Kennelly Kevin P, Holmes Toby M, Wallace Deborah M, O'Farrelly Cliona, Keegan David J
Cell Transplant. 2017 Jun 9;26(6):983-1000. doi: 10.3727/096368917X694697. Epub 2017 Jan 20.
Successful subretinal transplantation is limited by considerable early graft loss despite pharmacological suppression of adaptive immunity. We postulated that early innate immune activity is a dominant factor in determining graft survival and chose a nonimmunosuppressed mouse model of retinal pigment epithelial (RPE) cell transplantation to explore this. Expression of almost all measured cytokines by DH01 RPE cells increased significantly following graft preparation, and the neutrophil chemoattractant KC/GRO/CINC was most significantly increased. Subretinal allografts of DH01 cells (C57BL/10 origin) into healthy, nonimmunosuppressed C57BL/6 murine eyes were harvested and fixed at 1, 3, 7, and 28 days postoperatively and subsequently cryosectioned and stained. Graft cells were detected using SV40 large T antigen (SV40T) immunolabeling and apoptosis/necrosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Sections were also immunolabeled for macrophage (CD11b and F4/80), neutrophil (Gr1 Ly-6G), and T-lymphocyte (CD3-ɛ) infiltration. Images captured with an Olympus FV1000 confocal microscope were analyzed using the Imaris software. The proportion of the subretinal bolus comprising graft cells (SV40T+) was significantly (p < 0.001) reduced between postoperative day (POD) 3 (90 ± 4%) and POD 7 (20 ± 7%). CD11b+, F4/80+, and Gr1 Ly-6G+ cells increased significantly (p < 0.05) from POD 1 and predominated over SV40T+ cells by POD 7. Colabeling confocal microscopic analysis demonstrated graft engulfment by neutrophils and macrophages at POD 7, and reconstruction of z-stacked confocal images confirmed SV40T inside Gr1 Ly-6G+ cells. Expression of CD3-ɛ was low and did not differ significantly between time points. By POD 28, no graft cells were detectable and few inflammatory cells remained. These studies reveal, for the first time, a critical role for innate immune mechanisms early in subretinal graft rejection. The future success of subretinal transplantation will require more emphasis on techniques to limit innate immune-mediated graft loss, rather than focusing exclusively on suppression of the adaptive immune response.
尽管采用了药物抑制适应性免疫，但视网膜下移植的成功仍受到早期大量移植物丢失的限制。我们推测早期固有免疫活性是决定移植物存活的主要因素，并选择了一种非免疫抑制的视网膜色素上皮（RPE）细胞移植小鼠模型来进行探索。DH01 RPE细胞在移植物制备后，几乎所有检测的细胞因子表达均显著增加，其中中性粒细胞趋化因子KC/GRO/CINC的增加最为显著。将DH01细胞（源自C57BL/10）的视网膜下同种异体移植物植入健康、未免疫抑制的C57BL/6小鼠眼中，在术后1天、3天、7天和28天收获并固定，随后进行冰冻切片和染色。使用SV40大T抗原（SV40T）免疫标记检测移植物细胞，通过末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）检测凋亡/坏死情况。切片还进行了巨噬细胞（CD11b和F4/80）、中性粒细胞（Gr1 Ly-6G）和T淋巴细胞（CD3-ɛ）浸润的免疫标记。用Olympus FV1000共聚焦显微镜拍摄的图像使用Imaris软件进行分析。视网膜下团块中包含移植物细胞（SV40T+）的比例在术后第3天（90±4%）和第7天（20±7%）之间显著降低（p<0.001）。CD11b+、F4/80+和Gr1 Ly-6G+细胞从术后第1天开始显著增加（p<0.05），到术后第7天超过了SV40T+细胞。共聚焦显微镜联合标记分析显示，在术后第7天中性粒细胞和巨噬细胞吞噬了移植物，z轴堆叠共聚焦图像重建证实Gr1 Ly-6G+细胞内有SV40T。CD3-ɛ的表达较低，在各时间点之间无显著差异。到术后第28天，未检测到移植物细胞，炎症细胞也所剩无几。这些研究首次揭示了固有免疫机制在视网膜下移植排斥早期的关键作用。视网膜下移植未来的成功将需要更加强调限制固有免疫介导的移植物丢失的技术，而不是仅仅专注于抑制适应性免疫反应。
