Study On Clonal Generation Of Dendritic Cell From Human Peripheral Blood In Vitro And Its Biological And Immunological Characteristics

Dendritic cell-based cancer vaccines have been applied to clinical trials up to date. The sources of human dendiitic cells (DCs) were major from CD34~ hematopoietic stem cells in bone marrow, umbilical cord blood and spleen blood, or from peripheral momocytes directly under stimulation of ceitain cytokine cocktails. In order to prepare DCs with higher quality and quantity for vaccine use, we tried a new method to expand DCs from peripheral blood stem cells and hematopoietic precursor cells in vitro after stem cell mobilization in vivo, and studied their biological and immunological characteristics. Patients were pretreated in routine for stem cell mobilization, then their PBMNCs were separated by Blood Corpuscle Separator CS-3000. Stem cells and hematopoietic precursor cells were isolated after T lymphocytes wipe off by E- rosetting assay and monocyte depleted by adhesion to plastics. Then they were cultured in newborn calf serum supplemented with rhGM-CSF and rhIL-4 to generate SDC. After 2 weeks, the harvested SDC were identified for morphology characteristics by phase-contrast microscopy and electron microscope, surface antigen expression by flow cytometiy and T cell stimulatoiy ability by mixed lymphocyte reaction (MLR) and target cell killing assay. Approximately (0.5?)X iO of SDC could be obtained, which is 10-20 folds of the quantity of original ancestor cells. Phase-contrast microscopy and electron microscope detected that SDC displayed typical morphology. SDC expressed high level surface antigen of I-LLA-ABC(92.6%), 1-ILA-DR(21.5%), CD8O(9.7%) and CD86(64.00/o), and stimulated the proliferation of allo-T cells strongly. The stimulatory index(SI) is 4-5 folds higher than that of PBMNC. Another experiment proved that, SDC loaded with tumor lysate of LOVO cell line could induce antigen- specific CThs which kill LOVO cells specifically. Monocyte-derived DCs cannot be enlarged and usually express low level of HLA-DR, CD8O and CD86. While peripheral stem cell-derived DCs have no these defects. If Blood Corpuscle Separator is available, generating DCs from human peripheral blood stem and hematopoietic precursor cells of patients is a useful method for clinical application of tumor vaccine. We also studied the factors which lead SDC to maturation. Monocyte condictioned medium could raise the antigen-presenting molecule and costimulatoiy molecule expression of immature SDC. It's well known that tumor-associated glycolipid antigen plays important roles in the origin of cancer immune tolerance. Our experiment reveals that glycolipid antigen can be presented by DCs as well as polypeptide antigen , but the mechanism of presentation still needs further study.