The Pilot Study Of Obtaining The Dendritic Cell Low-expressing CD80,CD86 By IL-3

Aims Allograft rejection is the biggest hindrance in the field of organtransplantation. The survival of transplated organs has been significantly lengthended over the last two decades by immunosuppression drug. But cancer and infection derived from the side effects of immunosuppression limit the long-term successs in vascularized organ transplantation. The ultimate goal of clinical organ tansplatation is to induce immunological tolerance to the allograft.Dendritic cells (DCs) are essential antigen-presenting cells (APC) that initiate and regulate adaptive immune responses. They are able to capture and present antigens to T cells which results in the two opposite outcomes: potent activation (immunogenicity) or inhibition (tolerance) of the immune response. DCs derived from myeloid and lymphoid lineages, which derived from the same CD34 hematopoietic progenitor cell (HPC) of the bone marrow (BM). Myeloid DCs were named CD11c~+DCs or DC1 and correlative with monocyte/macrophage lineage. Lymphoid DCs subset were named plasmacytoid dendritic cells (PDCs), CD11c~- DCs or DC2, as the key cells which had the capacity to produce IL-10 and induce Th2 response and immunological tolerance.Now many manipulations of tolerance induction in transplantation had been widely developed, such as : CTLA4-Ig and antisense-Peptide nucleic acid(PNA) ,by which technique the expression costimulatory molecules could be blocked, and alloantigen specific T-cell hyporesponsiveness could be induced. But the inferior stabilization and permanence of these methods greatly limited the clinical therapy. GM-CSF is a key cytokine for myeloid DCs propagation. It is well known that mature DCs are cultured from bone marrow progenitors, peripheral blood monocytes (PBMC) with the stimulation of GM-CSF and IL-4. But in this culture system, just the myeloid DC was obtained. Lu reported that (CD80dim, CD86 )DC can be propagated from mouse bone marrow(BM) in low concentrations of GM-CSF. But costimulatory molecule expression on these DC and their sllostimulatory function are up-regulated upon maturation after cultured in GM-CSF+IL-4. DCs derived from plasmacytoid precursors rely on IL-3 for survival and proliferation.Ebner applied IL-3+IL-4 to culture PBMC, and obtained DC2 with abundant IL-3 receptor. PBMC was in haematopoietic terminate stage, with the characteristic of small quantity and low propagation, not suitable for DC2 propagation. This study focus on how to obtain PDC derived from bone marrow and obtain DC which could induce tolerance by substituting IL-3 for GM-CSF. This study applied IL-3+IL-4 and GM-CSF+IL-4 to culture mouse bone marrow derived DCs and compared IL-3 DCs with GM-CSF DCs on the aspect of morphology, yield, the phenotype and the ability of antigen capturing and presenting.Materials and Methods This study applied IL-3+IL-4(DCs inducedby IL-3+IL-4) and GM-CSF+IL-4(DCs induced by GM-CSF+IL-4) to culture mouse bone marrow derived DCs. IL-3 DCs and GM-CSF DCs were compared on the morphology, yields by phase-constrast microscope, scanning electron microscope, laser scanning confocal microscope (LSCM).DEC205, as a specific marker of DC, was detected by immunology histochemistry (IHC). Immunity phenotypesCMHC IK CD80>CD86>CD14) were detected by flow cytometer (FCM) and the endocytic activity was measured by FITC-DX. Immunostimulatory capacity in the mixed leukocyte reaction was detected by 3H-TdR incorporation assay.Results (D.IL-3 DC and GM-CSF DC consist of 1.0-1.3X107 progenitors obtained from the fresh isolated Balb/c mouse bone marrow were havested on the ninth day. The average yield of IL-3 DC was about 6.7 X 106 DC. The average yield of GM-CSF DC was about 3.3X106DC . ?.The expression rate of DC specific marker DEC-205 in IL-3 DCs and GM-CSF DCs was about 80% purity and 70% purity, respectively. ?-The morpholgy of IL-3 DC showed much dendritic outshoot and downy structures protruded from the cell membrane on the phase-contrast microscope and scanning electron microscop. HE staining showed that IL-3 DCs were oval or fan, with the eccentric nucleus. Cell cytoplasm was basophilic similar to the plasma cell. GM-CSF DCs had much short protrude, with the kidney-shaped nucleus. ?.The expression of CD80,CD86,CD14 in IL-3 DCs were negative, and the positive expression rate of major histocompability complex (MHC) class II was 68.5%. Addition of TNF- a into culture, resulted in high-expression of CD80, CD86 and keeping in the costimulatory molecule-deficient state.?. The positive expression rate of FITC-DX of IL-3 DC was 90.3%, average fluorescence intensity was 29.4 by FCM. The positive expression rate of FITC-DX of GM-CSF DC was 55.9%, average fluorescence intensity was 11.3 by FCM. ?.IL-3 DCs and GM-CSF DCs cocultured with C57BL/6 mouse spleen cell respectively lead to IL-3 DC weak allostimulatory activity, MLR which showed significant difference with GM-CSF DCs. (PO.05)Conclusion The findings show that IL-3 could substitute GM-CSF inDCs culture from mouse bone marrow cells. This innovative culture method comes to higher yield and purity of costimulatory molecule-deficient DCs(MHC class II+, CD80 ,CD86 ). These DCs had stronger endocytic activitythan GM-CSF DCs, and could induce alloantigen-specific hyporesponsiveness in vitro. The study of inducing bone marrow differentiateinto CD80 , CD86 DC by IL-3 had not been seen in recent relative report.Our study supply a new insight in transplant tolerance.