Kolen Sebastianus, Weijtens Mo, Hagenbeek Anton, van Spronsen Anke, Smulders Saskia, de Weger Roel, de Witte Theo, Dolstra Harry, van de Wiel van Kemenade Elly, Martens Anton
Department of Hematology, University Medical Center St. Radboud, 6500 HB Nijmegen, The Netherlands.
Hum Gene Ther. 2003 Mar 1;14(4):341-51. doi: 10.1089/104303403321208943.
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.
将单纯疱疹病毒胸苷激酶（HSV-Tk）自杀基因导入异基因T细胞，为在异基因骨髓移植（BMT）背景下控制正在发育的宿主反应性细胞提供了可能性。灵敏的定量检测方法是监测外周血和组织中基因修饰T细胞的先决条件，以便研究它们在移植物抗宿主病（GVHD）诱导的损伤中的作用，以及在更昔洛韦（GCV）诱导的自杀后它们的消失或持续存在情况。我们通过研究在WAG/Rij到完全不匹配的棕色挪威大鼠异基因BMT模型中，经HSV-Tk转导的T细胞在体内在外周血和组织中的分布及数量，监测了BMT后这些细胞的同种异体反应性。通过荧光激活细胞分选、免疫组织化学分析和实时定量聚合酶链反应（PCR）分析对外周血和组织中的基因修饰T细胞进行定量。尤其在脾脏和淋巴结中发现同种异体HSV-Tk(+) T细胞数量显著增加，并且在舌头、皮肤和肠道中也发现了大量此类细胞。在外周血中，HSV-Tk(+) T细胞数量增加紧接在GVHD临床症状之前出现。实时定量PCR被证明是一种快速且准确的工具，可用于定量血液和组织中的转导T细胞。这使得能够研究逆转录病毒转导细胞的体内同种异体反应性以及表达HSV-Tk的T细胞对GCV诱导的自杀疗法的反应。此外，我们展示了在涉及基因工程细胞的广泛动物和临床研究中研究特定因果关系的潜在用途。
