Lilienfeld Benjamin G, Crew Mark D, Forte Pietro, Baumann Bettina C, Seebach Jörg D
Laboratory for Transplantation Immunology, Department of Internal Medicine, University Hospital Zürich, Zürich, Switzerland.
Xenotransplantation. 2007 Mar;14(2):126-34. doi: 10.1111/j.1399-3089.2007.00378.x.
The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells.
Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca(2)PO(4)-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in (51)Cr-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays.
The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells.
Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.
猪内皮细胞（pEC）对人自然杀伤（NK）细胞的易感性与人类主要组织相容性复合体（MHC）特异性杀伤抑制受体无法识别猪MHC I类分子有关。本研究的目的是：（i）评估由经典的HLA-E结合肽抗原VMAPRTLIL、成熟的人β2-微球蛋白和成熟的HLA-E重链组成的HLA-E单链三聚体（SCT）的稳定表达对pEC免受异种NK介导的细胞毒性的保护作用；（ii）测试pEC和猪淋巴母细胞上HLA-E的表达是否影响人NK细胞的黏附。
通过Ca(2)PO(4)转染，随后进行有限稀释克隆，生成表达不同水平HLA-E SCT的猪EC系。通过流式细胞术测量HLA-E的表面表达。在(51)Cr释放细胞毒性试验中测试转染的pEC系对人NK细胞的易感性。在黏附和结合试验中进一步研究人NK细胞与HLA-E阳性pEC或猪淋巴母细胞之间的相互作用。
pEC免受人类NK介导的细胞毒性的保护水平与表面HLA-E表达的强度相关。此外，通过阻断HLA-E特异性NK抑制受体CD94/NKG2A可特异性逆转HLA-E SCT介导的保护作用。HLA-E表达既不影响人NK细胞对pEC的黏附，也不影响人NK与猪13271.10细胞之间的异源结合形成。
在没有外源性肽脉冲的情况下，实现了HLA-E在pEC上的稳定表面表达，并提供了对人NK细胞毒性的部分保护。虽然不足以完全抑制异种NK细胞反应性，但猪器官上的转基因HLA-E表达可能有助于结合其他保护策略成功应用临床异种移植。
Zotero 插件