Comparison Of The Phenotypes And Function Of Human Dendritic Cells Induced By IFN-? And IL-4 Combined With GM-CSF

Immunotherapy has become a new mode of tumor treatment besides surgery,radiotherapy and chemotherapy,among which treatment of tumor vaccine is one of the important methods.The development of tumor vaccine has been strengthened in recent years.However,the progress is slow.The main reason is that antigen-specific T cells are hard to be activated by vaccination in tumor patients.How to solve this problem has become the difficulty in the development of tumor vaccine.Tumor therapeutic vaccines need to be effectively presented by APC before activating antigen-specific T cells in the patient's body.DC is the most powerful APC,which can uptake,process and present exogenous antigens to CD4+ T cells in the formation of antigen peptide-MHC II molecule complexes and activate Thi and The effector T cells.In addition,DC can also present antigen peptide-MHC class I molecular complexes to CD8+ T cells,activate the immune response mediated by CTL and eliminate pathogens and tumor cells.IFN-DC induced by IFN-a combined with GM-CSF and IL-4-DC induced by IL-4 combined with GM-CSF are common APC used in immunotherapy.However the phenotypes and function of these two DC still need to be compared to confirm which one is more effective.DRibbles are composed mainly by autophagosome containing many tumor cell antigens.Some studies have shown that DRibbles could efficiently cross-prime antigen specific CTL in mice and activate human antigen-specific CTL in vitro.However,if DRibbles could activate antigen specific T cells in human has not been explored.In this study,human IFN-DC and IL-4-DC were produced by culturing monocytes in vitro.Then the phenotypes and function of these two DC were compared.After that,IFN-DC was combined with DRibbles extracted from hepatic tumor cell lines and injected into the area of inguinal lymph nodes of hepatocellular carcinoma patients.And the activation of antigen specific T cells was detected.This result is beneficial for the DC and DRibbles to be used to treat tumor patients.Objective:1.To compare the morphology,phenotypes and function between IFN-DC and IL-4-DC cultured from human peripheral blood monocytes in vitro.2.To investigate the effect of IFN-DC combined with liver cancer vaccine in activating the antigen-specific T cells in patients with hepatocellular carcinoma.Methods:1.The IFN-DC and IL-4-DC were cultured from human peripheral blood monocytes with cytokines GM-CSF plus IFN-a or GM-CSF plus IL-4 in vitro.Then TNF-a was added into the system to induce the maturation.The morphology of two DC were observed by light microscope and electron microscope.Flow cytometry was used to detect the phenotypes of DC.Two different DC loaded with CMVpp65 protein were used to stimulate the autologous T lymphocytes.The percentages of T cells secreting IFN-y were detected by flow cytometry intracellular staining.ELISA was used to determine the concentration of cytokines(IL-1?,IL-10,IL-12p70,IL-18,IL-23)secreted by two kinds of DC.2.6 patients with hepatocellular carcinoma were enrolled.PBMCs from patients were collected and IFN-DC was cultured according to the ways in the previous description.Then IFN-DC was injected subcutaneously and SMMC7721-DRibbles was injected into bilateral lymph nodes at the same time under the ultrasound detection.After 1 month,IFN-DC was loaded with SMMC7721-DRibbles and injected in the area of bilateral inguinal lymph nodes subcutaneously.PBMCs were collected before and after treatment and the proportion of T lymphocyte subsets in PBMCs was detected by flow cytometry.Then the activation of antigen-specific T cells was detected by ELISPOT and intracellular staining.Results:1.Both of the cultured cells had the typical morphology of DC.The spikes of IFN-DC were shorter and thicker,while IL-4-DC had longer and thinner spikes.Mature IFN-DC contained more organelles than mature IL-4-DC,while mature IL-4-DC contained more vacuoles.2.Monocytes expressed low level of HLA-DR,CD11c,CD80,CD83,CD86.Immature DC expressed higher levels of CD80,CD83,CD86 than monocytes,and the expression of HLA-DR,CD80,CD86 and CD83 of DC were up-regulated after maturation.Compared with IL-4-DC,IFN-DC expressed higher levels of CD80,CD83,CD86.The expression of HLA-DR were similar between IFN-DC and IL-4-DC,but mature IFN-DC expressed lower level of CD11c than mature IL-4-DC.3.For immature DC,both IFN-DC and IL-4-DC secreted few cytokines(IL-10,IL-1?,IL-18,IL-12p70,IL-23).After maturation,large amounts of the cytokines could be secreted by these two DC and mature IFN-DC could secret more cytokines than mature IL-4-DC in general.4.The percentage of autologous CD8+ T cells stimulated by immature IFN-DC loaded with CMVpp65 protein was higher than that by immature IL-4-DC[(0.28±0.07)%vs.(0.12±0.02)%](P<0.05).The percentage of CD8+ T cells activated by mature IFN-DC was also higher than IL-4-DC[(1.03±0.21)%vs.(0.35±0.14)%](P<0.05).5.The treatment of IFN-DC combined with SMMC7721-DRibbles was safe and has no serious adverse reaction.6.After the treatment of IFN-DC combined with SMMC7721-DRibbles,the proportion of CD3+ T cells in PBMC had no obvious change[(60.5±9.9%)VS(60.5±7.9%),P>0.05],the ratio of CD3+ CD4+ T cells to CD3+T cells decreased[(58.9±7.1%)VS(53.3±2.6%),P<0.05],the proportion of CD3+CD8+T cells in the whole CD3+ T cells increased[(28.7±5.9%)VS(35.7+5.6%),P<0.05],the percentage of CD3+CD56+ NKT cells had no obvious change[(5.6±2.6%)VS(6.1±3.3%),P>0.05].7.The treatment of IFN-DC combined with SMMC7721-DRibbles could induce the activation of antigen specific T cells in patients with primary liver cancer.Conclusion:1.Compared with IL-4-DC,IFN-DC could express higher levels of CD80,CD83,CD86,and secrete more cytokines.When loaded with CMVpp65 protein IFN-DC could activate human antigen-specific CD8+ T cells more effectively.IFN-DC might be more effective when used in immunotherapy than IL-4-DC.2.The treatment of IFN-DC combined with SMMC7721-DRibbles is safe and can induce the activation of antigen specific T cells in HCC patients.