In vivo protection of activated tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate

Jennifer L. Gori, Brian C. Beard, Nathaniel P. Williams, Christina Ironside, Debra Swanson, R S Mc Ivor, Hans Peter Kiem

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)233-241
Number of pages9
JournalJournal of Gene Medicine
Volume15
Issue number6-7
DOIs
StatePublished - Jun 1 2013

Fingerprint

Tetrahydrofolate Dehydrogenase
Methotrexate
Canidae
T-Lymphocytes
Genes
Transplantation
Dogs
Green Fluorescent Proteins
Tissue Donors
Hematopoietic System
Severe Combined Immunodeficiency
Hemoglobinopathies
Lentivirus
Aplastic Anemia
Hematopoietic Stem Cell Transplantation
Graft Rejection
Immunosuppressive Agents
Immunosuppression
Transplants
Antigens

Keywords

  • Animal model
  • Chemotherapy
  • Drug resistance
  • Gene therapy
  • Transplantation
  • Viral vector

Cite this

In vivo protection of activated tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate. / Gori, Jennifer L.; Beard, Brian C.; Williams, Nathaniel P.; Ironside, Christina; Swanson, Debra; Mc Ivor, R S; Kiem, Hans Peter.

In: Journal of Gene Medicine, Vol. 15, No. 6-7, 01.06.2013, p. 233-241.

Research output: Contribution to journalArticle

Gori, Jennifer L. ; Beard, Brian C. ; Williams, Nathaniel P. ; Ironside, Christina ; Swanson, Debra ; Mc Ivor, R S ; Kiem, Hans Peter. / In vivo protection of activated tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate. In: Journal of Gene Medicine. 2013 ; Vol. 15, No. 6-7. pp. 233-241.
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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.",
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T1 - In vivo protection of activated tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate

AU - Gori, Jennifer L.

AU - Beard, Brian C.

AU - Williams, Nathaniel P.

AU - Ironside, Christina

AU - Swanson, Debra

AU - Mc Ivor, R S

AU - Kiem, Hans Peter

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N2 - 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.

AB - 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.

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KW - Transplantation

KW - Viral vector

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