Preliminary study of the role of IL-18/IL-18BP in mediating cytotoxic ability of NK-92MI cells against endothelial cells from GTKO porcine models / 器官移植

Objective To evaluate the role and potential mechanism of interleukin (IL)-18/IL-18 binding protein (BP) in mediating the killing effect of natural killer (NK)-92MI cells upon endothelial cells from α-1, 3- galactosyltransferase gene-knockout (GTKO) porcine models. Methods NK-92MI cells were divided into the NK, NK+IL-18, NK+GTKO, IL-18+NK+GTKO and IL-18+IL-18BP+NK+GTKO groups. The messenger ribonucleic acid (mRNA) levels of inflammation-related genes in NK-92MI cells were detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The killing effect of NK-92MI cells on endothelial cells from GTKO porcine models was evaluated by lactate dehydrogenase (LDH) assay. The apoptosis of endothelial cells from GTKO porcine models was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The expression levels of proteins with killing effect and apoptosis-related proteins were determined by Western blot. Results Compared with the NK, NK+IL-18 and NK+GTKO groups, the expression levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-8, IL-3, IL-6 and granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA were up-regulated in NK-92MI cells in the IL-18+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the IL-18+NK+GTKO group, the expression levels of IFN-γ, TNF-α, IL-8, IL-3, IL-6 and GM-CSF mRNA were down-regulated in NK-92MI cells in the IL-18+IL-18BP+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the NK+GTKO group, the expression levels of perforin, granzyme B and IFN-γ proteins in NK-92MI cells were up-regulated, the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was enhanced, the apoptosis rate of endothelial cells from GTKO porcine models was increased, and the ratios of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax)/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were elevated in the IL-18+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the IL-18+NK+GTKO group, the expression levels of perforin, granzyme B and IFN-γ proteins were down-regulated, the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was decreased, the apoptosis rate of endothelial cells from GTKO porcine models was decreased, and the ratios of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were declined in the IL-18+IL-18BP+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Conclusions IL-18BP may block the expression of inflammation-related genes in NK-92MI cells induced by IL-18 and the killing effect of NK-92MI cells on endothelial cells from GTKO porcine models.

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5 / 生物工程学报

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Characterization of swine leukocyte antigen class I genes and homology analysis of the similarity to HLA in GGTA1 -/- Wuzhishan minipigs / 中国实验动物学报

Objective This study was aimed to characterize the swine leukocyte antigen( SLA) class I genes of GGTA1 -/ - Wuzhishan minipigs and compare their similarity to human leukocyte antigen( HLA) . It has important implica?tions for understanding the cellular rejection in xenotransplantation. Methods Specimens of ear tissue from six founding GGTA1 -/ - Wuzhishan minipigs were collected, and the SLA class I genes (SLA?1, SLA?3, SLA?2) were amplified by RT?PCR. Purified products were cloned into pEASY?T1 vectors and sequenced, followed by BLAST alignment and using bioin? formatc analysis to characterize the SLA class I genes and compare with the similarity to HLA. Results A total of six al?leles were detected, among them alleles were previously reported (SLA?1?0703,SLA?2?1102, SLA?3?0401, SLA?3?0403), and the other were novel (SLA?1?0401wz01, SLA?2?11wz01). The homology between alleles of SLA class I genes in Wuzhishan minipigs and HLA was from 70?5% to 72?1%. The homology analysis of critical amino acid residues on HLA binding with human CD8 + molecules showed that SLA?1?0401wz01, SLA?1?0703, SLA?2?11wz01, SLA?2?1102 and SLA?3?0401 occurred mutant at amino acid positions 225 and 228 ( T→S,T→M) , whereas the other loci were highly conserved. There was a high homology at amino acid level between SLA?2?11wz01, SLA?2?1102 and HLA class I genes which are NK cell KIRs binding sites. Conclusions The amino acid sequences of SLA class I genes of GGTA1 -/ -Wuzhishan minipigs have a high homology to HLA. From the point of view of cell?mediated xenograft rejection, the amino acid sequences of SLA class I genes of GGTA1 -/ - Wuzhishan minipigs have a high homology to HLA, therefore, Wzhishan minipigs may become a good potential donor for pig?human xenotransplantation.