Clinical-grade preparation of human natural regulatory T-cells encoding the thymidine kinase suicide gene as a safety gene
Clinical-grade preparation of human natural regulatory T-cells encoding the thymidine kinase suicide gene as a safety gene
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DOI：
10.1002/jgm.1220
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发表时间：
2008-08-01
影响因子：
3.5
通讯作者：
Lemoine, Francois M.
中科院分区：
文献类型：
--
作者：
Guillot-Delost, Maude;Cherai, Mustapha;Lemoine, Francois M.
背景人CD 4（+）CD 25（+）FOXP 3（+）天然调节性T细胞（nTreg）在诱导同种异体移植患者耐受或控制严重自身免疫性疾病方面具有巨大的治疗潜力。然而，nTreg的临床级生产仍然难以实现，因为缺乏真正特异性的表面标志物和它们的低频率，这意味着需要它们的离体扩增。此外，安全问题应考虑到由于任何不受控制的nTreg诱导的免疫抑制或自身反应性污染的T细胞，特别是在自身免疫context.Methods的不受控制的增殖的风险，我们比较了不同的临床级别的条件，免疫磁性选择和离体扩增的nTreg。为安全起见，扩增的细胞用共表达人CD 90和HSV 1胸苷激酶的逆转录病毒载体进行遗传修饰。结果（1）用CD 25微球可一步富集nTreg，nTreg具有功能抑制性，主要为FOXP 3（+）;（ii）使用抗-CD 28-和抗-CD 3-包被的珠粒、白介素-2和雷帕霉素，nTreg在3周后扩增150-200倍。（iii）在有效的逆转录病毒转导和CD 90选择后，nTreg保持其抑制活性;（iv）转导的nTreg在活化后可被更昔洛韦清除。结论本文报道的制备nTreg的有效方法，其安全性得到了保证，现在可用于进一步的临床试验。版权所有（c）2008约翰威利父子有限公司。
Background Human CD4(+)CD25(+)FOXP3(+) natural regulatory T-cells (nTreg) have a great therapeutic potential for the induction of tolerance in allo-transplanted patients or for the control of severe auto-immune diseases. However, clinical-grade production of nTreg remains difficult to achieve because of the absence of a truly specific surface marker and of their low frequency that implies a need for their ex vivo expansion. Furthermore, safety issues should be taken into consideration due to the risk of either uncontrolled nTreg-induced immunosuppression or uncontrolled proliferation of autoreactive contaminating T-cells particularly in an auto-immune context.Methods We compared different clinical-grade conditions for immunomagnetic selection and ex vivo expansion of nTreg. For safety, expanded cells were genetically modified with retroviral vectors co-expressing human CD90 and HSV1 thymidine kinase. The CD90 surface marker and thymidine kinase allow for selection and elimination of transduced cells by ganciclovir, respectively.Results We showed that (i) nTreg could be enriched in a one step using CD25 microbeads, were functionally suppressive and mainly FOXP3(+); (ii) using anti-CD28- and anti-CD3-coated beads, interleukin-2 and rapamycin, nTreg were expanded 150-200-fold after 3 weeks. Under these clinical-grade conditions, they remained suppressive, and no major alteration of the TCR repertoirc was observed; (iii) after efficient retroviral transduction and CD90 selection, nTreg maintained their suppressive activity; (iv) transduced nTreg could be eliminated by ganciclovir upon activation.Conclusions The efficient procedure reported here for the preparation of nTreg, whose safety has been ensured, is now applicable for further clinical trials. Copyright (c) 2008 John Wiley & Sons, Ltd.