Effect Of Carcinoma Supernatants On Killing Activity Of CTL Induced By Dendritic Cells

Bckground:Dendritic cells(DC),acting as professional and the most powerful antigen presenting cells,can effectively intake,process and present antigens,activate initial T cells ,and thereby work as the center of immune responses. Much is known of their important role in anti-tumor,resisting injection ,transplant repulsion and anti-self immuniy disease.It also have been confirmed that immature DC show significant capacity of intake,process antigen and transfer ability,but have low level expression of MHC- II and co-stimulate molecule on membrane,and low ability of activate T cells. On the contrast,mature DC lost their capacity of intake and process antigen,but efficiently prime antigen-specific lymphocyte activity.Therefore,DC play a center role of startup immunity T lymphocyte activity.Tumor can express self antigen like normal cells, and can express aberrant or unique antigens which can be recognize by immunity system, which is the foundation of tumor immunotherapy. DC-based cancer immunotherapy is one of important direction in tumor biologic therapy in the anti-tumor responses, DC entry tumor from peripheral blood and intake ,process tumor antigen, synthesize compound with MHC- II molecule and tumor antigen, then express on membrane.When DC circumfluence back to lymphoid tissue and embay T cells, which get tumor antigen presented by DC and be activated, initial T cells become especially cytotoxic T cells. DC mature gradually in the process of transference, and decline the capacity of intake antigen but increase activity of present antigen, also acquire representative burr cytokituber, increase MHC- II and B7 molecule expression. It have been known now, immunity function of DC in vivo of tumor patients beinfluence on differ approach and degree, cannot activate effective T cell immunity. When DC from tumor patients be cultured in vitro and pulsed ,then injected to patients, can activate effective T cell immunity. Madhav et al discovered that tumor responsive T cells which be activated DC pulsed tumor antigen in vitro were especial anti-tumor but not anti-self lyphocyte.At present DC be used for lot of tumor therapy, and positive results have been gotten in I / II phase clinical study and show capacious foreground in clinical application of tumor therapy. But some problem exist in clinical application. Though CTL activated by DC induced by tumor antigens can kill tumor cells significantly in vitro, it did not like this in vivo or in clinical application. It is not clear how tumor cells affect DC or DC bacterin now, and the inhibition that tumor cells affect DC may be one of reasons. It can be hope that if we get significant progress in the mechanisms between tumor cells and DC, we can exert the bacterin of DC better.Objective: We plan to establish a feasible method to isolate and induce of DC from human peripheral blood; study the proliferative ability and cytoxicity of T cells from human peripheral blood with DC; investigate the the effect of tumor cell supernatants on anti-cell reaction with CTL induced by monocytes-derived DC.Method: 1 > DC were derived from human PBMC in the presence of GM-CSF, IL-4 and TNF-a.The process of DC growth were observed with optics and electron microscope, then examined the express ratio of immune molecule HLA-DR, CD83, CD86 and CD la on DC membrane with FCM when DC were cultured in fifth and seventh day.2n Tumor cells antigens of LS174,HepG2,L02 cells were obtained with the method of freezing-fusing.3> DC pulsed by antigens were mixed with T cells from human peripheral blood in differ proportions, then induced CTL.4> CTL were mixed with target cells in differ proportions, then ratio of CTL killing target cells were examined for method of MTT.5 > In the process of cultiviting DC, carcinoma supernatants were put in for seven days, then examined the express ratio of immune molecule HLA-DR, CD83, CD86and CD la on DC membrane, compared with normal cultured DC.6> Carcinoma supernatants were put in with DC in first, fifth, seventh day in the process of cultivating DC and in the process of pulsed DC activating T cells respectively, ratio of CTL killing target cells of all groups were examined for method of MTT.Results: 1 -. After culture with GM-CSF,IL-4 and TNF-a,mature DC which have representative burr cytokituber were acquired with MTT and electron microscope.2, High ratio of CTL killing target cells were gotten with CTL activated by DC, which were pulsed with tumor antigens of LS174 cells with the method of freezing-fusing. When the proportions of available CTL and target cells were 25 '. 1,50 .' 1, the ratio of CTL killing target cells increased with the proportions of CTL and target cells. When the proportions of available CTL and target cells were 100 : 1, the ratio of CTL killing target cells were higher than other proportions, but did not increase with the proportions of CTL and target cells.3> Antigens with the method of freezing-fusing can pulse DC and especially killing effection of CTL were objected.4> The proliferative ability of DC cultured with carcinoma supernatants were low and DC displaying representative burr cytokituber were low. Ratio of immune molecule HLA-DR, CD83, CD86 and CD la on DC membrane were lower than that of DC cultured with normal culture medium.5 n The ratio of CTL killing target cells decreased when CTL which activated by DC cultured with carcinoma supernatants in first and thirst day of DC cultured, but not in fifth.Conclusions: Mature DC can be cultured with GM-CSF,IL-4 and TNF-a ;antigens with the method of freezing-fusing can pulse DC efficiently and especially killing effection of CTL can be induced; tumor-derived supernatants can decrease ability of DC to generate effective anti-tumor immune responses.