Our laboratory has demonstrated that most CD4-CD8- alpha-beta (double negative; DN) T cells generated in the mouse bone marrow are extrathymically derived and differ from the predominant DN T cells in the blood and spleen which are generated in the thymus. The proposed study is designed to further elucidate the differences in the marrow and thymus derived DN T cells including the patterns of surface markers expression, cytokine secretion, requirements for positive selection and function. A critical function of the marrow derived DN T cells is to suppress immune injury mediated by CD4+ and CD8+ (single positive; SP) T cells in models such as graft versus host disease (GVHD) and systemic autoimmune disease (murine lupus). After G-CSF administration the marrow DN T cells are mobilized into the blood along with hematopoietic progenitors. Although the marrow DN T cells (predominantly NK 1.1-) have little cytolytic activity, they can be activated in vitro to generate NK1.1+ T cells which kill tumor cells. A similar maturation occurs in human cells. In order to understand their biology, purified subsets of DN T cells from the normal mouse marrow, spleen, blood and G-CSF mobilized blood will be obtained by immunofluorescent staining and flow cytometry. These purified DN T cells will be studied for the expression of other T cell receptors by staining with monoclonal antibodies, for the secretion of cytokines after in vitro activation, and for their dependence on the beta2m molecule for positive selection. DN T cells will be tested for suppression of GVHD after allogeneic bone marrow transplantation, and for the suppression of systemic lupus induced by autoreactive SP transgenic T cells. The capacity of NK1.1- DN T cells to generate NK1.1+ T cells, which can mediate graft versus leukemia activity against the BCL1 tumor after marrow transplantation will be studied as well. Finally, we will determine the differences in surface markers, cytokine secretion and regulatory functions of DN T cells in human blood before and after administration of G-CSF. The mobilized DN T cells may have therapeutic application to treat GVHD, tumor relapse, and autoimmunity.
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