Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells.. Mus musculus

Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye. Keywords: Comparison Overall design: Comparison of gene expression pattern in Mouse Primary Cultured Cell Lines (RPE, IPE, CBPE). Adult (6- to 8-week-old) C57BL/6 purchased from CLEA Japan Inc. (Tokyo, Japan) were used as donors ocular pigment epithelium (PE). To cultivate iris PE (IPE) and ciliary body PE (CBPE), iris and ciliary body tissues in the eyes were dissected out, and then incubated in 1 mg/ml Dispase and 0.05 mg/ml DNase I (both from Boehringer-Mannheim, Mannheim, Germany) for 1 hr. To cultivate retinal PE (RPE) Eyes were cut into two parts along a circumferential line posterior to the ciliary process, creating a ciliary body-deficient posterior eyecup. The eyecup was then incubated in 0.2% trypsin (Biowhitaker) for 1 hr. These tissues were triturated to make a single cell suspension, and then re-suspended in the medium.

眼色素上皮（PE）细胞可促进调节性T细胞（T regulatory cells，Treg）的生成，即PE诱导型调节性T细胞（PE-induced Treg cells）。此外，经PE细胞处理的T细胞可获得通过转化生长因子-β（TGFbeta）抑制旁观者T细胞活化的能力。虹膜PE细胞表面的膜结合型转化生长因子-β（TGFbeta）可与T细胞表面的TGF-β受体结合，通过细胞接触依赖的机制将T细胞转化为CD8阳性调节性T细胞（CD8+ Treg）。与之相反，视网膜PE细胞分泌的可溶性转化生长因子-β（TGFbeta）则可通过不依赖细胞接触的方式将CD4阳性T细胞转化为调节性T细胞。
本研究通过寡核苷酸微阵列技术，检测了PE细胞中免疫调节因子（如TGFbeta、血小板反应蛋白、CD59、白细胞介素-1受体拮抗剂等）的表达情况。研究共检测到多种血小板反应蛋白结合分子，因此后续分析聚焦于血小板反应蛋白。经血小板反应蛋白1（TSP-1）介导的潜在型TGFbeta活化后，体外培养的虹膜PE细胞与视网膜PE细胞可诱导PE诱导型调节性T细胞的细胞命运转化。转化完成后，眼部PE细胞与PE诱导型调节性T细胞均表达TSP-1。当T细胞同时表达TSP-1时，调节性T细胞的生成得到增强。此外，PE诱导型调节性T细胞可通过TSP-1显著抑制旁观者T细胞的活化。上述结果强烈提示，眼部PE细胞与PE诱导型调节性T细胞抑制旁观者T细胞活化的能力，取决于其产生TSP-1的能力。因此，由眼部实质细胞与调节性T细胞共同产生的眼内TSP-1，对于眼部的免疫调控至关重要。
关键词：对比；整体实验设计：比较小鼠原代培养细胞系（RPE、IPE、CBPE）的基因表达模式。
实验所用的供体眼部色素上皮（PE）细胞，取自购自日本CLEA公司（东京，日本）的6~8周龄成年C57BL/6小鼠。为培养虹膜PE细胞（IPE）与睫状体PE细胞（CBPE），我们分离了眼球中的虹膜与睫状体组织，随后将其置于1mg/ml Dispase与0.05mg/ml DNase I（均购自德国Boehringer-Mannheim公司，曼海姆，德国）中孵育1小时。为培养视网膜PE细胞（RPE），我们将眼球沿睫状突后方的环形切线切成两半，得到不含睫状体的后部眼杯。随后将眼杯置于0.2%胰蛋白酶（Biowhitaker公司产品）中孵育1小时。将上述组织研磨制成单细胞悬液，并重悬于培养基中。