Transplant Division, Department of Surgery, University of Kentucky, College of Medicine, Lexington, KY.
Department of Pathology and Laboratory Medicine, University of Kentucky, College of Medicine, Lexington, KY.
Transplantation. 2019 Apr;103(4):705-715. doi: 10.1097/TP.0000000000002495.
Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel phase I/II clinical trial with ex vivo expanded autologous Treg cells as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen.
The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux, and suppressor function of the cells. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway profiles of treated cells were analyzed by Western blot and cell bioenergetic parameters by extracellular flux analysis.
EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of protein kinase B compared with RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA.
Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.
实验和临床前证据表明，过继转移调节性 T（Treg）细胞可能是一种合适的治疗策略，可以诱导移植患者的耐受并提高移植物的存活率。肯塔基大学移植服务线正在开发一项新的 I/II 期临床试验，使用体外扩增的自体 Treg 细胞作为接受依维莫司（EVR）为基础免疫抑制方案的肾移植受者的过继细胞治疗。
本研究的目的是确定 EVR 在开发基于功能健全的 Treg 细胞的移植中作为适应性免疫治疗的作用机制和疗效，将常见的 EVR 为基础的方案整合到体内（患者）和体外（自体 Treg 细胞的扩增）。使用 GMP 兼容的细胞分离系统从白细胞分离物中选择 CD25 Treg 细胞，并在含有 EVR 或雷帕霉素（RAPA）的 5 天（短）或 21 天（长）培养中培养。多参数流式细胞术分析用于监测细胞的扩增率、表型、自噬流和抑制功能。通过 Western blot 分析处理细胞的磷酸肌醇 3-激酶/蛋白激酶 B/雷帕霉素靶蛋白信号通路谱，通过细胞生物能分析测定细胞的能量参数。
与 RAPA 处理的细胞相比，EVR 处理的细胞生长速度较慢、代谢率较低、蛋白激酶 B 磷酸化程度较低。尽管存在这些差异，但在 EVR 培养中长期 Treg 细胞的扩增率、表型和抑制功能与 RAPA 相似。
我们的结果支持 EVR 用于扩增具有临床应用潜力的功能健全的 Treg 细胞。
