Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
Department of Ophthalmology & Visual Science, Tokyo Medical and Dental University Graduate School of Medicine and Dental Sciences, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2018 Apr 1;59(5):1719-1731. doi: 10.1167/iovs.17-22703.
To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can suppress natural killer (NK) cell activation.
iPS-RPE cells were cocultured with peripheral blood mononuclear cells (PBMCs) or purified NK cells from healthy donors after stimulation with cytokines. To confirm expression of NK cell-specific markers, flow cytometry and quantitative RT-PCR (qRT-PCR) were performed. NK cells (or PBMCs) cocultured with iPS-RPE cells were assessed for proliferation by Ki-67 expression with flow cytometry, and NK suppression by RPE cells was assessed for granzyme B production with ELISA. Human leukocyte antigen (HLA) expression including HLA-E on iPS-RPE cells was evaluated with flow cytometry and qRT-PCR. The effect of HLA-E downregulation was also investigated using small interfering RNA (siRNA) systems. Following iPS-RPE cell transplantation in vivo, we evaluated NK cell invasion in the retina with immunohistochemistry.
Activated NK cells expressed NK-related markers such as CD16, CD56, and CD11b, and NK cells produced cytotoxic agents such as granzyme B, perforin, and TNF-α. Human iPS-RPE cells inhibited cell proliferation and production of these cytotoxic agents by activated NK cells in vitro. iPS-RPE cells constitutively expressed HLA-E and suppressed NK cell activation through an interaction between HLA-E and CD94/NKG2A. Moreover, immunohistochemical evaluation of monkey RPE transplantation into in vivo immune rejection models showed no NK cell invasion in the retina in allografts or xenografts except for one xenografted eye.
Cultured iPS cell-derived RPE cells greatly suppress NK cell activation. Thus, NK cells might be inactivated when exposed to this type of retinal cell.
确定人诱导多能干细胞（iPS）衍生的视网膜色素上皮（RPE）细胞（iPS-RPE）是否可以抑制自然杀伤（NK）细胞的激活。
将 iPS-RPE 细胞与来自健康供体的外周血单核细胞（PBMC）或纯化的 NK 细胞在细胞因子刺激后进行共培养。为了确认 NK 细胞特异性标志物的表达，进行了流式细胞术和定量 RT-PCR（qRT-PCR）。通过流式细胞术评估 Ki-67 表达来评估与 iPS-RPE 细胞共培养的 NK 细胞的增殖，通过 ELISA 评估 RPE 细胞对 NK 抑制的颗粒酶 B 产生。通过流式细胞术和 qRT-PCR 评估 iPS-RPE 细胞上包括 HLA-E 的人类白细胞抗原（HLA）表达。还使用小干扰 RNA（siRNA）系统研究了 HLA-E 下调的影响。在体内进行 iPS-RPE 细胞移植后，我们通过免疫组织化学评估了 NK 细胞在视网膜中的浸润。
活化的 NK 细胞表达 NK 相关标志物，如 CD16、CD56 和 CD11b，并且 NK 细胞产生细胞毒性物质，如颗粒酶 B、穿孔素和 TNF-α。人 iPS-RPE 细胞在体外抑制活化的 NK 细胞的细胞增殖和这些细胞毒性物质的产生。iPS-RPE 细胞组成性表达 HLA-E，并通过 HLA-E 与 CD94/NKG2A 的相互作用抑制 NK 细胞的激活。此外，对猴 RPE 移植到体内免疫排斥模型的免疫组织化学评估显示，除了一个异种移植眼外，在同种异体和异种移植物中，视网膜中均未发现 NK 细胞浸润。
培养的 iPS 细胞衍生的 RPE 细胞可显著抑制 NK 细胞的激活。因此，当暴露于这种类型的视网膜细胞时，NK 细胞可能会失活。
