A study of soluble HLA-G1 protecting porcine endothelial cells against human natural killer cell-mediated cytotoxicity.
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Zeng
M H
MH
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.
Fang
C Y
CY
Wang
S S
SS
Zhu
M
M
Xie
L
L
Li
R
R
Wang
L
L
Wu
X W
XW
Chen
S
S
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects / Q000276:immunology*; D006680:HLA Antigens / Q000235:genetics / Q000494:pharmacology*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D023421:Models, Animal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection; D014183:Transplantation, Heterologous / Q000276:immunology
D018076:DNA, Complementary; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I