The long term objective of this proposal is to determine the requirements for induction of T cell tolerance to transplanted hematopoietic stem, cells. Results of the experiments proposed here will have application in clinical medicine. The definition of the immunogenicity of purified allogeneic hematopoietic stem cells will guide in selecting the type and intensity of the immunosuppressive therapy required by recipients of purified stem cell transplants from normal donors and might suggest new strategy for induction of transplantation tolerance. Hematopoietic stem cell transplants may be useful for induction of specific tolerance to solid organ grafts.
Specific aims of this project are: 1) define the conditions required for induction of T cell unresponsiveness to allogeneic MHC molecules presented by hematopoietic stem cells. A human model recently developed in our laboratory will be used to study the induction of T cell hyporesponsiveness to HLA-DR antigens presented by an erythroleukemia cell line (HEL). Exposure of T cells to HEL-DR+ cells results in the induction of specific hyporesponsiveness to restimulation with a B lymphoblastoid cell line autologous with HEL (H-LCL). In contrast, T cells preincubated with a DR- variant of HEL(HEL-DR-) respond normally to H-LCL. This observation is consistent with the hypothesis that occupancy of the T cell antigen receptor in the absence of costimulatory signals is not sufficient for inducing T cell proliferation but instead results in a state of energy, detected when the T cell is rechallenged to specific antigen presented by a competent antigen presenting cell (APC). In order to define the costimulatory molecule(s) on the surface of the APC required for T cell sensitization, a cDNA library will be generated from H-LCL mRNA after subtraction from HEL mRNA. Then, cDNA clones will be transfected into HEL-DR+ cells and the transfectants will be assayed for antigen presenting function.
Specific aim 2) is to define the antigen presenting function of purified normal hematopoietic stem cells. Human precursors of colony-forming cells will be isolated from bone marrow by selection of CD34+ cells by avidin-biotin immunoabsorption and fluorescent cell sorting using a CD34-specific antibody. Antigen presenting function will be tested in primary and secondary T cell proliferation and cytolytic T cell precursor assays and compared to the function of unseparated mononuclear cells from peripheral blood or marrow. Susceptibility of purified hematopoietic precursors to lysis by specific CTL effectors will be tested in colony- forming assays.
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