The Immune Tolerance Promoting Effect And Mechanism Of Immature DC Induced By PD-L1-IgG1 Fusion Protein In Cardiac Transplantation

At present, organ transplantation has become the only effective method to treat the end-stage diseases of various organs. Through the transplantation of allogeneic organs, many patients have recovered from the diseases. However, after the foreign organs enter the body, they will inevitably stimulate the immune systems in the receptor, leading to the immune responses and transplant rejection to the transplanted organs. There have found several new immunosuppressive drugs for clinical application to reduce the incidence of transplant rejection, but there are still two problems:first, immune inhibitors cannot completely suppress the rejection reaction; second, long-term use of immunosuppressive agents have many adverse reactions, including excessive drug toxicity, inhibition causing infection and immunity tumor angiogenesis. Therefore, it is necessary to make some researches on how to inhibit allograft rejection and immune tolerance to donor antigen in the body, while making the body maintaining the normal immune response against microbial pathogens infection and tumor occurrence. Seeking for the best solution to the rejection of organ transplantation has become a hot topic in the fields of transplant research.DC (dendritic cells) are very powerful and they are the only antigen-presenting cells that can activate naive T cells. They play an important role in identifying and presenting antigen, starting immune responses and inducing transplant rejection. As DC is a class of heterogeneous cell population with different subsets and different functional status, it not only plays an important role in enhancing immune responses, but also has the potential to inhibit the rejection and induce specific immune tolerance. Research has shown that because immature DC is lack of costimulatory molecules CD80 (B7-1) and CD86 (B7-2) expression, T cells in vitro that can induce antigen-specific anergy get malfunctioned. Inside the body, the immature DC possesses an intrinsic mechanism that is mature naturally and after organ transplantation, a variety of inflammatory mediators produced by recipients, such as proinflammatory cytokines, LPS, can promote DC maturation, so it has strong immunogenicity. Therefore, some measures, such as immunosuppressive molecule 1,25-dihydroxy vitamin D co-culture or IL-10 (interleukin 10), TGF-β (transforming factor β), can make DC at a stable immaturity and enhance their tolerogenic. It is an important way to reduce graft rejection and induce specific immune tolerance. Thus it has great potential clinical value.The most representative costimulatory factor is a member of the B7 family and the earliest B7 family members include B7-1 (CD80), B7-2 (CD86), which can activate naive T cells generating interleukin 2 (IL-2) through stimulates CD28. With the progress of the research, a new member of the B7 family has been found. It is B7-H1 (also known as PD-L1), identified as a ligand to PD-1 (programmed death-1, immune inhibitory receptor programmed death ligand protein). Based on many lab reports, PD-L1 has two effects on T cell responses:stimulation and inhibition and it is still controversial since the mechanism has not been fully elucidated. Scholars have pointed out that after T cells are stimulated for 5-15 minutes, B7-H1 and B7-DC will be combined with the second unknown activated receptor first, upregulate the expression of T cell surface CD40L and further promote the activation of T cells; and 24 hours later, T cells begin to express PD-1, PD-L/PD-1 pathway plays a dominant role, secretion mediated by T cell apoptosis and down-regulation of cytokine, inhibited the proliferation of T cells. Lymphocyte proliferation and cytokine secretion of PD-L1/PD-1 costimulatory pathway can inhibit T cell receptor mediated, phenotypes of B7-H1 mice also showed that B7-H1 has a important negative regulatory role in the body. PD-L1/PD-1 pathway plays an important role in transplantation immunity. PD-L1/PD-1 pathway inhibits GAD (graft arterial disease). Studies have also shown that, PD-L1/PD-1 pathway for T cell signal, affect the acute GVHD (graft-vs-host disease) mortality rate.In view of the important role of PD-L1 in the regulation of DC maturation and immune function, we envision the use of new genetic engineering technology sensitizing immature DC to slow down or stop the mature process of DC. Transfusion in vivo is helpful to reduce the rejection, and possibly may induce stable, effective donor specific immune tolerance.This research intends to build yeast strains with mPD-L1-IgG1 fusion protein, purify the building of mPD-L1-IgG1 fusion protein, culture DC in vitro that expand and gather mouse bone marrow resources, use the fusion protein sensitization of immature DC, to study the function and effect of low reactivity of antigen specific T cells induced by changes in phenotype and function of immature DC sensitized by mPD-L1-IgG1 and in vitro; through the establishment of heterotopic heart transplantation in mouse allogeneic, we tend to observe how mPD-L1-IgGl-DC reduces rejection in vitro, immune tolerance to cardiac allografts induced by effect, and to explore the possible mechanism.Part Ⅰ The expression, purification and identification of mPD-L-IgG1 fusion protein1. The construction of PD-L-IgG1 fusion protein in yeast strainsObjective:To construct PD-L1-IgG1 fusion protein in yeast strains with high expression.Methods:The mPD-L1 gene and Ig gene were cloned in order into the yeast pPIC9k vector to construct pPIC9k-mPD-L1-Ig fusion gene expression plasmid by electroporation of expression plasmids; yeast competent bacteria GS115, nutrition screening by MD board, and G418+ resistance screening high-copy strains of Saccharomyces cerevisiae.Results:The successful construction of the high expression of PD-L1-IgG1 fusion protein in yeast strains.Conclusion:The construction of the mPD-L1-IgG1 fusion protein lays a good foundation for further purification study.2. The expression, purification study of mPD-L1-IgGlFc fusion proteinObjective:To purify and identify PD-L1-IgG1 fusion protein in yeast strains.Methods:To explore different induction time and the content of dissolved oxygen on the fermentation yield of fusion protein expression; to explore and optimize the preparation conditions of purifying yeast in the supernatant of the fusion protein.Results:The successful establishment of fermentation conditions for yeast strains and fusion protein purification methods was determined. The results confirmed the fermentation conditions for yeast strain is fermented 72h, methanol concentration is 8ml/h, optimized the purification method of fusion protein established a purification process of mPD-L1-IgG1 fusion protein, finally got the purity of >95%mPD-L1-IgG1 fusion protein. Endotoxin content is less than 0.03pg/μg protein.Conclusion:Preparation of the biological function of the experimental requirements of the mPD-L1-IgG1 fusion protein lays a good foundation for further function study.Part Ⅱ PD-L1-IgG1 fusion protein maintained DC immature state and induced the low reactivityObjective:To establish PD-L1-IgG1 fusion protein sensitized DC method by studying the immature state of fusion protein DC and low reactivity DC induced.Methods:With the mPD-L1-IgG1 fusion protein sensitized in vitro amplification fifth days BALB/c mouse bone marrow-derived immature DC, change the phenotype before and after LPS stimulation was detected by flow cytometry in sensitized DC; BALB/c LPS-DC, imDC and mPD-L1-IgG1-DC source, for mixed lymphocyte reaction with different proportion of mice with C57BL/6 derived T cells (MLR) determination of content, and cytokine response in the supernatant; BALB/c mice were sources of imDC and mPD-L1-IgG1 sensitized DC were injected through caudal vein of the mice of C57BL/6 (2×106 cells/mouse),7 day taking the recipient spleen, MLR detection of the recipient spleen T cell and the donor and unrelated third spleen lymphocytes.Results:Due to high sensitivity DC surface expression of mPD-L1-IgG1 fusion protein, sensitized DC can effectively resist LPS stimulation while maintaining low mature state, expression of the surface CD80, CD86 and HLA-DR molecules; the ability of sensitized DC stimulation of allogeneic T cell proliferation in the MLR reaction was significantly lower than that in the control group, the secretion of Thl factor (IL-2, IFN-γ) decreased significantly, Th2 factor (IL-4, TGF-β) increased significantly; sensitized DC immunized spleen T lymphocyte contact with donor antigen, showed low apparent reactivity.Conclusion:mPD-L1-IgG1 can effectively sensitize immature DC, maintain DC phenotype and function remains immature, low reactivity induced by DC.Part Ⅲ Mouse model of heart transplantation with acute rejectionObjective:To build a mouse model of heart transplantation for the research of acute transplantation rejection.Methods:The donors and acceptors in the experiment group were BALB/c and C57BL/6 mice respectively(n=20), and those in the control group were 20 pairs of BALB/c mice. The "cuff" vascular anastomosis technique was used for heterotopic heart transplantation at the neck of mice. The tissues of transplanted hearts from 10 subjects of each group were taken for pathologic observation on Day 7. The survival time of the transplanted hearts were observed in the rest of subjects.Results:The success rate of the operation was 92.5%. At 7 days after translation, the pathologic examination revealed that the rejection level were 3.55±0.44 in the experiment group and 0.40±0.32 in the control group, and there was a statistically significant difference between them(P<0.05). The survival time of the transplanted hearts in the experiment group was (8.22±0.67) d, and that in the control group was more than 100 d, and there was a statistically significant difference between them (P<0.05).Conclusion:A stable mouse model of heart transplantation from BALB/c to C57BL/6 has been built by using the "cuff" method. This method has a high operative success rate and is worthy of being extended.Part Ⅳ The immune tolerance effect and mechanism of PD-Ll-IgGl-DC induced in mice heart transplantationObjective:To build PD-L1-IgG1-DC mouse heart transplantation immune tolerance induced in vivo model, to explore the mechanism of negative immune function.Methods:Select the C57BL/6 mice as receptor, in 7 days before transplantation, mice was injected BALB/c source Day5-DC and mPD-L1-IgGl-DC via caudal vein (2× 106 cells/mouse), group PBS as the control group, were of heterotopic cervical heart transplantation, observe the cardiac allograft survival; 7 days after operation to observe the pathological changes of cardiac allograft, and the serum levels of cytokines were assayed in vivo; analysis of the recipient spleen T cells of donor antigen stimulation and CTL effect.Results:mPD-L1-IgG1-DC group of cardiac allograft survival (24.2±3.29) d, than in the Day5-DC group and the control group were significantly prolonged (P<0.01); pathological changes of sensitized DC group of cardiac allograft was lighter than that in the other 2 groups, Th1 serum cytokines (IL-2, IFN-y) level than the other 2 groups decreased, while the elevated levels of TGF-0, Th2 cytokine IL-4 levels have no significant difference;mmPD-L1-IgGl-DC group of spleen lymphocytes of donor antigen stimulation decreased significantly (p<0.01), the CTL reaction of target cell killing activity was weaker than that of Day5-DC group and the control group (p< 0.01); mPD-L1-IgG1-DC group immunized mice with CD4+CD25+Foxp3+Treg regulator T cells increased significantly.Conclusion:mPD-L1-IgG1-DC can effectively induce mouse heart transplantation immune tolerance, strong negative immune response performance of control. The negative immune regulation function is mainly due to PD-L1.SummarymPD-L1-IgG1 fusion protein can effectively sensitize immature DC and construct mPD-L1-IgG1-DC. Such new DC can inhibit allogeneic T cells proliferation in vitro, and the inhibition of Thl cytokine production in mice; BALB/c from mPD-L1-IgG1-DC into C57BL/6 mice through caudal vein, antigen-specific hyporesponsiveness induced the recipient to donor. Application of mice heart heterotopic transplantation model in allogeneic transplantation confirmed,7 days before intravenous injection of mPD-L1-IgG1-DC can effectively prolong the survival time of cardiac allograft, significantly reducing pathological changes of heart transplantation. The analysis indicates that the effect of mPD-L1-IgG1-DC in vivo by PD-Ll effectively induced regulatory T cells to maintain immune tolerance state. This research has studied the effect of mPD-L1-IgG1 fusion protein sensitization of DC induced resistance as a primary transplantation tolerance induction effect and provided a new experimental basis to further explore the immune negative factors regulating subgroup DC prevention of allograft rejection...