Theil Anke, Tuve Sebastian, Oelschlägel Uta, Maiwald Anja, Döhler Diana, Oßmann Dennis, Zenkel Anja, Wilhelm Carmen, Middeke Jan Moritz, Shayegi Nona, Trautmann-Grill Karolin, von Bonin Malte, Platzbecker Uwe, Ehninger Gerhard, Bonifacio Ezio, Bornhäuser Martin
DFG Center for Regenerative Therapies, Technische Universität Dresden, Dresden, Germany.
Medical Clinic I, University Hospital Dresden, Technische Universität Dresden, Dresden, Germany.
Cytotherapy. 2015 Apr;17(4):473-86. doi: 10.1016/j.jcyt.2014.11.005. Epub 2015 Jan 6.
Mouse models indicate that adoptive transfer of regulatory T cells (Treg) may suppress graft-versus-host-disease (GvHD) while preserving graft-versus-leukemia reactions. We aimed to develop a protocol for the efficient isolation and in vitro expansion of donor-derived Treg and to establish the proof-of-concept for the clinical application of ex vivo-generated Treg preparations in five patients with otherwise treatment-refractory chronic GvHD (cGvHD).
Allogeneic Treg were isolated from unstimulated leukapheresis products of the corresponding human leukocyte antigen-matched donors by use of clinical-grade magnetic-activated bead sorting. To increase the amount and purity, Treg were cultivated for 7-12 days and infused after a median time of 35 months after allogeneic hematopoietic cell transplantation.
Final products contained Treg with a median purity of 84.1% CD4(+)CD25(high)CD127(low)FOXP3(+)of CD45(+) cells and a mean quantity of 2.4 × 10(6) Treg per kg body wt. All isolated cell products showed in vitro suppressive activity. On transfusion, two of five patients showed a clinical response with improvement of cGvHD symptoms. The other three patients showed stable cGvHD symptoms for up to 21 months. In four of five patients, increased counts of Treg were detectable on Treg transfusion, immunosuppressive treatment could be reduced and suppression of CD69 activation marker expression on T-effector cells was observed. However, one patient had development of malignant melanoma and another patient had Bowen skin cancer 4 months and 11 months after Treg transfusion, respectively.
We demonstrate a feasible and reproducible approach of isolating functional Treg in high quantity and purity for clinical application and show opportunities and risks of adoptive Treg transfer into patients with cGvHD.
小鼠模型表明，调节性T细胞（Treg）的过继转移可能会抑制移植物抗宿主病（GvHD），同时保留移植物抗白血病反应。我们旨在制定一种方案，用于高效分离和体外扩增供体来源的Treg，并为5例其他治疗方法难治的慢性移植物抗宿主病（cGvHD）患者应用体外生成的Treg制剂建立概念验证。
通过临床级磁激活珠分选，从相应人类白细胞抗原匹配供体的未刺激白细胞单采产品中分离出同种异体Treg。为了增加数量和纯度，将Treg培养7 - 12天，并在异基因造血细胞移植后中位时间35个月后进行输注。
最终产品中Treg的中位纯度为CD45(+)细胞的84.1% CD4(+)CD25(高)CD127(低)FOXP3(+)，平均数量为每千克体重2.4×10(6)个Treg。所有分离的细胞产品均显示出体外抑制活性。输血后，5例患者中有2例出现临床反应，cGvHD症状改善。另外3例患者的cGvHD症状在长达21个月内保持稳定。5例患者中有4例在输注Treg后可检测到Treg计数增加，免疫抑制治疗可减少，并且观察到T效应细胞上CD69激活标志物表达受到抑制。然而，1例患者在输注Treg后4个月发生恶性黑色素瘤，另1例患者在输注Treg后11个月发生鲍温皮肤癌。
我们展示了一种可行且可重复的方法，用于大量且高纯度地分离功能性Treg以用于临床应用，并展示了将Treg过继转移到cGvHD患者中的机会和风险。
