Role of cytokines in lymphopoiesis, in fetal liver and bone marrow: from multipotent progenitors to mature lymphocytes – lymphopoiesis

Regulation of lymphocyte development is required for efficient immune responses and is essential to prevent autoimmunity and leukemic transformation. Lymphoid production is done in fetal liver, thymus and in the bone marrow by the successive restriction in the differentiation potential of progenitor cells, with loss of self-renewing capacity. This process involves regulated changes in the transcription factor networks functioning within the differentiating progenitors. Gene expression profiles are influenced by signals delivered by surface receptors that interact with factors produced in the microenvironment. However, the degree to which differentiation is intrinsically programmed or regulated by external factors remains controversial. Our aim is to identify the influence of cytokines produced by the microenvironment in transcription factor regulation that determine lymphocyte commitment. The study of fetal liver microenvironment is particularly relevant as this is the only hematopoietic organ where hematopoietic stem cells (HSC) expand. By understanding the rules for lineage commitment we will get new information on the processes involved in maintenance of the undifferentiated state in HSC. The potential role of cytokines in regulating the levels of transcription factors required for lineage commitment is a novel paradigm in hematopoiesis, since they function as modulators of the differentiation potential of developing progenitor cells. Our project is concentrated on three axes: 1. Identification of the role of IL-7, Flk2/Flt3 ligand and TSLP in the generation of LMPP, CLP and committed lymphocytes in embryonic and adult lymphopoiesis. Mutant mouse strains for the three main cytokines involved in lymphoid development, IL-7, TSLP and Flt3 ligand are being intercrossed to obtain IL-7/ TSLP-/-, Flt3 ligand/IL-7-/- and TSLP/IL-7-/- mutants. These mutants lacking combinations of cytokines will allow the definition of the role of each at different stages of the differentiation pathway. We propose to identify and functionally characterize HSC, LMPP, CLP and pro-B cells in these mutants during adult life in the bone marrow and in fetal liver. These experiments will indicate the differential requirements for lymphocyte differentiation of cytokine signaling between adult and embryonic development and will indicate which progenitors are affected by the absence of one or combinations of lymphoid cytokines. The role of these different cytokines in T cell development will be studied by identification of the earliest thymic progenitors in these different mutants. We will do grafts under the kidney capsule of mutant and wild type thymic lobes in different combinations. These experiments will allow us to define the role of cytokines in regulating the compartment of thymic immigrants, in bone marrow or fetal liver or in thymic differentiation. 2. Modulation of transcription factor expression by cytokine signaling. The identification and isolation of lymphoid progenitors at different stages of differentiation that developed in the absence of one or more cytokine signals will allow to define the main modulator effects of cytokines in the transcription profile of lymphoid progenitors. We will thus isolate HSC, LMPP, CLP and B ell precursors from fetal liver and BM of wild type and single or double mutant mice. We will measure the levels of transcription factors that are essential for lymphocyte development. In a second approach we will use microarrays of transcription factors to define variations in progenitors of cytokine mutants. Variations in the levels of transcription factors will be correlated with differences in the potential to generate mature progeny and will be complemented by knock-down (si RNA) or over-expression in vitro. 3. The identification of the stromal compartment that is essential in the production of cytokines in fetal liver and bone marrow. We have obtained a mutant mouse strain that expresses GFP under the control of the regulatory sequences of Flk1. Flk1 is a VEGF receptor expressed in endothelial cell. We will use Flk1-GFP embryos, to isolate in the fetal liver the non-hematopoietic compartment and to distinguish between the endothelial and the stromal and hepatocyte progenitor compartments. We will do direct PCR analysis of freshly sorted cells that will be studied for the expression of mRNA for IL-7, TSLP and Flt3 ligand. Luminex technology will complement these experiments by allowing the characterization at the protein level of secreted cytokines. An important aspect of our study is the identification of the fetal liver compartment that regulates hemtopoiesis. The fetal liver is a unique site for HSC compartment increases in numbers. The identification and gene expression analysis of the cells that determine this process will be a required step on devising strategies to maintain and expand HSC, in vitro.