Experimental Investigation On The Generation Of Tolerogenic Dendritic Cells And Their Role In Transplant Tolerance Induction

Objective To investigate the mechanism of inducing tolerogenic dendritic cells; to investigate the role of tolerized DCs in the induction and maintenance of transplant tolerance.Methods (1) Retroviral Foxp3 gene transfer was applied to na?ve CD4+CD25-T cells. Then the expression level of co-stimulatory molecules CD80 and CD86 on the membrane of dendritic cells were analyzed by FACS. The immunological function of tolerized DCs was studied through mixed lymphocyte reaction (MLR). Also the transwell co-culturing system was applied to study the contact impact of DCs-T cells. (2) The leukemic cell line K562 was to be transduced with HLA-G constructs and to be used to co-culture with DCs. Then their membrane molecules such as CD80, CD86, ILT3 and ILT4 expression levels were detected by flow cytometry. Allogeneic proliferation of peripheral blood mononuclear cells (PBMC) was detected by mixed lymphocyte reaction (MLR). (3) The DNA sequence of the ILT4 promoter area was amplified with the human genomic DNA by PCR. And then the segment was cloned into the eukaryotic expression vector pGL3-Basic. The recombinant was then testified by the double endonuclease digestion and sequenced. Plasmid pGL3-ILTP was then transfected into Human acute monocytic leukemic cell line THP-1 cells by lipofectamine. The activity of luciferase was detected, and the effect of the ILT4 promoter was studied. After culturing with IL-10 for a limited time, THP-1 cells were harvested and their ILT4 expression was detected via FACS analysis. Meanwhile, the mRNA was extracted form THP-1 cells for further RT-PCR test. The dual-luciferase reporter assay system was applied to study the ILT4 promoter activity with or without IL-10 inducing. (4) The peripheral blood mononuclear cell (PBMC) from renal transplant donors were collected before the operation, and then part of the donor derived PBMC were cryopreserved. PBMC from every subject were separated by density gradient centrifugation. CD8+ T cells were positively selected from PBMC by anti-CD8 antibody-coated magnetic beads according to the manufacturer's instructions. Human DCs were derived from the adherent fraction of donor PBMC by culturing 7 days in defined medium containing human GM-CSF and IL-4. Total RNA from CD8+ T cells and DC were extracted using the RNA extracted with Trizol RNA isolation according to the manufacturer's instructions. During cells co-culturing, CD8+T cells were treated with 20μg/ml mitomycin C at 37℃for 30 minutes and then incubated with DCs for 18 hours. The cells were then stained with antibodies and analyzed by FACS.Results (1) The ectopic expression of Foxp3 was detected as stable and continuous. The results showed that Foxp3 transduced CD4+CD25-T cells could function on the expression level of co-stimulatory molecules, down-regulate CD80 and CD86 on the membrane of dendritic cells. It was showed by in vitro lymphocyte proliferation test that forced expression of Foxp3 on na?ve CD4+CD25-T cells could suppress the immune activity of dendritic cells. The experiment also showed that CD4+CD25-T cells with forced expression of Foxp3 fulfill their regulatory function on dendritic cells was cell contact dependent. (2) It showed that CD80 and CD86 expression on DCs were down-regulated, while ILT3 and ILT4 expression were up-regulated after co-culturing with K562-HLA-G cells. The DCs were less able to stimulate the allogeneic PBMC. (3) The ILT4 promoter sequence was cloned and inserted to the pGL3-Basic vector. By double endonuclease digestion and DNA sequencing, the sequenced DNA in the recombinants was identified to that Genbank had reported and the segment was inserted in the right direction. The activity of luciferase was significantly increased in the pGL3-ILTP transfected THP-1 cells over ten times than that of pGL3-Basic transfected THP-1 cells. After culturing with IL-10, FACS analysis showed that ILT4 expression of THP-1 cells was up-regulated. Meanwhile the mRNA level of ILT4 was clearly higher than that of the control group. To further discover the impact of IL-10 on the promoter activity of ILT4, The dual-luciferase reporter assay system showed that ILT4 promoter got much more activity after IL-10 treated. (4) CD8+T cells from PBMC of renal transplant recipients in quiescence showed an increase in the Foxp3 mRNA level compared with those collected before renal transplant. After co-culturing with CD8+T cells from PBMC of renal transplant recipients in quiescence, the donor derived DCs showed an increase in both the ILT3 and ILT4 expression levels.Conclusion (1) Foxp3-transduced CD4+CD25-T cells suppress function of dendritic cells, inducing tolerogenic DCs. The tolerized DCs have their specific phenotype. Induction of T cells anergy may be involved in the fulfillment of their inhibitory function. (2) The membrane-bound HLA-G could up-regulate inhibitory receptors ILT3 and ILT4 expression, inducing tolerogenic DCs in vitro, which may provide a novel strategy for transplant tolerance inducing. (3) The eukaryotic luciferase expression vector, pGL3-ILTP containing human immunoglobulin-like transcript promoter was constructed successfully. It might render a specific technique/method for the study of regulatory mechanism of ILT4 expression. FACS analysis and RT-PCR study both had shown that IL-10 could up-regulate the ILT4 expression levels in the transcriptional and translational level. The IL-10 also showed its positive role in the augment of ILT4 promoter activity as detected via dual-luciferase reporter assay system. (4) CD8+Foxp3+T cells subgroup have been augmented during the inducing and maintenance of transplant tolerance. This could be proved by the fact that Foxp3 mRNA level of CD8+ T cells subgroup was clearly up-regulated, indicating an augment of CD8+Foxp3+T cells subgroup in the PBMC of recipients. CD8+Foxp3+T cells could induce tolerogenic DCs both in vitro and in vivo. The induced tolerant DCs were charactered as high ILT3/ILT4 expression levels. This tolerance induction function of CD8+Foxp3+T cells was MHC-I restricted, for only the APC from their respective donors could be induced to express ILT3/ILT4 by CD8+Foxp3+T cells from recipients. However, the APC from unrelated persons could not be induced to be ILT3+ILT4+ DCs.