Tiberghien P, Reynolds C W, Keller J, Spence S, Deschaseaux M, Certoux J M, Contassot E, Murphy W J, Lyons R, Chiang Y
Biological Carcinogenesis Development Program, Program Resources, Inc, Frederick, MD.
Blood. 1994 Aug 15;84(4):1333-41.
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.
异基因骨髓移植（BMT）与一种严重并发症——移植物抗宿主病（GVHD）相关。尽管体外T淋巴细胞骨髓清除可有效预防GVHD，但不幸的是，这会增加移植物排斥反应并降低移植物抗白血病（GVL）效应。在将T细胞与造血干细胞一起输注之前，将单纯疱疹胸苷激酶（HS-tk）自杀基因体外转移到T细胞中，如果随后发生GVHD，就可以用更昔洛韦（GCV）在体内选择性清除这些T细胞。因此，对于未发生严重GVHD的患者，可以保留T细胞对植入和肿瘤控制的有益作用。为了获得被GCV特异性清除的T细胞，我们用含有HS-tk和新霉素抗性（NeoR）基因的逆转录病毒载体转导原代T细胞。通过将PHA +/- CD3 - 或同种异体抗原刺激的纯化T细胞与经辐照的逆转录病毒载体生产细胞系共培养，或通过将T细胞在生产细胞的上清液中孵育来进行基因转移。随后在G418中培养1周可选择转导的细胞。用白细胞介素-2反应性转导并选择的细胞进行GCV处理导致生长抑制超过80%，而对对照细胞进行GCV处理则没有效果。同样，HS-tk转导细胞的同种异体反应性被GCV特异性抑制。将转导和未转导的T细胞混合未显示旁观者效应，这意味着所有被GCV抑制的细胞确实都被转导了。最后，涉及HUT-78（T淋巴瘤）细胞系转导的研究表明，在没有G418的情况下，HS-tk的稳定表达在体外可维持3个月以上。总之，我们已经确定了生成HS-tk转导的T细胞以便随后与造血干细胞一起进行体内转移的可行性，并且如果发生GVHD，在异基因BMT受者中通过体内GCV诱导特异性T细胞清除的可行性。
