Li T, Ma R, Zhu J Y, Wang F S, Huang L, Leng X S
Department of Hepatobiliary Surgery, Peking University People's Hospital, Beijing, China.
Department of Surgical Intensive Care Unit, Peking University People's Hospital, Beijing, China.
Transplant Proc. 2015 Jan-Feb;47(1):165-70. doi: 10.1016/j.transproceed.2014.10.043.
Programmed death-1/PD-1 ligand-1 (PD-1/PD-L1) costimulatory signals may play an important role in T-cell-induced immune response. The aim of this study is to investigate the role of the PD-1/PD-L1 costimulatory pathway on immunotolerance induction in mouse pancreatic islet transplantation.
Full-length mouse PD-L1 cDNA was subcloned into pShuttle-GFP-CMV(-) shuttle plasmid. The product was cut by certain restriction endonuclease and ligated with pAdxsi vector. The adenovirus bone plasmid was transformed into DH5α-competent bacteria. After linearization, the recombined adenovirus DNA was transfected into 293 cells for package and amplification. Streptozotocin was injected intraperitoneally into C57BL/6 (H-2(b)) mouse to induce diabetic model recipient. Recipients were randomly divided into 3 groups. Group A was the control. Group B and group C were injected with Ad-EGFP and Ad-PD-L1 through the tail vein, respectively, 1 day before islet transplantation. The 300 to 400 islets of DBA/2 (H-2(d)) were transplanted into the renal subcapsular space of the diabetic model recipient. We monitored and analyzed the blood glucose levels and the survival time of grafts after transplantation.
Recombinant adenovirus Ad-PD-L1 had high efficiency expression of PD-L1 in recipient mouse. The blood glucose concentration of mice in the Ad-PD-L1 gene treatment group was obviously lower than that of the control and Ad-EGFP treatment groups and was stable and kept within the normal range at post-transplant 21 days. The survival time of grafts in the Ad-PD-L1 group (27.6 ± 3.5 days) was significantly longer than in the control (7.8 ± 0.33 days) and Ad-EGFP groups (7.6 ± 0.59 days), P < .01. Mixed lymphocyte response showed a specific decrease reaction of recipient lymphocyte vs donor lymphocyte. Flow cytometry detection showed that unsplit cells occupied 90% of recipient mouse lymphocytes, but unsplit cells among normal C57BL/6 mouse lymphocytes without Ad-PD-L1 gene treatment were 51 in the control group. The differences between them were significant (P < .01).
Recombinant adenovirus Ad-PD-L1 has been successfully constructed. In mouse pancreatic islet transplantation, it can suppress the activation of recipient T lymphocyte through the PD-1/PD-L1 costimulatory pathway, and significantly prolong the survival time of grafts.
程序性死亡蛋白1/程序性死亡蛋白1配体1（PD-1/PD-L1）共刺激信号可能在T细胞诱导的免疫反应中发挥重要作用。本研究旨在探讨PD-1/PD-L1共刺激通路在小鼠胰岛移植免疫耐受诱导中的作用。
将全长小鼠PD-L1 cDNA亚克隆到pShuttle-GFP-CMV(-)穿梭质粒中。产物用特定限制性内切酶切割，与pAdxsi载体连接。腺病毒骨架质粒转化到DH5α感受态细菌中。线性化后，将重组腺病毒DNA转染到293细胞中进行包装和扩增。将链脲佐菌素腹腔注射到C57BL/6（H-2(b)）小鼠中诱导糖尿病模型受体。受体随机分为3组。A组为对照组。B组和C组在胰岛移植前1天分别通过尾静脉注射Ad-EGFP和Ad-PD-L1。将300至400个DBA/2（H-2(d)）胰岛移植到糖尿病模型受体的肾被膜下间隙。我们监测并分析了移植后血糖水平和移植物存活时间。
重组腺病毒Ad-PD-L1在受体小鼠中高效表达PD-L1。Ad-PD-L1基因治疗组小鼠的血糖浓度明显低于对照组和Ad-EGFP治疗组，且在移植后21天稳定并保持在正常范围内。Ad-PD-L1组移植物的存活时间（27.6±3.5天）明显长于对照组（7.8±0.33天）和Ad-EGFP组（7.6±0.59天），P<0.01。混合淋巴细胞反应显示受体淋巴细胞对供体淋巴细胞有特异性降低反应。流式细胞术检测显示未分裂细胞占受体小鼠淋巴细胞的90%，但未接受Ad-PD-L1基因治疗的正常C57BL/6小鼠淋巴细胞中的未分裂细胞在对照组中为51%。它们之间的差异具有统计学意义（P<0.01）。
成功构建了重组腺病毒Ad-PD-L1。在小鼠胰岛移植中，它可通过PD-1/PD-L1共刺激通路抑制受体T淋巴细胞活化，并显著延长移植物存活时间。
