Unit: Lymphocyte Development - Inserm U668

Director: CUMANO Ana

The work carried out by the Unit for Lymphocyte Development aims at understanding the mechanisms involved in the development of the Immune System: 1. The establishment of the hematopoietic system, during embryogenesis; 2. The physiology and development of the γT lymphocytes; 3. The ontogeny, repertoire and physiology of regulatory T cells, involved in the homeostasis of the Immune System

Establishment of the hematopoietic system, in the mouse embryo. (A. Cumano).

Hematopoietic stem cells (HSC) are important in hematopoiesis throughout life. The HSC or their immediate precursors are not generated in the hematopoietic organs but have an exogenous origin. We established that the HSC are generated, between day 9 and 12 of gestation, in the mouse, from precursors in the intraembryonic mesoderm, in a region called splanchnopleura (Sp) and later in the AGM (Aorta, Gonads, Mesonephros) in the dorsal aorta. We identified surface markers in HSC which allowed us to isolate them to virtual homogeneity. We analyzed the expression of genes involved in the hematopoietic development, in these cells. Immunocytochemistry analysis allowed postulating that the HSC are generated in the sub-aortic mesenchyme in novel structures called the sub-aortic patches. In this region of the intra-embryonic mesoderm there are other progenitors that might play a role in the generation and/or survival of the HSC. We are currently identifying the different cell types in this region and their relationship with the emerging HSC.

Development of B lymphocytes in the IL-7-/- mouse. (P. Vieira and A. Cumano).

In the absence of interleukin 7 (IL-7), the number of T and B lymphocytes in the peripheral lymphoid organs is 10 fold reduced. Our work showed that B lymphopoiesis is abrogated in the bone marrow of IL-7-/- mice, after seven weeks of age, demonstrating the essential role of this cytokine in B cell production. The effect of IL-7 is already detectable in Common lymphoid progenitors (CLP) where it is necessary for the maintenance of their B cell differentiation potential by modulating the expression of EBF (Early B cell factor), a transcription factor essential for B lineage commitment. We identified "Thymic stromal derived lymphopoietin" (TSLP) as the cytokine responsible for the fetal B cell lymphopoiesis, in IL-7-/- mice. We further showed that TSLP has also a role in the expansion of B lymphocytes after pre-BCR expression in the bone marrow.

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 TCRg 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 Jg segments.

Ontogeny and repertoires of natural regulatory FOXP3+ CD25+ CD4 T cells. (A.Bandeira).

Natural regulatory CD25+CD4+ T cells are essential in the maintenance of peripheral tolerance to self-antigens, and in the regulation of adaptive and innate immune responses against pathogens. We showed that these cells are a key element in the systemic homeostatic regulation of peripheral lymphocyte numbers.

It has been proposed that regulatory CD25+ CD4 T cells are only significantly exported from the thymus after the 4th day of post-natal life. We demonstrated that the peripheral immune system of day-3 old newborns is already endowed of a remarkable regulatory potential, with a well-represented compartment of Foxp3-expressing CD25+CD4 T cells. This potential can fully develop in the absence of further thymic supply, with an over-representation of the Foxp3-expressing CD25+CD4 T cell compartment in the adult. These cells were as efficient as their equivalents of normal mice in preventing IBD and in controlling homeostatic expansions of CD4 T cells. These results were now confirmed by the analysis of FOXP3 at the single cell level. Our studies indicate that there is no differential developmental program controlling the generation of regulatory CD25+ and naïve CD4 T cells. Altogether, they explain the variable incidence/severity of autoimmune following neonatal thymectomy, compatible with a very good survival of these animals, a phenotype that sharply contrasts with that of Foxp3 ko mice.

We also show the TcRαβ repertoire of the natural CD25high CD4 T cells is proportionally as high as that of naïve CD4 T cells, but 10-fold smaller due to the difference in size between the two compartments. Moreover, the two repertoires are virtually non-overlapping. Our studies demonstrate the very high potential of ligand recognition of the natural regulatory cells and establish qualitative and quantitative differences with the naïve repertoire that impact on the understanding of the development of both cell types and on the regulation of immune responses.

Keywords: hematopoiesis, lymphocyte development, gamma delta T cells, regulatory T cells, grafts

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