| Dendritic cell (DC) has been considered to be the most potent professional antigen-presenting cell (APC) that initiates primary immune responses. Its ability to stimulate and regulate T- and B-cell responses makes DC ideally suited to serve as an adjuvant for thepurpose of cancer immunotherapy. DC also can be involved in the pathogenesis of graft versus host disease or host versus graft disease after transplantation as well as immunization against viral infections and immunosuppression in autoimmune diseases. One possible reason for losing effective anti-tumor immunity is that tumor antigens can not be appropriately presented by DC. Thus, a novel approach to vaccination against cancer is to exploit DC as "nature adjuvant" and actively immunize cancer patients with their own DC pulsed with tumor antigens.Purpose : To establish a method to generate DC from leukapheresis products (mobilized peripheral blood mononuclear cells) of patients with solid tumor in vitro and to identity the phenotypes and the biological functions of DC, then pulsing DC with hepatocarcinoma cell 1\'sates to investigate the ability of DC to induce tumor-specific killer.Methods:1. Generation of DC in vitroAfter the pretreatment for stem cell mobilization, leukapheresis products were obtained by cell separator. The obtained cells (mono-nuclear cell enrichment) were purified by density gradient and polystyrene adhesion. They were then cultured in vitro in cytokine-enriched medium (GM-CSF and IL-4) with different sources of serum: 10% fetal calf serun (PCS) or 2% autologous plasm (AP) of patients. On d5 of culture, cells were washed and suspended in mediumcontaining TNF-a to generate mature DC, which was identified by morphological features, surface antigens expression (analyzed by FCM ) and the ability to stimulate allo-T cells.2. Effector cells culture and cytotoxiciry assay in vitroNon-adherent MNC were resuspended in complete medium supplemented with IL-2 and cultured at 37 C and 5% CO: ( therein called as L ). Culture medium was refreshed every 4 days. HepG2 antigen pulsed DC and L were co-cultured, and the obtained cells were named as Hep-DC-L. DC-L was DC co-cultured with lymphocytes only. Cytotoxicity of Hep-DC-L. DC-L and L were determined by MTT assay. In brief, effector cells ( Hep-DC-L, DC-L, L ) and target cells ( HepG2, SMMC7721. K.562 ) were co-cultured in 96-well plates at different E:T ratios.Results:I.Characterization of DCAfter induction, the cultured cells displayed typical morphology with elongated dendritic processes viewed by inverted microscope as well as electron microscope. DCs obtained from patients were identical to those from healthy donors in terms of phenotypes and the abilities to stimulate proliferation of allo-T cells. Both DCs expressed high level surface antigens- includingCD11c ( 92.27±4.88 )%, CDla( 45.30±5.40 )%, CD86 ( 61.57±6.88 ) %, CD80 ( 54.63±4.25 ) %, HLA-DR ( 82.88±2.32 ) % ( DCderived from patients ); CD11c (88.19±1.35) %, CD la (44.43±6.12)%, CD86( 64.43±4.90)%, CD80 ( 51.91 ±2.95 )%, HLA-DR ( 88.51 ±2.68 ) % ( DC derived from healthy donors ), respectively. The results of mixed lymphocyte reaction ( MLR ) showed that both DCs had the ability to stimulate high proliferation of allo-T cells. DC differentiated in autolgous plasm demonstrated more mature behavior than did DC cultured in PCS.2. Results of cytotoxicity activityAt different E:T ratios ranged from 5:1 to 40:1, the cytotoxic activities of Hep-DC-L to HepG2 cells were 23. 6±2. 77 (5:1), 30. 9± 1. 97 10:10 , 11.2±2. 19 (20:1), 52. 3 ±2. 84 (40:1). respectively. In contrast to Hep-DC-L, there was only minimal lysis of HepG2 targeted by DC-L or L. There was no significant lysis of other cancer cell lines SMMC7721 or K562 at the E:T ratios of 20:1 and 40:1, respectively.Conclusions:1. Mature DC could be generated from enriched MNC of patients with solid tumor, which presents the feasibility for further clinical application in the immunotherapy of cancer.2. DC pulsed by an...
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