Larsson, Eva Lena Birgitta; (1991) An "in vitro" analysis of murine thymocyte-stromal cell interactions. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis is directed towards two separate but closely related goals; one involves the biology of precursor cells in T-cell development and the second involves the use of retroviral constructs for the establishment of thymic stromal cell lines and its application for studying differentiation of T-cells in vitro. Using the mouse foetal thymic organ culture system, a quantitative assay was devised in which the relative abundance of T-cell progenitors among different populations was estimated by seeding decreasing numbers of precursors into alymphoid thymic rudiments. This limiting dilution approach combined with serial transfer of successfully recolonised precursor cells in organ culture, provided evidence that certain populations seeding the thymus have the potential for extensive cell divisions (up to 10- 12 weeks). Stromal cells from adult and foetal sources were transformed by a temperature sensitive (ts) mutant of SV40 and Ela 12S in order to study interactions between the developing thymocytes and specific elements of the microenvironment. Eighty four cell lines were generated and have been shown to contain properties related to their counterparts in vivo. The thermolabile transforming agent (large T) allowed inactivation of the immortalising gene when the cells were switched to the nonpermissive temperature. Properties like the expression of MHC antigens and the ability to bind thymocytes could be induced by growing ts-derived clones at the nonpermissive temperature, which suggests that although cell proliferation had ceased, the cells were still metabolically active and had reverted to a more 'normal' non-transformed phenotype allowing certain molecules to be expressed at the cell surface. Close interaction of the developing thymocytes with the stroma is essential for the development of functionally mature T-cells. Using a rosette assay unfractionated adult thymocytes were found to bind unstimulated Ela-derived clones resembling epithelial cells (15.5 and 15.18) and also to several other established clones after temperature switching and/or IFNγ treatment. In co-culture with a multipotent bone-marrow derived stem cell clone (A4) it was shown that both proliferation and differentiation into certain myeloid lineages were supported. Furthermore, in co-culture with CD4'CD8' thymocytes, with and without interleukin-7, the growth of this subset could be sustained for ~20 days. In addition, phenotypic changes of thymocytes in these cultures suggest that some of these lines may have the potential to induce differentiation of early precursors. These findings indicate that established cell lines could be useful tools for studying maturation of T-cells in vitro and furthermore, to investigate distinct events in T-cell ontogeny governed by different stromal cell types.

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