Contassot E, Ferrand C, Certoux J M, Reynolds C W, Jacob W, Chiang Y, Cahn J Y, Hervé P, Tiberghien P
Laboratoire de Thérapeutique Immuno-moléculaire, Etablissement de Transfusion Sanguine, Besançon, France.
Hum Gene Ther. 1998 Jan 1;9(1):73-80. doi: 10.1089/hum.1998.9.1-73.
We have demonstrated in previous studies that retrovirus-mediated transfer of the herpes simplex thymidine kinase (HS-tk) and neomycin phosphotransferase (neo) genes in CD3/IL-2 stimulated primary T lymphocytes followed by G418 selection resulted in T cells retaining both interleukin-2 (IL-2) and alloresponsiveness and specifically inhibited by ganciclovir (GCV). A clinical trial examining the therapeutic potential of such gene-modified donor T cells after allogeneic bone marrow transplantation is presently underway. In the present study, we have investigated the feasibility and consequences of replacing polyclonal stimulation of T cells by an allogeneic stimulation prior to retrovirus-mediated gene transfer. Exposure of allostimulated primary donor T lymphocytes to retrovirus-containing supernatant resulted in T cells resistant to G418 while maintaining a strong, GCV-sensitive, allogeneic response when subsequently restimulated with the initial allogeneic cells. Control nontransduced cells identically stimulated exhibited a weaker, GCV-insensitive, allogeneic proliferative response. The transduced T cells were also capable of GCV-sensitive alloreactivity when exposed to third-party cells with, however, a lower proliferative response than that seen with the allogeneic cells used for stimulation at the time of transduction. Importantly, this difference in the proliferative responses was not observed with control nontransduced cells identically stimulated. A similar response pattern was observed with respect to pre-cytotoxic T lymphocyte (CTL) frequencies. Overall, retrovirus-mediated gene transfer after an allogeneic stimulation can lead to efficient transduction and the pattern of alloreactivity of the HS-tk-expressing cells is consistent with the preferential transduction of alloantigen-specific dividing T cells. Such an approach could be used to generate cells both strongly alloreactive and GCV-sensitive for in vivo therapeutic use.
我们在先前的研究中已证明，在经CD3/IL-2刺激的原代T淋巴细胞中，通过逆转录病毒介导转移单纯疱疹胸苷激酶（HS-tk）和新霉素磷酸转移酶（neo）基因，随后进行G418筛选，可使T细胞保留白细胞介素-2（IL-2）和同种异体反应性，并受到更昔洛韦（GCV）的特异性抑制。目前正在进行一项临床试验，研究这种基因修饰的供体T细胞在异基因骨髓移植后的治疗潜力。在本研究中，我们调查了在逆转录病毒介导的基因转移之前，用同种异体刺激替代T细胞多克隆刺激的可行性及后果。将经同种异体刺激的原代供体T淋巴细胞暴露于含逆转录病毒的上清液中，可使T细胞对G418产生抗性，并且当随后用初始同种异体细胞再次刺激时，能维持强烈的、对GCV敏感的同种异体反应。同样受到刺激的对照未转导细胞表现出较弱的、对GCV不敏感的同种异体增殖反应。当转导的T细胞暴露于第三方细胞时，它们也能够产生对GCV敏感的同种异体反应性，不过其增殖反应低于转导时用于刺激的同种异体细胞所引发的增殖反应。重要的是，同样受到刺激的对照未转导细胞未观察到这种增殖反应的差异。关于细胞毒性T淋巴细胞（CTL）前体细胞频率，也观察到了类似反应模式。总体而言，同种异体刺激后通过逆转录病毒介导的基因转移可实现高效转导，并且表达HS-tk的细胞的同种异体反应模式与同种异体抗原特异性分裂T细胞的优先转导一致。这种方法可用于生成体内治疗用的、既具有强烈同种异体反应性又对GCV敏感的细胞。
