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 Engineering a Thymus for Efficient Production and Study of Murine T Cell Development in a Thymic Organoid

Deborah J. Marshall, James Bagley, Michael Rosenzweig
Immunotherapy, Cytomatrix, Woburn, MA, 01801

Fetal thymic organ cultures (FTOC) have been used extensively as the sole source to study thymopoiesis ex vivo. The manufacture of T lymphocytes in vitro has proven problematic in part because of the obligate requirement that T cell generation is dependent on three-dimensional (3-D) interactions with thymic stromal cells. We have developed a novel artificial thymus culture system comprised of a stroma covered, three-dimensional, bio-compatible Cytomatrix™ cell growth matrix. We have previously demonstrated that the Cytomatrix™ provided the obligate three dimensionality required for T cell differentiation; sustained the long-term growth of both murine thymic stromal cells and human hematopoietic stem cells (HSC); and fostered the natural differentiation of T cells in the absence of exogenous factors (i.e. cytokines or serum). We are using this ex vivo culture system to generate T cells from hematopoietic progenitor cells isolated from murine thymus (Sca+Lin-) to induce the differentiation of HSC to functional T cells as a tool to understand the steps involved in thymopoiesis and central tolerance. Briefly, murine thymus was cultured in the Cytomatrix for 2 weeks. Sca+Lin- cells isolated from 4-6 week old murine thymocytes were then co-cultured on the stroma in the absence or presence of cytokines for an additional two weeks. FACS analysis demonstrated the development of CD4+CD8+ double positive as well as CD4+ and CD8+ single positive T cells, the majority of which were CD45RA+ (naive) and TCRab+. In addition, we have been able to demonstrate the maintenance of negative selection events to a superantigen (SEB) and the maintenance of positive and negative selection events to peptides using transgenic mouse models and peptide antigens within this microenvironment.