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PDF Version      Lymphocyte Population Biology - URA CNRS 1961


  Director : FREITAS Antonio (afreitas@pasteur.fr)


  abstract

 

The main research objectives of the " Unité de Biologie des Populations Lymphocytaires " are :

a) To study the homeostatic mechanisms that control the number of B and T lymphocytes.

b) To study lymphocyte population dynamics. To quantify the rates of cell production and death as well as the rates of cell renewal. To study the mechanisms of lymphocyte survival.

To study the role of lymphocyte competition in the selection and control of the immune responses. To study the mechanisms implicated in immune tolerance. To study the mechanisms of immune memory induction and persistence.



  report

cale

The role of Treg cells in the control of CD4 T cell homeostasis(Afonso Almeida).

We have continued to study the characteristics and relevance of the regulatory CD4+CD25+ T cell subpopulation which control peripheral T cell homeostasis. After establishing that these cells require IL2 in the peripheral pools for survival and/or function, we have investigated the possible sources of the IL2 used by the CD4+CD25+ T cells. We have also investigated the homing proprieties of the CD25- and CD25+ CD4+ T cell subpopulations, as well as the steady-state kinetics of the populations originated after transfer into T cell deficient hosts. We have extended our studies to the effects of CD4+CD25+ cells in the expansion of CD8+ T cells or CD8+ T cell subsets. We have also investigated the mechanisms responsible for the CD4+CD25+ regulatory T cell action. Some of the results and conclusions mentioned were published recently (JI).

Role of the IL- 7/ IL-7 receptor axis on T cell homeostasis (Jaime Franco).

The size of the peripheral T cell pool is set by homeostatic mechanisms not yet fully understood. Interleukin (IL)-7 has been involved in the generation and survival of T cells, and probably is also determining the population size. However, the quantitative relation between IL-7 level and T cell number is unknown. We will quantify the availability of IL-7 in different chimeric mice, and assess its effects on T cell homeostasis. As an alternative approach, we will reconstitute mice with bone marrow (BM) mixtures containing variable proportions of competent and knockout cells. We will rescue irradiated mice with dosed mixtures of IL-7+/IL-7-/- BM cells to grade the body IL-7 production. This will allow us to quantify the IL-7 required in T cell survival in the peripheral pools. We will also generate CD4+ T cells producing alternatively IL-7 or soluble IL-7 receptors (sIL-7R), by infecting mature T cells or BM stem cells with modified retroviral vectors. These cells will be then studied to know whether they can act as regulatory T cells by altering the local available levels of IL-7.

Kinetics and Homeostasis of Th1 and Th2 Cell Populations (Vanesa Guajardo).

We investigate the kinetics, the survival requirements, the competitive capacity and the mutual cell interactions of Th1 and Th2 (CD4+ T cells) populations in the absence or presence of antigenic stimulation. We aim to elucidate the mechanisms which regulate the final outcome of a T cell response following a given infection. We use Stat4-/- and Stat6-/- deficient mice, on a Balb/c genetic background, as precursors for the Th1 and Th2 T cells. The Stat4 and Stat6 genes are involved in phosphorylation cascades as a result of specific cytokine stimulation on CD4+ T cells. In the absence of Stat4 the response to IL-12 is impaired and T cells tend to a Th2 development. In contrast, the absence of Stat6 leads to a propensity towards Th1, as it blocks T cells response to IL-4 stimulation.

Competition and survival among CD4 memory T cells (Sylvie Garcia).

The survival properties of CD4 memory T cells are one of the parameters that control the composition of the memory compartment. Two aspects of these mechanisms will be explored. The competition between different memory subsets will be analyzed by co-transferring several types of TCR transgenic memory CD4 T cells into adoptive immune-deficient hosts. The capacities of newly generated CD4 T cell memory subpopulation to compete with preexistent CD4 memory pool will be assessed by sequential transfers of different TCR transgenic memory and naïve CD4 T cells into adoptive immune-deficient hosts. In both cases, the fate of each memory subsets will be studied regarding to the specificity, function (Th1 vs Th2 memory) and age of memory cells. These studies should help to understand the rules that govern the generation and maintenance of the memory CD4 T cell pool during acute (i.e. vaccination) or chronic (i.e. HIV infection) antigenic stimulations.

CD8 T cell survival (Nicolas Legrand).

In order to test the influence of MHC class I presence or absence on the survival and lymphopenia-driven proliferation of CD8+ T cells, we have developed a new adoptive transfer system of T lymphocytes into hosts with different MHC class I expression patterns. Donors T cells, isolated from mouse strains transgenic for the TCR in a RAG-deficient genetic background (MoaHY, MoP14 and MoOT-1 mice), were separately transferred into different types of recipient animals: T-cell deficient mice intact (CD3-/-), partially deficient (CD3-/-xH-2Db-/- and CD3-/-xH-2Kb-/-) or totally deficient for the expression of MHC class I molecules ("TetraKO" mice: CD3-/-x2m-/-xH-2Db-/-xH-2Kb-/-). After transfer into MHC class I molecules intact hosts, T cells show a maintenance without division (MoaHY) or strong lymphopenia-driven proliferation capacity (MoP14, MoOT-1), depending on the hierarchy of TCR cross-reactivity (MoOT-1>MoP14>MoaHY). After transfer into H-2Db-deficient mice, MoaHY and MoP14 T cell populations, both H-2Db-restricted, dramatically disappear in less than 2 days. In the case of the highly cross-reactive OT-1 TCR expressing lymphocytes (H-2Kb-restricted), transfer into H-2Kb-deficient mice interestingly shows accumulation of highly proliferating T cells. The usage of "TetraKO" mice should allow us to precise the controversial role of MHC class I molecules in the survival of memory CD8+ T cells.

B cell homeostasis and selection (Emmanuelle Gaudin).

We studied the development of hen egg lysozyme (HEL)-specific B cells in presence of varying amounts of HEL neo self-antigen. We found that both the quantity of antigen and the avidity of the B cell determine the fate of the self-reactive B cells. High doses of antigen delete or functionally impair "high and low avidity" B cells. Low quantities of antigen, allow "high avidity" B cells to escape negative selection and positively select "low avidity" self-reactive B cells that express few HEL-specific BCRs. A fraction of these B cells is activated and secretes self-reactive IgMs, which form immune complexes in the serum with circulating self-antigen. These results suggest that natural serum antibodies may be the result of the selection and activation of low avidity self-reactive B cells by self-antigen.

Notch signaling in lymphocyte development and survival (Alix de la Coste).

Using a system of retroviral infection we have recently developed, in collaboration with Yacine Laabi, UDL, Pasteur, a new strategy for the over-expression of the Delta1 and Jagged2 Notch ligands. We have reconstituted lethally irradiated Rag-/- or B6 mice with foetal liver cells infected with the control vector or the vector expressing the Delta like 1 ligand. We have studied the reconstituted mice at 3 or 7 weeks after FL transfer. Mice reconstituted with FL cells expressing Delta 1 showed significant changes in lymphocyte development. In the BM we observed a strong inhibition of B cells differentiation accompanied by a marked infiltration by DP CD4CD8 cells. Most peripheral lymphoid organs showed similar infiltration by DP CD4CD8 T cells. We will now attempt to characterize in detail all modification of lymphoid development induced by different Notch ligands.

Independent Attached Group (Paolo Truffa-Bachi).

Characterization of two genes expressed by T-cells activated in presence of cyclosporin A (Paolo Truffa-Bachi, Laurent Mascarell, Jean Kanellopoulos, Rodolphe Auger, Iris Motta, Andrés Alcover, Josyane Ragimbeaud & Véronique Cadet-Daniel)

We have cloned the cDNA encoded by 2 genes of unknown functions. These two genes are overexpressed in T lymphocytes activated by Concanavalin-A or the specific peptide in presence of immunosuppressors such as cyclosporin (CSA) or FK506 . The first encodes two proteins sharing the same C-terminal and different N-terminal regions. These proteins are located in the mitochondria and are probably implicated in cell apoptosis. The second gene induced by the immunosuppressors encodes for the synaptotagmine-2 like protein. CSA provokes new alternative spliced mRNA molecules. One of these mRNA is made by the assembly of the nucleotide sequences of two contiguous exons and of the corresponding intron (1545 nucleotides). This mRNA shares with the synaptotagmin the C-terminal sequence but differs in the N-terminal region. These data strongly argue that CSA and FK506 control the spliceosome machinery using different splicing sites, an hypothesis reinforced by the recent finding that cyclophilin, a receptor of CSA is a member of the spliceosome.

Photo: Mitochondrial localization of the T cell activated and cyclosporin A-induced protein. Human fibroblasts were transfected with the cDNA encoding this protein fused to GFP. Left panel: cells were labelled with mitotracker only. Middle panel:  localization of the GFP-fusion protein. Right panel: colocalization of the two fluorochromes

Keywords: B cells, T cells, lymphocyte homeostasis, lymphocyte survival, immunological memory



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  personnel

  Office staff Researchers Scientific trainees Other personnel
  VOUGNY Marie-Christine (mcvougny@pasteur.fr) FREITAS Antonio IP (Pr,afreitas@pasteur.fr)

GARCIA Sylvie, IP (CR,sygarcia@pasteur.fr)

AGENES Fabien, Inserm (CR2,fagenes@pasteur.fr)

TRUFFA-BACHI Paolo, CNRS (DR2,ptbachi@pasteur.fr)

DE LA COSTE Alix, post-doc

BORGHANS Josephina, post-doc

MUNOZ FRANCO Jaime, post-doc

ALMEIDA Afonso, PhD student

LEGRAND Nicolas, PhD student

GAUDIN Emmanuelle, PhD student

SANCHEZ GUAJARDO Vanesa, PhD student

MASCAREL Laurent, PhD student

MAILHE-LEMBEZAT Marie-Pierre (tech sup labo,mpmailhe@pasteur.fr)

CADET-DANIEL Véronique (tech sup labo,vcadet@pasteur.fr)

VOUGNY Marie-Christine (Sec direction,mcvougny@pasteur.fr)

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