PDL1Ig gene-loaded BMSCs Induce liver transplantation immune tolerance.

Abstract

We investigated the effects of BMSCs, which was modified by programmed death ligand-1 immunoglobulin (PDL1Ig) gene, on the immunological rejection of orthotopic liver transplantation (OLT) in rats. Rat BMSCs were cultured and modified by recombinant adenovirus pAdEasy-1/PDL1Ig for 72h. The total protein was extracted, and the protein expression of PDL1Ig after transfection was detected by Western blotting. Mixed lymphocyte reaction was applied to detect the inhibitory effect of BMSCs, including pre-transfection and post-transfection, on the cell activity of T lymphocytes in peripheral blood. The male Wistar rats were used as donors, and the male Sprague-Dawley (SD) rats were used as recipients. The improved cuff method was computed in OLT to establish the rat orthotopic liver transplantation model of acute rejection. The rats were randomly divided into 4 groups, including a control group (10 pairs), BMSCs treatment group (10 pairs), BMSCs/GFP (green fluorescent protein) treatment group (10 pairs), and BMSCs/PDL1Ig treatment group (10 pairs). 5 rats in each group were randomly collected and euthanized at day 7 after the operation. The peripheral blood was gathered to detect levels of 3 types of cytokines, including interferon gamma (IFN-g), interleukin-4 (IL-4), and IL-2. In addition, the liver function of rats was checked, and the pathological changes of liver transplantation were observed under the optical microscope. The remaining five rats in every group were used for measuring the survival situation and survival time. After BMSCs were modified by a recombinant adenovirus pAdEasy-1/PDL1Ig for 72h, the expression of PDL1Ig in the BMSCs was detected. The inhibitory effects of BMSCs/PDL1Ig on the cell activity of lymphocytes were stronger than that of BMSCs/GFP. The levels of IL-4 in BMSCs/PDL1Ig group were significantly higher than those in the other 3 groups, and the levels of IFN-g and IL-2 were significantly decreased (p<0.05). The liver function of 4 groups was measured at day 7 after transplantation. Our results showed that the liver function in BMSCs/PDL1Ig group was most significantly improved, and the levels of ALT, AST, and TBil almost recovered to normal. The differences were statistically significant compared to the control group, BMSCs treatment group, and BMSCs/GFP treatment group. Results of pathological examination of liver tissue showed that the control group underwent severe rejection of liver transplantation. The BMSCs treatment group and BMSCs/GFP treatment group also rejected the liver transplantation, but the degree was lighter when compared to the control group. In addition, there was almost no rejection of liver transplantation in the BMSCs/PDL1Ig group. The recipient survival time of most rats was more than 100d, which was significantly longer than the other 3 groups (p<0.05). PDL1Ig-modified BMSCs can inhibit the rejection of liver transplantation in rats, induce the formation of the immune tolerance of liver transplantation, and the effect was more significant than that of BMSCs alone.