Immunological Tolerance Of Normal Human Hepatocytes Cell Line L02 Induced By CTLA4Ig Gene Modification

Hepatocytes transplantation has become an important treatment of patients with end stage liver failure and cirrhosis. Potential advantages of cell transplantation include a simpler, safer, less invasive and costly procedure, and more efficient use of donor organs compared to liver transplantation for treatment of liver diseases. However, hepatocytes transplantation still face immunological processes in liver transplantation.Cytotoxic T-lymphocyte associated with antigen 4-immunoglobulin (CTLA4-Ig) could block the CD28/B7 co-stimulating pathway, inhibit T cell activation, and thus induce the immunological tolerance to specific antigen. There are massive data indicating that tolerance of graft could be induced by systemic administration of CTLA4-Ig protein or transduction with CTLA4-Ig gene in vivo. However, systemic administration of CTLA4Ig has the potential to inhibit the immune system extensively and cause unwanted systemic adverse effects (e.g., susceptibility to infections and malignancy). In this study, we expected to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation. Main methods and results are as follows:1. Biological characteristics of L02 cells transducted by Ad-CTLA4Ig-EGFP.Normal liver cells L02 were transducted by Ad-CTLA4Ig-EGFP; gene transferring efficiency was detected through flow cytometry; immunocytochemistry, Western Blotting and ELISA were used to detective the expression of CTLA4Ig by L02 cells; changes in biological characteristics were observed through cell proliferation curve and urea synthesis.1.1 Green fluorescence could be observed in the endochylema within 6 hours post-transduction, which reached the highest fluorescence intensity at 48~72 hours post-transduction, and lasted for more than 4 weeks. Gene transferring efficiency reaced 88.0% when MOI=600. Immunocytochemistry, and Western Blotting indicated that CTLA4Ig were highly expressed in cytoplasm and could be secreted to culture supernatant. The peak concentration of CTLA4 in supernatant was (16.55±1.20)ng/ml at D7 post-tansducted.1.2 Cells growth rate stepped down after gene transfer, but with no statistical significance. However, urea synthesis was enhanced after gene transfer(P<0.01).2. L02 modified by CTLA4Ig gene (CTLA4-L02) induced immunological tolerance both in vitro and vivo.2.1 Proliferation of rat splenocytes was inhibited when co-cultured with CTLA4-L02 (P<0.05), inhibition rate reaced 36.8%; the inhibited splenocytes kept a low proliferation when co-cultured with normal L02 cells, but showed a high proliferation when co-cultured with Hela cells(P<0.01).2.2 CTLA4-L02 cells were splenically injected into SD rats which were performed 2/3 hepatic lobectomy, survival L02 cells which expressed human ALB could be observed in experimental group at the 3rd and 4th week post-transplantation, while none was detected in control group. Percentage of CD4+ and CD4+CD69+ T cells in transducted group were (24.48±1.65)% and (45.11±2.83)%, which were significantly decreased compared to (35.30±2.05)% and (55.22±2.79)% in control group at day 7 post-transplantation (P<0.01). Meanwhile, IL-2 level was also in lower level in transducted group (204.15±1.10 pg/ml) than that in control group (223.52±2.76 pg/ml, P<0.01).Conclusions1. CTLA4Ig gene could be transferred into L02 cells high efficaciously mediated by adenovirus vector. After transducted, L02 cells could express CTLA4Ig in cytoplasm and secreted to culture supernatant, which lasted for more than 4 weeks. No obvious negative change of biological characteristics was observed.2. CTLA4-L02 cells appeared immunological inhibition activity in vitro, which could inhibit the proliferation of rat splenocytes, and induce immunological tolerance, and this tolerance appeared antigen specificity to some extent. After transplanted into rat, CTLA4-L02 could inhibit the proliferation and activation of CD4+T cells, decrease the level of IL-2 and induce immunological tolerance of itselves to survive in rat liver.