Carry An Antibody Of Cd40 Single Dendritic Cells Of Tumor Immunotherapy And Cd4 ~ + T Cell Surface Expression Of Cd40 And Action Research

Part I Cancer immunotherapy using dendritic cells carryingagonist anti-CD40scFvObjective:The combination of CD40stimulation and interleukin-2(IL-2) leads to synergisticantitumor responses in several models of advanced metastatic disease. However,although this combination can effectively promote the primary immune response, it candiminish the secondary immune response. Because CD4~+T cells are essential for thegeneration and maintaining of CD8memory T cells, the apoptosis of CD4~+T cellsinduced by IFN-γ is thought to be the reason for the reduced CD8~+T cell memoryresponses. Macrophages activated by anti-CD40antibody could secret large amount ofIFN-γ, which might affect the survival of CD4~+T cells. Therefore, we first comfirmedthe differential effects of anti-CD40antibody stimulation on DC and macrophages.Then we cloned the scFv of the anti-CD40agonist antibody and transfected the DCsthrough lentiviral infection. We further evaluated the anti-tumor effects of the DCcarrying anti-CD40single chain antibody variable fragment (scFv) through in vitro andin vivo experiments.Methods:（1）To analyse the proliferation and apoptosis by flow cytometry of the CD4~+Tcells which were cocultured with DCs or macrophages for24h, then determine differenteffects on CD4~+T cells and the secretion of IFN-γ after the stimulation of DCs ormacrophages in the present of the agonist anti-CD40antibody.（2）To clone scFv of the agonist anti-CD40antibody by RT-PCR and overlapextension PCR and construct lentiviral shuttle plasmid of recombinant scFv of agonist anti-CD40antibody. Infect the293T cells through the superphosphate infection to getthe pseudotype virus, then to infect DCs.（3）To analyse the activation of DCs carrying the Venus empy vector or the scFvof agonist anti-CD40antibody by flow cytometry from different perspectives to ensureDC carrying the scFv of agonist anti-CD40antibody can be activated effectively.（4）To evaluate the anti-tumor effects of the genetically modified DCs. Thetumor-bearing mice were divided into five groups, which are PBS group, DC infectedwith Venus empty vector group, DC infected with Venus-scFv group, DC infected withVenus empty vector pulsed with antigen group and DC infected with Venus-scFv pulsedwith antigen group, respectively.Results:（1） Macrophages secrete more IFN-γ and induce more CD4~+T cellsapoptosis comparing with DCs after agonist anti-CD40antibody stimulation.（2） The scFv of agonist anti-CD40antibody is cloned and the DCs carryinganti-CD40scFv were prepared through lentiviral infection.（3） The DCs carrying the scFv of agonist anti-CD40antibody can beactivated effectively.（4） DCs carrying anti-CD40scFv pulsed with tumor antigen can inhibittumor growth and stimulate tumor-specific immune responses.Conclusion:In this study, DC carrying anti-CD40scFv pulsed with tumor antigen can inhibittumor growth and stimulate tumor-specific immune responses. Part II The expression and function of CD40on CD4~+T cellsObjective:Intercellular communication is important for molecular information transfer fromcells to cells through transfer or exchange of their membrane proteins. CD40isconstitutively expressed on DCs. Activated T cells can also express CD40. We proposedto elucidate the source of CD40expressed on activated CD4~+T cells and the phenotypiccharictersitics of the CD40~+CD4~+T cells. Finally we would study whether the CD40onthe surface of the activated CD4~+T cells can transfer signal intracellularly withanti-CD40stimulation.Methods:（1） To detect the expression of CD40on CD4~+T cells by flow cytometry.（2） To demonstrate CD40is transferred from DC to CD4~+T cells by RT-PCR.To detect the activation and memory phenotypes of the resulting CD4hiT cells andCD4lowT cells，CD40~+CD4~+T cells and CD40-CD4~+T cells.（3） To analyse the cell proliferation and apoptosis of CD40~+CD4~+T cells andCD40-CD4~+T cells. To study whether anti-CD40agonist antibody can further stimulateCD40~+CD4~+T cells.Results:（1）CD4~+T cells stimulated alone did not express CD40. However, the activatedCD4~+T cells express high level of CD40when cocultured with DC. Furthermore, themore DCs were added, the higher level of CD40expression. The results of RT-PCRshowed that CD40expressed on the activated CD4~+T cells were transferred from DCs.（2）CD4hiT cells express more CD40and show more activated phenotypes withhigher percent of memory cells compared with CD4lowT cells.（3）CD40~+CD4~+T cells show more activated phenotypes with higher percent ofmemory cells compared with CD40-CD4~+T cells.（4）CD40~+CD4~+T cells proliferate faster and have lower apoptosis rate comparedwith the CD40-CD4~+T cells. Anti-CD40treatment has no effect on the proliferation and apoptosis of the CD40~+CD4~+T cells.Conclusion:The activated CD4~+T cells acquired CD40from DC are within CD4hipopulation,which showed more activated phenotypes and has higher percent of memory cells. Inaddition, the CD40~+CD4~+T cells have the ability to proliferate faster and survive longerthan the CD40-CD4~+T cells. The CD40~+CD4~+T cells can not be further activated byanti-CD40agonist antibody stimulation, suggesting that transferred CD40on the T cellsurface can not transfer signals intracellularly.