G-CSF mobilized peripheral blood mononuclear cell products (G- PBMC), specifically CD14+ monocytes and CD4+ T cells, differ in function from comparable populations isolated either from aspirated marrow or from normal blood. Studies indicate that the CD14 cells in this population have reduced expression of HLA-DR as well as B7.2, a co-stimulatory molecule needed for T-cell activation. In addition, the CD14+ cells secrete significantly increased amounts of interleukin-10 (IL-10) which inhibit T-cell proliferation and gamma-interferon production. The ration of CD14+ cells to CD4+ cells in G-PBMC is increased 5-fold. Studies indicate that this increased population of CD14+ cells reduces the ability of CD4 cells to utilize the CD28 signaling pathway and respond to alloantigen. Based on these observations we hypothesize that the monocytes contained within the G-PBMC may provide immunosuppressive effects that can inhibit both graft-versus-host and host-versus- graft reactions, thereby facilitating alloengraftment. To test this hypothesis we have used an established canine transplant model involving DLA-identical donor/recipient pairs in which the endpoint of transplantation, stable mixed hematopoietic chimerism, is obtained with non-myeloablative condition regimen. Currently this is achieved using 200 cGy of total body irradiation (TBI) plus 35 days of cyclosporine and mycophenolate mofetil (MMF). However, a further decrease in the irradiation dose or the elimination of MMF results in donor graft failure. This is a unambiguous result easily documented by VNTR analysis. Using this model we have determined that unfractionated G-PBMC can replace the requirement of MMF for establishing long-term stable mixed chimerism. Importantly, the donor chimerism achieved in the marrow is greater than 70% and there was no evidence of graft-versus-host disease. Studies proposed in this application will test the hypothesis that monocytes are responsible for this graft facilitating effect. The effective G-PBMC population will be identified by selective depletion of the product prior to its use in the aforementioned transplantation model. Once this is accomplished, the G-PBMC product will be optimized and used to replace TBI. Our final goal is to use the G-PBMC product to establish a non-toxic conditioning regimen to achieve stable mixed chimerism in haploidentical donor/recipient pairs.
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