In vivo protection of activated Tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate

Abstract

Background: Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy for decreasing rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX-resistant dihydrofolate reductase (Tyr22-DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment.

Methods: To evaluate MTX resistance of Tyr22-DHFR(+) T lymphocytes in vivo, we transplanted dogs with autologous CD34(+) cells modified with yellow fluorescent protein (YFP) and DHFR-green fluorescent protein (GFP) lentivirus vectors. Dogs were then treated with a standard MTX regimen days 1, 3, 6 and 11) following immune activation with a foreign antigen as a surrogate assay to mimic early transplantation.

Results: DHFR-GFP(+) gene marking was maintained in CD3(+) CD25(+) and CD4(+) T lymphocytes after MTX treatment, whereas the level of T lymphocytes that expressed only a fluorescent reporter (YFP(+) ) decreased. These data show that Tyr22-DHFR expression protects T lymphocytes from MTX toxicity in dogs, highlighting a clinically relevant application for preserving donor T lymphocytes during post-transplantation immunosuppression.

Conclusions: The findings of the present study have implications for the clinical translation of MTX-resistant T cells to facilitate engraftment of allogeneic cells following nonmyeloablative conditioning and to minimize the risk of rejection. In summary, Tyr22-DHFR expression in T lymphocytes provides chemoprotection from MTX-mediated elimination in the context of immune activation in vivo.

Keywords: animal model; chemotherapy; drug resistance; gene therapy; transplantation; viral vector.