Gene gun mediated CTLA4-Ig gene transfer for immunological tolerance induction

为了诱导供体抗原特异性免疫耐受，抑制辅助性T细胞激活所需的共刺激信号是一个重要的问题。我们研究了供体器官本身通过CTLA4-Ig基因转移抑制共刺激信号的可能性。为了避免病毒载体引起的反应，我们使用了非病毒载体pCAGGS。利用基因枪系统代替逆转录病毒载体或腺病毒载体，将小鼠CTLA4基因胞外区与人IgG1或EGFP基因连接到pBluescrip载体中，然后将mCTLA4hIgGI或mCTLA4EGFP片段插入到pCAGGS载体中。以5μg DNA/mg金颗粒包被pCAGGS/mCTLA4hIgG1或绿色荧光蛋白。在400磅/平方英寸的压力下，用基因枪(Helios，Biorad，USA)将BALB/c小鼠的皮肤、肌肉或肝脏转移。将pCAGGS/CTLA4-EGFP轰击的同种异体BALB/c皮肤移植，获得绿色荧光蛋白…荧光显微镜检测到的更多。将pCAGGS/CTLA4-Ig注射的BALB/c小鼠同种异体皮肤移植于C57BL/6小鼠的侧腹。将pCAGGS/2B4TCR-Ig轰击的皮肤作为对照移植，CTLA4-EGFP在真皮中的表达很难检测到，因为包裹在真皮中的金颗粒被包裹在真皮的脂肪层。在基因枪击伤小鼠的肝组织中，由于基因枪轰击造成的组织损伤，表皮中央未见EGFP的表达，而中心区周围有较强的表达，即使在第1天，肝脏中也检测到CTLA4-EGFP的表达，但轰击对肝脏的损伤很小。肝脏未见中枢性损伤，与受撞击的表皮相同。第5天肌肉中检测到绿色荧光蛋白的表达，但在表皮和肝脏中表达较少。CTLA4-EGFP在表皮和肌肉中表达1周，而在肝脏中表达2~5周，提示基因枪介导的基因转移在肝脏中诱导了特别稳定的基因表达。移植皮片中CTLA4-EGFP在真皮中的表达模式和持续时间没有改变。尽管基因枪能有效地将基因转移到真皮中，但注射CTLA4-Ig的同种异体皮片的存活时间(MST±SD=10.8±0.9)与对照皮片(MST±SD=9.8±0.4)相比无明显延长。在同种异体皮片中观察到的高效基因枪介导的基因表达与在天然皮肤中的表达相同。然而，与对照组相比，注射CTLA4-Ig的同种异体皮肤移植物的存活时间并没有延长。一些报告表明，CTLA4-Ig在心脏、胰岛和肝移植中诱导免疫耐受，但在皮肤移植物中不诱导。我们的结果表明，除了皮肤特异性抗原外，基因枪轰击引起的炎症反应也是导致同种异体皮片存活失败的另一个因素。应该进行进一步的实验，以最大限度地减少组织损伤。较少

To induce the donor antigen specific immunological tolerance, it is an important issue to inhibit the costimulatory signal required for helper T cell activation. We examined the possibility that donor organs themselves had the performance to inhibit the costimulatory signal by CTLA4-Ig gene transfer. To avoid viral vector inducing responses, we used non-viral pCAGGS vector. In addition, we used gene gun system instead of retro-or adeno-viral vector to achieve higher gene transfer frequency.The extra-cellular domain of mouse CTLA4 gene was ligated to human IgG1 or EGFP gene in pBluescript vector, and then mCTLA4hIgGI or mCTLA4EGFP fragment was inserted to the modified pCAGGS vector. Gold particles were coated with pCAGGS/mCTLA4hIgG1 or EGFP at 5μg DNA/mg gold particles. The skin, muscle, or liver of BALB/c mouse was transferred with the gene gun (Helios, BIORAD, USA) at the pressure of 400 psi. Syngeneic BALB/c skin bombarded with pCAGGS/CTLA4-EGFP was transplanted, and EGFP expression … More was detected by fluorescence microscopy. C57BL/6 mice were engrafted on the flank with the pCAGGS/CTLA4-Ig injected allogeneic skin of BALB/c mice. Skin bombarded with pCAGGS/2B4TCR-Ig was transplanted as control graft.In vivo expression of CTLA4-EGFP was hard to detect in dermis due to the trapment of the plasmid coated gold particles in its fatty layer. In epidermis, there was no expression of EGFP in the central portion of gene gun bombarded-area, because of tissue damage by bombardment, whereas there was strong expression in the area around the central portion, even on day 1, CTLA4-EGFP expression in liver was also detected on day 1 with little bombardment-induced damage. Bombarded liver showed no central damage as per bombarded epidermis. EGFP expression in muscle was detected on day 5, but less than in epidermis and liver. CTLA4-EGFP expression in epidermis and muscle were observed for 1 week, while it was observed in liver for 2 to 5 weeks, suggesting that gene gun mediated gene transfer induces particularly stable gene expression in the liver. Pattern and duration of CTLA4-EGFP expression in epidermis was not changed in the grafted skin.Despite efficient gene gun mediated gene transfer into epidermis, survival of the CTLA4-Ig injected allogeneic skin graft (MST±SD=10.8±0.9) was not significantly prolonged compared with the control grafts (MST±SD=9.8±0.4). The efficient gene gun mediated gene expression observed in the syngeneic skin graft was identical to that in the native epidermis. However, survival of allogeneic skin grafts injected with CTLA4-Ig was not prolonged compared with the controls. Several reports showed that CTLA4-Ig administration induced immunological tolerance in cardiac, islet, and liver transplantation, but not in skin grafts. In addition to the skin specific antigen, our results suggested that the inflammatory response induced by gene gun bombardment was another factor that contributed to the failure of allogeneic skin graft survival. Further experiments to minimize tissue damage should be carried out. Less