title: Monitoring of developing graft-versus-host disease mediated by herpes simplex virus thymidine kinase gene-transduced T cells.
abstract: Introduction of the HSV-Tk suicide gene into allogeneic T cells offers the possibility to control developing host-reactive cells within the context of allogeneic bone marrow transplantation (BMT). Sensitive quantitative detection methods are a prerequisite to monitor genetically modified T cells in peripheral blood and tissues to study their involvement in graft-versus-host disease (GVHD)-induced lesions as well as their disappearance or persistence after ganciclovir (GCV)-induced suicide. We monitored the alloreactivity of HSV-Tk-transduced T cells after BMT by studying their in vivo distribution and quantity in peripheral blood and in tissues in a WAG/Rij into Brown Norway fully mismatched rat allogeneic BMT model. Genetically modified T cells were quantified in blood and tissues by fluorescence-activated cell sorting, immunohistochemical analysis, and real-time quantitative polymerase chain reaction (PCR) analysis. A significant increase in the number of allogeneic HSV-Tk(+) T cells was found in particular in spleen and lymph nodes and large numbers were found in tongue, skin, and intestines. In blood, an increase in HSV-Tk(+) T cells closely preceded clinical symptoms of GVHD. Real-time quantitative PCR proved to be a fast and accurate tool by which to quantify transduced T cells both in blood and tissues. This enables the study of the in vivo alloreactivity of retrovirus-transduced cells and the response of HSV-Tk-expressing T cells to GCV-induced suicide therapy. Furthermore, we showed the potential use to study specific cause-effect relationships in a broad range of animal and clinical studies involving genetically engineered cells.
authors.affiliation: Department of Hematology, University Medical Center St. Radboud, 6500 HB Nijmegen, The Netherlands.
authors.forename: Sebastianus
authors.initials: S
authors.lastname: Kolen
authors.forename: Mo
authors.initials: M
authors.lastname: Weijtens
authors.forename: Anton
authors.initials: A
authors.lastname: Hagenbeek
authors.forename: Anke
authors.initials: A
authors.lastname: van Spronsen
authors.forename: Saskia
authors.initials: S
authors.lastname: Smulders
authors.forename: Roel
authors.initials: R
authors.lastname: de Weger
authors.forename: Theo
authors.initials: T
authors.lastname: de Witte
authors.forename: Harry
authors.initials: H
authors.lastname: Dolstra
authors.forename: Elly
authors.initials: E
authors.lastname: van de Wiel van Kemenade
authors.forename: Anton
authors.initials: A
authors.lastname: Martens
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D002478:Cells, Cultured; D006086:Graft vs Host Disease / Q000175:diagnosis / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D018189:Immunomagnetic Separation; D008979:Moloney murine leukemia virus / Q000235:genetics; D051381:Rats; D011914:Rats, Inbred BN; D020800:Receptor, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*; D013937:Thymidine Kinase / Q000097:blood / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D020800:Receptor, Nerve Growth Factor; D013937:Thymidine Kinase