Mechanism And Application Of Recombinant Yeast Regulation Of Intestinal Immune Dendritic Cells(CD11c~+DCs)

Dendirtic cells （DCs） are the most efficient antigen present cells. As immune sentinels,DCs are ideally positioned throughout the entire body, sample the environment pathogens,transport antigens from the periphery to lymphoid tissues and trigger the immune response.And Saccharomyces cerevisiae, historically used in food precessing, has been demonstratedto enhance human immune system. Based on these observations, we designed series ofexperiments to uncover how the yeast reprogramed intestinal DCs global gene expressionupon uptaken into the DCs. We also engineered the yeast to deliver a functional gene（β-catenin） into DCs and examined the effect of its expression within DCs on immuneresponse. We finally tested the capability of recombinant yeast delivery of protein antigen andfunctional genes into DCs.We demonstrated for the first time how yeast reprogramed intestinal DCs geneexpression.Transcriptome analysis of isolated intestinal CD11c⁺DCs after oral administrationof yeast indicated that yeast up regulated1558genes and down regulated754genes. The GOand KEGG analysis of the DEGs showed that genes involved in the receptor pathway and thecellular compents were significantly influenced by the oral adminstration of the yeast.Thebiological function analysis of these genes indicated that the immune system process wassignificantly changed.We next engineered the yeast to deliver functional gene into intestinal DCs. β-catenin isan essential protein in Wnt/β-catenin pathway and plays an important role in maintaining theimmunity balance in intestine. We constructed a recombinant yeast containing mammaliangene expression cassette β-catenin-HA and β-catenin-HA-GFP driven by CMV promoter, andnamed them as JMY-Pcat and JMY-Pcat-GFP, respectively. Then mouse C57BL/6wasimmunized with recombinant yeast stains JMY-Pcat and JMY-Pcat-GFP by oraladiminstration of the yeast for5days. By analysing the CD11c⁺DCs cells isolated from theintestine with CD11c⁺magnetic activated cell sorting, we confirmed that β-catenin wasexpressed in a higher level in the group JMY-Pcat-GFP. The expression of co-stimulatorymolecules CD80, CD83and CD86of DCs were increased by qRT-PCR detection inJMY-Pcat group, while the expression of MHCⅡ was decreased compared with other groups.We also detected the expression of β-catenin or GFP in intestinal tract, lymph, thymus glandand spleen tissue of JMY-Pcat group or JMY-Pcat-GFP through the immunohistochemical experiment, indicated that the DCs were sitmulated and migrated into the immune tissues. Atthe same time, the concentration of IL-12, TNF-α and IFN-γ which related with immuneresponse were lower in group JMY-Pcat-GFP group, while the concentration of immunetolerance related cytokines IL-6, and IL-10were higher in the JMY-Pcat group orJMY-Pcat-GFP group, suggesting that the over expression of β-catenin in DCs triggeredimmune tolerance in mice.We tested the capability of the recombinant yeast mediated delivery of myostatin proteinantigen into DCs and examined its immune response. During conducting the experiments, wedeveloped a new method for protein extraction from yeast Saccharomyces cerevisiae cellsusing LiAc/NaOH and tested the application of the protein prepared with this new method inWestern Blot. To develop a vaccine grade of recombinant yeast, we constructed a MSTNgene integrated yeast strain. With the oral delivery of this recombinant yeast strain for aperiod of12weeks, the body weight increased significantly compared with the control group.The new MSTN integrated yeast strain can be cultured in YPD medium. Establishment ofsuch yeast strain is a step further toward transformation of yeast cells into edible vaccine togene therapy and feed additives.Finally, we established a hybridoma-free monoclonal antibody production method forhApoB100medicated by SV40T/p53transgenic mice and recombinant yeast oralimmunization technology. The expression plasmid JMB88-OVA-HA-hApoB100wasconstructed by inserting hApoB into JMB88-OVA-MSTN, and used for transformation ofyeast strain JMY1with CuSO₄induction for expression of hApoB100. Then the recombinantyeast JMY1-OVA-HA-hApoB was used to orally immunize SV40T/p53transgenic mice.Then we isolated splenocytes, cultured them in the medium supplemented with Dox forimmortalization, and subsequently subcloned by limited dilution method to obtain the cellclones that could secrete the hApoB100antibody and be immortal. We showed thatsplenocytes derived from transgenic mice harboring a simian virus40large T antigen andmouse p53gene under the control of Tet inducible promoter were conditionally immortalafter doxycycline induction and could produce monoclonal antibodies. This novel approachmay become a method of choice for production of both polyclonal and monoclonalantibodies.