Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?
Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus-leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV-induced T-cell depletion in allogeneic BMT recipients.
Tiberghien
P
P
Biological Carcinogenesis Development Program, Program Resources, Inc, Frederick, MD.
Reynolds
C W
CW
Keller
J
J
Spence
S
S
Deschaseaux
M
M
Certoux
J M
JM
Contassot
E
E
Murphy
W J
WJ
Lyons
R
R
Chiang
Y
Y
D001853:Bone Marrow / Q000276:immunology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015774:Ganciclovir / Q000494:pharmacology*; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects; D008212:Lymphocyte Depletion / Q000379:methods*; D018139:Simplexvirus / Q000201:enzymology*; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D013937:Thymidine Kinase / Q000096:biosynthesis*; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection
D005839:Gentamicins; D007376:Interleukin-2; C010680:antibiotic G 418; D013937:Thymidine Kinase; D015774:Ganciclovir