PROJECT 2: IDENTIFICATION OFCANINE MINOR HISTOCOMPATIBILITY ANTIGENS Project 1 has developed an approach at DLA-identical canine hematopoietic cell transplantation (HCT) that results in stable mixed donor-host chimerism. Persistent host hematopoiesis can serve as an experimental model of persistent hematologic malignancy seen in some patients transplanted under Projects 3 and 4. Conversion of mixed to all-donor chimerism can be achieved with injection of donor lymphocytes that have been sensitized to host minor histocompatibility antigens expressed on peripheral blood mononuclear cells (PBMC), however at the price of often fatal graft-vs.-host disease (GVHD). T-cell responses directed against ubiquitously expressed minor antigens are thought to be responsible for GVHD, while T-cell responses against a combination of ubiquitous and hematopoietic-specific minor antigens contribute to elimination of residual host hematopoietic cells in a manner analogous to the graft-vs.-leukemia effect observed in human patients. The identification of minor antigens restricted to hematopoietic cells therefore holds great promise for improving allogeneic HCT outcomes. That knowledge would facilitate the development of sensitization strategies that target host hematopoietic cells while sparing GVHD target tissues. However, while the dog model of allogeneic HCT is optimal for preclinical development of novel HCT therapies, no canine minor, histocompatibility antigens have been described to date, and existing methodologies for minor antigen identification are inefficient. To address this problem, we propose a novel approach to minor antigen discovery in the dog. This approach utilizes next generation sequencing technology to define protein coding variations unique to the recipient and expressed in PBMC which will be used for sensitizing the HCT donor. Sensitized donor T-cells will then be injected into the respective recipients with the aim of converting mixed to full-donor chimerism and causing GVHD. After conversion has been accomplished, T-cells will be harvested from recipients and tested for responses against candidate minor antigens using a novel, high- throughput T-cell assay. Positive responses would define genuine minor histocompatibility antigens. Using qRT-PCR, we will then identify those minor antigens that are highly expressed in hematopoietic cells but not in GVHD target tissues. Next, relevant minor antigen peptides will be used to sensitize donor T-cells with the aim of converting mixed to full-donor chimerism without GVHD (Project 1). Eventually, this concept will be tested in a canine model of acute leukemia in Project 1. Benefits to Public Health: Taken together, the studies proposed in this Project and the in vivo studies proposed in Project 1 have the potential of developing new and effective approaches benefiting patients with persisting/relapsing malignancies treated by allogeneic HCT under Projects 3 and 4.
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