Center for Vascularized Composite Allotransplantation, Linkou Chang Gung Memorial Hospital, Gueishan, Taoyuan, Taiwan.
Department of Plastic and Reconstructive Surgery, Linkou Chang Gung Memorial Hospital, Gueishan, Taoyuan, Taiwan.
PLoS One. 2018 Sep 7;13(9):e0203624. doi: 10.1371/journal.pone.0203624. eCollection 2018.
Tipping the balance toward regulatory T cells (Tregs) through adoptive cell therapy has shown promise to induce transplantation tolerance. Although such strategy has been explored in many mice organ transplantation studies, less knowledge was available in rat systems. Furthermore, the behaviors of the transferred cells have not been well studied in real-time fashion.
Tregs from naïve LEW rats were purified in two steps with the autoMACS system. Immunosuppression potential of these cells was examined with mixed lymphocyte reaction. Following stimulation by the alloantigen in vitro, the purified Tregs were infused into the recipients of vascularized composite allotransplantation (VCA). Secondary allogeneic skin grafting challenge was performed on the recipients with long-term survived VCA. Live optical imaging was performed to track luciferase-expressing Tregs following infusion to the VCA recipients. Expression of relevant molecules was studied by flow cytometry or quantitative RT-PCR.
Rat Tregs were enriched following two-step cell sorting and showed immunosuppressive capacity. Upon infusion into the VCA recipients that have been treated with antilymphocyte serum and short-term Cyclosporin A, the antigen-stimulated Tregs significantly prolonged VCA survival and induced donor-specific tolerance. Tracking of the infused bioluminescent Tregs showed their specific homing to lymph nodes, and then to the VCAs. Following secondary skin grafting, Tregs specifically gathered at the donor-derived skin that was not rejected by the recipient. The in vivo migratory pattern coincided with the altered expression of cell surface molecules of CD62L, CD103, CD134, and CD278, following donor-antigen stimulation. Elevated expression of CCR4 and CCL22 in allograft may also participate in recruiting Tregs for maintenance of VCA survival and promoting donor-specific tolerance.
Sorted Tregs induced donor-specific tolerance to VCA in rats. Live cell tracking demonstrated that activated CD4+CD25+FoxP3+ Tregs targeted primarily to the lymph nodes and VCA. The Tregs migrated to the secondary grafted donor skin and contributed to the maintenance of donor-specific tolerance. These behaviors were associated with phenotypic changes induced by donor antigen stimulation. Increased expression of CCR4 and CCL22 in VCA skin may also be relevant.
通过过继性细胞治疗使调节性 T 细胞（Tregs）倾斜平衡以诱导移植耐受已显示出前景。尽管这种策略已在许多小鼠器官移植研究中进行了探索，但在大鼠系统中知识较少。此外，转移细胞的行为尚未实时进行很好的研究。
使用自动 MACS 系统对来自 naive LEW 大鼠的 Tregs 进行两步纯化。通过混合淋巴细胞反应检查这些细胞的免疫抑制潜能。在体外经同种抗原刺激后，将纯化的 Tregs 输注到血管化复合同种异体移植（VCA）的受者中。对长期存活的 VCA 受者进行二次同种异体皮肤移植挑战。对 VCA 受者进行活体光学成像以跟踪输注后的荧光素酶表达 Tregs。通过流式细胞术或定量 RT-PCR 研究相关分子的表达。
大鼠 Tregs 经两步细胞分选后得到富集，并表现出免疫抑制能力。在接受抗淋巴细胞血清和短期环孢素 A 治疗的 VCA 受者中输注抗原刺激的 Tregs 后，VCA 存活时间显著延长，并诱导供体特异性耐受。对输注的生物发光 Tregs 的跟踪显示，它们特异性归巢到淋巴结，然后归巢到 VCA。进行二次皮肤移植后，Tregs 特异性聚集在未被受者排斥的供体衍生皮肤处。体内迁移模式与供体抗原刺激后细胞表面分子 CD62L、CD103、CD134 和 CD278 的改变表达一致。移植物中 CCR4 和 CCL22 的表达升高也可能参与招募 Tregs 以维持 VCA 存活并促进供体特异性耐受。
分选的 Tregs 在大鼠中诱导 VCA 的供体特异性耐受。活细胞跟踪显示，激活的 CD4+CD25+FoxP3+Tregs 主要靶向淋巴结和 VCA。Tregs 迁移到二次移植的供体皮肤，并有助于维持供体特异性耐受。这些行为与供体抗原刺激诱导的表型变化有关。VCA 皮肤中 CCR4 和 CCL22 的表达增加也可能相关。
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