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.
The dorsal aorta develops from the intra-embryonic mesoderm, called the Splanchnopleura (Sp), the anlage of the mesonephros, mesentery, and gonads develop in this site after the 10th day of gestation and this region is then called AGM (Aorta, Gonads, Mesonephros).
We have previously established that the HSC are generated from hematopoietic precursors in the intra-embryonic mesoderm are the only ones capable of long-term reconstitution of the hematopoietic compartment of adult NK-deficient hosts. They can thus be called HSC. The yolk sac-derived (YS) precursors are unable of long-term engraftment of recipient mice. We identified surface markers that allowed the isolation of a population of multipotent hematopoietic precursors, in the AGM, represented at a frequency of one in three cells. 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. This population is already at this stage strictly engaged in hematiopoietic fate and is unable to give rise to neural precursors. We identified a pipulation of primitive macrophages in the YS and AGM and we are actively pursuing the characterization of the origin and function 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.
Ontogeny and physiology of CD4+ regulatory T cells. A. Bandeira
Regulatory T cells belonging to the natural pool of CD25+CD4+ T cells play a fundamental role in the maintenance of peripheral tolerance to self-antigens and in the regulation of adaptive and innate immune responses against pathogens. Due to their capacity of efficiently inhibit T cell proliferation, these cells are a key element in the systemic homeostatic mechanisms that control total lymphocyte numbers. It has recently been demonstrated that the transcription factor Scurfin, encoded by the forkhead/winged helix (Foxp3) gene, is a specific marker for regulatory activity and is essential for the development and function of regulatory CD25+CD4+ T cells.
At least part of the regulatory CD25+CD4+ T cells are produced in the thymus. However, it has been proposed that they are only significantly exported to the periphery after the third day of post-natal life. This hypothesis is based on the observation that mice thymectomized between days 2 to 4 after birth, develop organ-specific autoimmune disorders, due to the lack/paucity of regulatory CD25+CD4+ T cells.
Our recent studies show that the spleen of day 3 old newborn BALB/c mice, a strain sensitive to thymectomy-induced disease, is already endowed of a remarkable regulatory potential, with a well-represented CD25+CD4+ T cell population, expressing high levels of Foxp3 mRNA This regulatory potential develops considerably in the absence of thymic supply. Indeed, adult day 3-thymectomized mice contain an over-represented Foxp3-expressing CD25+CD4+ T cell compartment, independently of the development of autoimmune gastritis. These cells are able to control inflammatory bowel disease and CD4+ T cell expansions in vivo.
Altogether, our studies demonstrate that the genesis of peripheral regulatory and naïve CD4+ T cells is not differentially controlled. They explain why the incidence/severity of autoimmune diseases following day 3 neonatal thymectomy vary among animals of the same strain and why these animals do not develop some types of inflammatory diseases such as inflammatory bowel disease (IBD), and keep a long lasting state of lymphopenia.
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. On the other hand, different TCRγ chains compete unequally for the same TCRδ chain or for other components of the TCR-CD3 complex with Vγ1 chains dominating over Vγ4 chain and both dominating over Vγ2 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, gamma delta T cells, regulatory T cells, grafts