Unit: Lymphocyte Development - CNRS URA 1961

Director: CUMANO Ana

In the Unité du Développement des Lymphocytes we study the major events implicated in the establishment of a functional immune system: 1.Establishment of the hematopoietic system, during embryonic development; 2. Development and physiology of a sub-population of T cells, the γ T cells; 3.Ontogeny, repertoire analysis and physiology of regulatory T cells, involved in the homeostatic regulation of the immune system.

Generation of hematopoietic stem cells, in the mouse embryo. A. Cumano.

Hematopoietic stem cells (HSC) guaranty the production of blood cells throughout life. HSC are not generated in the hematopoietic organs but are produced exogenously.

We have previously established that the HSC are generated from mesodermal precursors present in the dorsal aorta and are the only ones capable of long-term reconstitution of the hematopoietic compartment of adult NK-deficient hosts. This region of the intra-embryonic mesoderm is called the Splanchnopleura (Sp) or AGM (Aorta, Gonads, Mesonephros) after the 10th day of gestation also contains progenitors for other cell types such as stroma and muscle.

We identified surface markers that allowed the isolation of a population of multipotent hematopoietic precursors, in the AGM. We sorted these precursors and analyzed the pattern of expression of genes involved in hematopoietic differentiation. This analysis allowed us to postulate that HSC are generated in the sub-aortic mesenchymal patches. We pursue the identification of different cell types that co-exist in this region to establish a lineage relationship between them and the HSC and to get insights in the generation process of these cells.

B cell development in the absence of interleukine 7. P. Vieira and A. Cumano.

In the absence of interleukin 7 (IL-7), the numbers of T and B lymphocytes are ten fold reduced in the peripheral lymphoid organs. Our analysis showed that B lymphopoiesis is completely abrogated in the bone marrow of adult IL-7-/- mice starting at the 7th week of age, illustrating the essential role of IL-7 in bone marrow lymphopoiesis. We showed that the majority of B lymphocytes in these mice were of fetal or neonatal origin. We established that this production is strictly dependent on the "Thymic stromal lymphopoietin" (TSLP).

TLR4 gene expression during B cell ontogeny. P. Vieira.

We showed that TLR4, a gene product involved in the innate response to bacterial products such as LPS, is monoallelic expressed in B cells from the stage of pre-B cells, and in bone marrow granulocytes. The pattern of expression resembles the inactivation of the X chromosome. We are presently studyng the role of TLR receptors on the surface of B cells. We further showed that TSLP as an additional role of expanding B cell precursors after expression of the pre-BCR, in the bone marrow.

Allelic and isotypic exclusion of TCRγ genes. Laurent Boucontet, Pablo Pereira

Most lymphocytes express only one antigen receptor at cell surface. This phenomenon, known as allelic exclusion, results from different mechanisms depending of the locus involved. We have developed an experimental system allowing the analyses of TCRγ and TCRδ gene rearrangements in progenies of γ T cell with high efficiency and analyzed close to 250 γ thymocyte clones selected by their expression of different TCRγ chains at the cell surface. Several important conclusions emerge from these analyses. First, our results suggest a hierarchy in the probabilities at which different Jγ and Vγ gene segments are engaged in a recombination process. Second, although an important fraction of γT cells contain two or more functionally rearranged TCRγ chains, only one of these TCRγ chains is expressed at detectable levels at the cell surface. This is the result of at least two different phenomena. On one hand, several TCRγ chains only pair with a restricted number of TCRδ chains. Third, less than 1 % of mature γ thymocytes contain both alleles of the same TCRγ chain functionally rearranged, suggesting that both alleles are not open to recombinase action at the same time. Fourth, precursors of γT cells do not attempt to rearrange all Jγ segments.

Keywords: hematopoiesis, lymphocyte development, ?? T cells, regulatory T cells, grafts

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