Unit: Immunorégulation

Director: ROGGE, Lars

The Immunoregulation group pursues two main axes of research: the analysis of the molecular mechanisms that control the differentiation of naive T helper cells into polarized Th1 and Th2 effector cells (directed by Lars Rogge) and the study of a novel regulator of ubiquitination processes, the COP9 signalosome (directed by Elisabetta Bianchi).

Epigenetic modifications determine Th1 cell-specific expression of the signalling subunit of the IL-12 receptor, IL-12Rβ2 (Fabrice Letimier)

Protective immune responses against pathogens are orchestrated by functionally distinct subsets of antigen-specific T helper cells, termed Th1 and Th2 cells. Th1 cells promote cell-mediated immunity and are necessary to clear the organism from intracellular pathogens but can cause chronic inflammatory diseases. On the other hand, Th2 responses are essential to combat extracellular pathogens but are associated with allergies and asthma. This indicates that the development of Th1 and Th2 cells must be tightly controlled and that therapeutic modulation of immune reponses may have an impact on human diseases.

The main interest of the Immunoregulation laboratory is to analyze the mechanisms that control the differentiation of naive CD4+ T lymphocytes into polarized Th1 and Th2 cells. We have established an in vitro cell culture system based on human cord blood leukocytes, which allows the generation of Th1 and Th2 lines with strongly polarized cytokine profiles. Using this system, we identified the first cell surface marker for Th1 cells (the signalling component of the IL-12 receptor, IL-12Rβ2). We have shown that expression of IL-12Rβ2 is not detected in naive CD4+ T cells but is induced during differentiation of T cells along a Th1 pathway. In contrast, IL-12Rβ2 expression is not induced in Th2 lineage-committed cells. The molecular basis for Th1-specific expression of IL-12Rβ2 is unknown.

To identify the elements that determine Th1-specific expression of IL-12Rβ2, we have analyzed DNAse I hypersensitive sites in the IL-12Rβ2 gene in cells differentiating along a Th1 or a Th2 pathway. This strategy allowed us to identify enhancer elements that determine Th1 lineage-restricted expression of the human IL-12Rβ2 gene. We have also analyzed epigenetic changes at the IL-12Rβ2 locus that occur during T helper cell differentiation using chromatin immunoprecipitation (ChIP) assays for the analysis of histone acetylation and methylation. We could demonstrate that histone H3 acetylation and lysine 4 methylation increased at specific sites within the IL-12Rβ2 locus very early during Th1 cell differentiation but not in developing Th2 cells. Furthermore, we have established a ChIP assay for the signal transducer and activator of transcription (Stat)4 and could demonstrate that Stat4 is bound to the enhancer of the IL-12Rβ2 gene in vivo in developing Th1 but not in Th2 cells. Our results indicate a critical role for Stat4 and of chromatin remodelling complexes very early during induction of IL-12Rβ2 gene expression in Th1 cells. Current work addresses the nature of the chromatin remodelling complexes recruited to the IL-12Rβ2 locus during Th1 cell development.

Lineage commitment versus lineage plasticity: The role of chromatin remodeling during T helper cell development (Sona Gasparian)

Recent studies have indicated that progressive chromatin remodeling and DNA demethylation play a role in defining T helper cell lineage fate. The current debate centers on the question whether progressive silencing of genes expressed in pluripotent cells or whether sequential activation of genes directly responsible for the differentiated phenotype determine differentiation of T helper cells. We are addressing this issue by analyzing the role of the two "master regulators" of T helper cell development (T-bet and GATA-3) in shaping the gene expression program and remodelling of the chromatin structure of T helper cell subsets. The T-box-containing transcription factor T-bet is expressed in Th1 cells and not in Th2 cells and promotes Th1 differentiation. Conversely, the transcription factor GATA-3 plays a central role in Th2 cell development.

We are studying whether these "master" transcription factors act via activation of the genes that determine the phenotype of a specific Th subset or the silencing of the genes typical for the opposite subset in naïve CD4+ T cells, and in differentiated effector Th1 or Th2 cells. We have generated lentiviral constructs encoding the T helper lineage-specific transcription factors T-bet and GATA-3. We will perform a global analysis of gene expression profiles to study whether expression of the master regulators GATA-3 and T-bet is sufficient to completely reverse the gene expression pattern of Th1 to that of Th2 cells and vice versa. To analyze the molecular basis for the plasticity of the Th phenotype, we will analyze changes in the chromatin structure (DNAseI hypersensitive sites, DNA methylation and histone acetylation/methylation) at selected Th subset-specific loci. We envision that the proposed study will allow us to define the role of transcriptional changes and chromatin remodeling in determining the balance between plasticity and commitment during development of T cell-dependent immune responses.

Molecular profiling of CD4+ T cell populations during IL-2 therapy of HIV-infected individuals (Fabrice Letimier and Denise Martin)

HIV infection results in a severe loss of CD4+ T cell number and function, which is largely responsible for the pathological features of the disease and HIV-related mortality. Recent studies have established that intermittent interleukin-2 (IL-2) therapy in combination with highly active antiretroviral therapy (HAART) leads to a substantial increase of CD4+ T cell numbers in HIV-infected individuals. IL-2 immunotherapy induces a novel subset of CD4+ T cells expressing high levels of the IL-2 receptor alpha chain (CD25) in the peripheral blood of patients. This subset can account for up to 70% of the total CD4+ T cell pool of treated patients and persists for a long time after IL-2 treatment. Despite of the extensive use of IL-2 treatment in cancer therapy and numerous studies in HIV-infected individuals, a molecular characterization of CD4+ T cell populations during IL-2 therapy has never been performed.

The main goal of of this project is to determine the mode of action of IL-2 in HIV-infected individuals. We are analyzing the long-term effects of intermittent IL-2 therapy on CD4+ T cells in HIV-infected individuals and characterize at the molecular level the CD4+/CD25+ T cell subset that appears in patients treated with HAART and IL-2. We are generating a multi-parameter representation of CD4+ T cell populations before, during, and after IL-2 therapy of HIV-infected patients, integrating the analysis of their phenotypic and functional characteristics with the study of their gene expression profile using high-density oligonucleotide arrays. The project is part of a " Grand Programme Horizontale " at Institut Pasteur and is performed in collaboration with the team of Yves Levy at the Hôpital Henri Mondor and is a nested study of the ANRS 118-ILIADE phase II/III clinical trial aiming at the evaluation of the effects of IL-2 on the physiology and the survival of CD4+ T cells under optimal HAART regimen and after antiviral drugs withdrawal in HIV-infected individuals.

A novel regulator of ubiquitination processes: the COP9 signalosome (Elisabetta Bianchi, Simona Denti and Emmanuel Sechet)

Ubiquitin-mediated proteolysis plays an important role in many fundamental cellular processes, including cell cycle regulation, differentiation, modulation of growth factor receptors, and gene transcription. It is therefore not surprising that alterations in this pathway contribute to the pathogenesis of several diseases, among which several malignancies. The COP9 signalosome (CSN) complex has been recently identified as a novel upstream regulator of ubiquitination processes. Studies in vitro and in model organisms (yeast, plants) have indicated that the CSN may control the activity of the SCF complex, an ubiquitin ligase responsible for the degradation of many cell-cycle and transcriptional regulators (such as inhibitors of cell-cycle dependent kinases, p27Kip1 and p21, cyclins and the inhibitor of NF-kB, IkB). The CSN is conserved in mammalian cells: our laboratory and others have established a role for the human CSN in signal transduction and transcriptional control. However, the mechanisms and the targets of CSN activity in mammalian cells are still largely unknown.

We propose to study the biochemical and molecular functions of the human CSN and its role in regulating the ubiquitination processes that control cell proliferation and gene expression in normal and transformed cells. In Arabidopsis the CSN functionally interacts with the transcriptional repressor atCOP1. We have recently cloned the human COP1 orthologue and have demonstrated that huCOP1 is a novel ubiquitin-ligase that represses c-Jun transcriptional activity. We will further investigate the molecular basis of huCOP1 function and its relationship to the CSN in mammalian cells. The aim of our studies is to provide a comprehensive map of the cellular pathways that are controlled at the transcriptional level by the CSN and to broaden our comprehension of how ubiquitination processes are regulated.

Mediators of LFA-1 integrin signalling: RanBPM (Elisabetta Bianchi and Simona Denti)

Integrins adhesion receptors can act as signaling receptors that transmit information from the extracellular environment to the interior of the cell, affecting many fundamental cellular processes, such as cell motility, proliferation, differentiation and survival. The identification of molecules that interact with the cytoplasmic domain of integrins has been the focus of research aimed to elucidating the mechanistic basis of integrin signal transduction. We have identified RanBPM as a novel interactor of the beta-2 integrin LFA-1. We demonstrate that RanBPM is a phosphorylated peripheral membrane protein and that integrins and RanBPM interact in vitro and in vivo and co-localize at the cell membrane. Transfection of RanBPM synergizes with LFA-1-mediated adhesion in the transcriptional activation of an AP-1 dependent promoter, indicating that the two proteins interact functionally as well. We suggest that RanBPM may constitute a molecular scaffold that contributes to coupling LFA-1 and other integrins with intracellular signaling pathways.

Keywords: T helper cell differentiation, chromatin remodelling, epigenetic modifications, HIV, CD4+ T cell subsets, ubiquitination, transcription, COP9 signalosome, SCF complex

Activity Reports 2003 - Institut Pasteur

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