T CELL TOLERANCE TO ALLOGENIC HEMATOPOIETIC STEM CELLS

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The long term objective of this proposal is to determine the requirements for induction of peripheral T cell tolerance to allogeneic hematopoietic stem cells. Definition of the immunogenicity of purified hematopoietic stem cells may suggest new approaches to achieve specific immunological tolerance. Results of these studies may guide how to best use hematopoietic stem cell transplants as a means for inducing specific tolerance to solid organ grafts. Specific aim 1: Define the antigen presenting function of human marrow CD34+ hematopoietic progenitor cells. CD34+ cells from normal human marrow are almost entirely HLA-DR+, B7- 1/B7-2 negative. CD34+/CD18lo cells are small and agranular, have LTC-IC activity and do not stimulate proliferative response of allogeneic T cells. In contrast, CD34+/CD18+ cells are large and somewhat more granular, have minimal LTC-IC activity and stimulate allogeneic T cells. Experiments will determine whether expression of CD18, the beta2 leukointegrin, and one of the associated alpha chains (CD11a, b, or c) is a prerequisite for antigen presenting function. Aim 2. Define the antigen presenting function of peripheral blood CD34+ hematopoietic progenitor cells mobilized by stimulation with hematopoietic growth factors. PBMC from G-CSF-treated donors are less efficient in stimulating allogeneic T cells than PBMC from unstimulated donors. Purified CD34+ cells from blood of G-CSF-treated donors can stimulate allogeneic T cells. The hypothesis to be tested is that G-CSF treatment of normal donors may decrease activity of classical APCs or induce a functional inhibitor(s). B cell, monocytes and dendritic cells will be tested individually in bulk and LDA, and inhibitory activity will be ruled out by mixing experiments. Aim 3. Define the costimulatory molecules required for T cell response to allogeneic MHC molecules presented by an erythroleukemia cell line HEL. Lack of adequate expression of the appropriate adhesion or costimulatory molecules could be the reason why an HLA-DR+/CD11alo/LFA-3+/ICAM-1+ erythroleukemia cell line (HEL-DR+ does not stimulate allogeneic T lymphocytes to proliferate. Resting CD4+ purified from adult blood were activated by the HEL-DR+/B7+ line to express CD69 and IL-2 receptor alpha. Native CD4+ T cells purified from umbilical cord blood, however, did not proliferate unless autologous APC was added to the culture, indicating that other adhesion receptors or costimulatory molecules from a fully competent APC were required for activation. We will test whether antigen presenting function of HEL-DR+ cells will be reconstituted by transfection of cDNA encoding putative costimulatory molecules including CD11a, b, or c.