Mature Dendritic Cells Transfected With Carcinoembryonic Antigen MRNA And SW480 Total RNA Induce Specific Anti-tumour Effects In Vitro

Immunotherapy served as a part of cancer therapy besides surgery, chemotherapy and radiationtherapy receives increasing attention now. Dendritic cells(DCs) are the most powerful professional antigen-presenting cells, with the ability of initiating and maintaining primary immune responses. Mature DCs express high levels of major histocompatibility complex (MHC) classⅠandⅡand costimulatury molecules. They can also secrete cytokine and chemokines which involved in T-cell activation. They play an important role in tumor, autoimmune disease, transplantation Immunology. Recently, DCs pulsed with different tumor antign that break up tumor protecing mechanism, stimulate specific anti-tumor effect are becoming an attractive tumor vaccination strategy.Tumour-associated antigen(TAA) and tumour-specific antigen(TSA) can be used as targets for immunotherapy. Rectenly, DC-based vaccine which loaded with tumor RNA can overcome many shortcomings of using other antigen and become a promising strategy. RNA can be rather easily obtained and there is no risk for intergration of delivered genetic material in host genome and also no need for using complicated transcripition mechanisms in order to have it properly expressed. Besides, RNA have a higher transfection efficiency contrast to DNA. RNA may encode multiple epitopes corresponding to many HLA alleles, hence, RNA transfected DCs may be used to stimulate T-cell responses in patients without need to determine their HLA haplotype. One significant advantage of using RNA as source of the tumor antigen is that small amounts of tumor are necessary to extract sufficient quantities of RNA and this can be readily amplified using techniques such as polymerase chain reaction. DCs can be procured in two functional states as immature and mature DCs. Until now, the study about DC-based vaccine selective immature DCs and utilize their function of internalizing antigen leading to maturation to induce specific anti-tumor responses, but there are some questions. 1 Immature DCs fail to express costimulatory molecules and thereby can tolerize rather than immunize T cells. 2 They can also induce regulatory T cells that inhibit anti-tumor immunity. 3 Because immature DCs need 24h to mature after transfection before they can be used for vaccination, the intracellular expression of the tumor Ag is very low or absent in this period and result in a lower and shorter Ag presentation on DCs surface ,this could reduce the effects of the anti-tumor. Hence, mature DCs are the better choice as antigen-prestenting cells. Since antigen internalization are not a function of mature DCs and RNA is rapidly degraded by RNases, current studies using RNA transfection are limited by low efficiency of transfection into mature DCs and may result in suboptimal T cell priming. Finding a method which can introducing RNA into mature DCs effectively and stimulate specific anti-tumor response is cricul.Our research pulsed mature DCs with tumor antigen comprise in vitro-transcribed-CEA mRNA and total tumour RNA extracted from the CEA-expressing colon adenocarcinoma cell line SW480 via electroporation and observed the effect of anti-tumor. The aim of this study was to investigate the following questions. First, we tested whether electroporation might be suitable for transfecting the tumor RNA into mature DCs as regards the efficiency and influence on DCs. Sencond, the DCs loaded with in vitro-transcribed-CEA -mRNA and total tumor RNA can elicit the antigen-Specific and tumor-Specific T cell response.Finally, whether the DCs pulsed with total tumor RNA are able to evoke T-cell response against multiple antigens,instead of targeting a single defined antigen.There are four parts in our investigation.1 We acquired monocyte suspensions by Ficoll density gradient centrifugation. The generationg of human DCs from peripheral blood monocytes using GM-CSF and IL-4 and matured with TNF-α. Assessment of DCs was characterized by morphology, phenotype and their T-cell stimulating capacity.2 CEA gene was cloned by RT-PCR method from the CEA-expressing colon adenocarcinoma cell line SW480 and subcloned into the plamsid pMD18-T then cloned into the XbaI and ECORI sites of plamsid pcDNA3.1 to create in vitro transcription plasmid pcDNA3.1-CEA. Identified by enzyme digestion and DNA sequencing, linearization with XbaI followed by transcription with T7 RNA polymerase in vitro.3 Transfecting CEAmRNA into mature DCs and examining the ability of DCs loaded with antigen to stimulate anti-CEA responses.4 Introduced the whole tumour RNA from SW480 into mature DCS, compare the effect of tumor antigen-specific cytotoxic T Lymphocytes with DCs loaded with CEAmRNA.The results are as follows:1 Mature DCs obtained and induced from human peripheral blood mononuclear by GM-CSF,IL-4 and TNF-αin vitro, respectively revealed the typical morphology, phenotype, function and were well be used to the operation of tumor mRNA vaccine during this study.2 The transcription plasmid pcDNA3.1-CEA was constructed using genetic engineering recombination technique. It was determined by DNA sequencing analysis and enzyme digestion that CEA gene cDNA has been successfully inserted into pcDNA3.1 (+), the direction and sequence is entirely correct. Then CEAmRNA was transcripted in vitro .This established the experimental basis for future research on creating CEAmRNA-DC vaccine.3 CEAmRNA and SW480 total RNA could be transfected into mature DCs by Electroporation and express assesed by flow cytometry and immunocyte staining. Electroporation didn't influence the morphology,relevant surface markers observed by optical microscope and flow cytometry.4 DCs loaded with CEAmRNA could stimulate proliferative response of the allogeneic T-cell and induce CEA-specific CTL.The secretion of IFN-γincreased significantly in contrast to control group.5 Specific cytotoxic activity assay confirmed DCs with total tumor RNA could initate CEA-specific CTLs and tumor-specific CTLs.This polyvalent vaccine was able to generate protective responses to some unidentified tumor antigens other than CEA alone.In a word, our results demonstrated that mature DCs electroporated with RNA coding for a TAA or even whole tumour RNA were able to induce antigen and tumour-specific T-cell responses, the latter delivered technique may prime directed more broader potent epitopes. It provided a promising immunotherapy stratergy.