Xia Guliang, He Jie, Zhang Zheng, Leventhal Joseph R
Department of Surgery-Organ Transplantation, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Transplantation. 2006 Dec 27;82(12):1749-55. doi: 10.1097/01.tp.0000250731.44913.ee.
Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice.
Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry.
By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection.
Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.
天然CD4CD25调节性T（Treg）细胞已被证明在体外和体内均能抑制同种异体反应性。我们推测，使用体外扩增的天然Treg进行免疫治疗可以预防小鼠急性移植排斥反应。
通过自动磁性细胞分选仪从幼稚小鼠中新鲜纯化天然CD4+CD25+Treg，并使用抗CD3/CD28单克隆抗体（mAb）包被的磁珠在体外进行扩增。通过混合淋巴细胞反应在体外测定抑制作用，并通过靶向心脏移植排斥反应在体内进行测定。通过流式细胞术追踪体内过继转移后Treg或效应T（Teff）细胞的存活情况，并通过组织学和免疫组织化学检查所有同种异体移植物。
在培养的第9天，新鲜天然Treg与抗CD3/CD28 mAb包被的磁珠和白细胞介素-2共培养可实现26.6±5.3倍的扩增。体外扩增的Treg在体外对同种异体抗原的抑制作用比新鲜Treg更强，并且在Rag-/-小鼠中可预防CD4 Teff介导的心脏移植排斥反应，但仅延迟了CD4+/CD8+ Teff介导的心脏移植排斥反应。长期存活的同种异体移植物未显示急性或慢性排斥反应的迹象，移植浸润的Treg表达CD25和FoxP3。注入的Treg在体内长期持续存在并扩增，并通过外周淋巴组织迁移。CD25表达在体内是动态变化的：Treg上持续的CD25表达表明同种异体抑制作用得以保留，而CD4+效应T细胞上显著增强的CD25表达很可能与T细胞扩增和移植排斥反应有关。
体外扩增的天然CD4+CD25+Treg的治疗应用可能是控制移植排斥反应或诱导移植耐受的一种可行且无毒的方法。
