Zeng M H, Fang C Y, Wang S S, Zhu M, Xie L, Li R, Wang L, Wu X W, Chen S
Institute of Organ Transplantation, Key Laboratory of Organ Transplantation (HUST), Ministry of Education, Key Laboratory of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Transplant Proc. 2006 Dec;38(10):3312-4. doi: 10.1016/j.transproceed.2006.10.179.
Human natural killer (NK) cells, which can mediate direct lysis of porcine endothelial cells, play an important role in xenograft rejection. HLA-G, which is a critical molecule in maintaining maternal immune tolerance of semi-allogenic fetus, is able to protect susceptible target cells from lysis induced by NK cells. In this study, we investigated whether soluble HLA-G1 (sHLA-G1) protected porcine xenogeneic cells against human NK cell-mediated lysis.
The human sHLA-G1 genomic DNA (pcDNA3-sHLA-G1) was transfected into a B lymphoblastoid cell line 721.221 (LCL721.221) by nucleofector. The sHLA-G1 expression of the transfected LCL721.221 cells was identified by RT-PCR and Dot-ELISA. The sHLA-G1 protein was purified by affinity chromatography on anti-HLA-ImAb W6/32 coupled to cyanogen-bromide-activated Sepharose 4B from culture supernates of transfectants. Various concentrations of sHLA-G(1) protein (0, 2, 4, 6, or 8 microg/mL) were added to a NK cell-mediated xenogenic cell lysis system with either NK92 cells or fresh human peripheral blood mononuclear cells (PBMCs) cocultured with the porcine endothelial cells line. A LDH release assay was used to evaluate NK cell-mediated cytotoxicity.
sHLA-G1 provided significant protection of porcine endothelial cells against human NK-mediated cytotoxicity in a dose-dependent manner. The rates of NK92 cell-mediated cytotoxicity were reduced to 83.4 +/- 5.7% (2 microg/mL), 56.6 +/- 9.3% (4 microg/mL), 39.3 +/- 10.2% (6 microg/mL), and 31.2 +/- 4.9% (8 microg/mL) versus 96.9 +/- 3.0% in the control group (P < .01). Similarly, adding 6 microg/mL sHLA-G1 reduced the mean rate of PBMC-mediated cytotoxicity (n = 4) to 5.8 +/- 1.6% from 23.9 +/- 1.3% in the control group (P < .01).
These results indicated that sHLA-G1 protected xenogeneic porcine endothelial cells against attack by human NK cells, thus providing a new approach to overcome NK-mediated immunity to xenografts.
人类自然杀伤（NK）细胞可介导对猪内皮细胞的直接裂解，在异种移植排斥反应中起重要作用。HLA-G是维持母对半同种异体胎儿免疫耐受的关键分子，能够保护易感靶细胞免受NK细胞诱导的裂解。在本研究中，我们调查了可溶性HLA-G1（sHLA-G1）是否能保护猪异种细胞免受人类NK细胞介导的裂解。
通过核转染将人类sHLA-G1基因组DNA（pcDNA3-sHLA-G1）转染到B淋巴母细胞系721.221（LCL721.221）中。通过RT-PCR和斑点ELISA鉴定转染的LCL721.221细胞的sHLA-G1表达。通过在与溴化氰活化的琼脂糖凝胶4B偶联的抗HLA-ImAb W6/32上进行亲和层析，从转染子的培养上清液中纯化sHLA-G1蛋白。将各种浓度的sHLA-G(1)蛋白（0、2、4、6或8μg/mL）添加到NK细胞介导的异种细胞裂解系统中，该系统由NK92细胞或新鲜的人类外周血单个核细胞（PBMC）与猪内皮细胞系共培养。使用乳酸脱氢酶释放试验评估NK细胞介导的细胞毒性。
sHLA-G1以剂量依赖性方式为猪内皮细胞提供了对人类NK介导的细胞毒性的显著保护。NK92细胞介导的细胞毒性率分别降至83.4±5.7%（2μg/mL）、56.6±9.3%（4μg/mL）、39.3±10.2%（6μg/mL）和31.2±4.9%（8μg/mL），而对照组为96.9±3.0%（P<.01）。同样，添加6μg/mL sHLA-G1可将PBMC介导的细胞毒性平均率（n = 4）从对照组的23.9±1.3%降至5.8±1.6%（P<.01）。
这些结果表明，sHLA-G1保护异种猪内皮细胞免受人类NK细胞的攻击，从而为克服NK介导的异种移植免疫提供了一种新方法。
