined to plasmacytoid DC (pDC), is enhanced by exposure to hapten, and requires CD4+ T cells. Finally, in vivo depletion of pDC abrogated oral tolerance and restored hapten-specific CD8+ T cell and CHS responses. Thus, pDC are tolerogenic and play an essential role in oral tolerance. Toxoplasma gondii infected TAP1 deficient mice display impaired NK cell IFN-gamma production leading to defective CD4+ T cell priming and increased mortality (collaboration with Dr. Alan Sher, NIAID; in press in Journal of experimental Medicine). To investigate if Transporter Associated with Antigen Processing (TAP)1 is required for CD8+ T cell mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP1-/-, CD8-/- and wild-type (WT) mice to infection with the parasite. As expected, both TAP1-/- and CD8-/- animals vaccinated with an attenuated strain of T. gondii failed to develop protective immunity to lethal tachyzoite challenge consistent with absence of effector CD8+ T cells. Surprisingly, however, TAP1-/- mice displayed greater susceptibility than either CD8-/- or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP1-/- mice correlated with a reduction in the frequency of activated (CD62Llow CD44hi) and interferon gamma (IFN-gamma)-producing CD4+ T cells. Interestingly, infected TAP1-/- mice showed a reduced frequency of IFN-gamma producing natural killer (NK) cells relative to that of WT controls, and after NK cell-depletion both CD8-/- and WT mice succumbed to infection with the same kinetics as TAP1-/- animals and displayed impaired CD4+T cell IFN-gamma responses. Together, these results reveal a previously unappreciated role for TAP1 in the induction of IFN-gammaproducing NK cells and provide the first demonstration of the function of this cell population in the priming of CD4+ T lymphocyte responses to T. gondii infection

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National Cancer Institute (NCI)
Intramural Research (Z01)
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National Cancer Institute Division of Basic Sciences
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Stewart, C Andrew; Trinchieri, Giorgio (2009) Reinforcing suppression using regulators: a new link between STAT3, IL-23, and Tregs in tumor immunosuppression. Cancer Cell 15:81-3
Lyakh, Lyudmila; Trinchieri, Giorgio; Provezza, Lisa et al. (2008) Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans. Immunol Rev 226:112-31
Gerosa, Franca; Baldani-Guerra, Barbara; Lyakh, Lyudmila A et al. (2008) Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells. J Exp Med 205:1447-61
Trinchieri, Giorgio; Sher, Alan (2007) Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 7:179-90
Trinchieri, Giorgio (2007) Pillars of immunology: The birth of a cell type. J Immunol 178:3-4
Hasan, Uzma A; Caux, Christophe; Perrot, Ivan et al. (2007) Cell proliferation and survival induced by Toll-like receptors is antagonized by type I IFNs. Proc Natl Acad Sci U S A 104:8047-52
Trinchieri, Giorgio (2007) Interleukin-10 production by effector T cells: Th1 cells show self control. J Exp Med 204:239-43