The mechanisms controlling the initial aspects of thymocyte development (including rearrangement and expression of TCR genes), when pre-T cells first enter the thymus from the bone marrow or fetal liver (FL), are unknown. The broad, long-term objective of these studies is to define the mechanisms by which pre-T cell development is regulated. We found that interleukin-7 (IL7), a pre-B cell and thymocyte growth factor, stimulates growth of FL cells, expression of TCR genes, and expression of T cell- related antigens (Thy1 and PgP1) in the murine FL in vitro. Stem cell factor (SCF), a growth factor for myeloid and erythroid progenitor cells also stimulates FL cell growth and expression of cell surface TCR antigens.
The specific aims of this proposal are to 1) further examine the ability of IL7 and SCF to induce: expression and rearrangement of TCR genes, intracellular and cell surface expression of T cell antigens, and cell proliferation and to determine the relationship between gamma/delta cells induced by IL7 and gamma/delta cells in the fetal thymus and peripheral organs; 2) determine whether responsiveness to IL7 and SCF is mediated directly by these factors; 3) determine whether IL7 plays a role in development of T cells in utero. These studies will establish a system for further investigation of the molecular mechanisms which regulate pre-T cell development. The experiments described herein will utilize: assays of thymic repopulation to test if IL7 and SCF affect pre-T cell activity; in situ hybridization, immunohistochemistry, and autoradiography to determine if the cells which proliferate in response to IL7 or SCF are the same cells which express T cell genes and antigens; reverse transcription and polymerase chain reaction followed by sequencing, as well s Southern blot analysis to determine which gamma and delta variable regions are expressed in response to IL7 or SCF and whether these genes are rearranged. The importance of IL7 in pre-T cell development in the fetus will be evaluated by assessing the previously mentioned endpoints following in utero injection of anti-IL7 antibodies or fetal thymus organ culture with anti- IL7 antibodies.
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