Study On The Biological Characteristics Of A Novel CD8?? Tregs Differentiation From CD4~+T Cells

Dendritic cells(DC)has been a promising antitumor strategy and has been extensively focused.Polyethylene glycol(PEG)has been widely used for inducing cell fusion.The fusion cells(FCs)express tumor antigens which are all known and unknown,and thus it is more effective in stimulating T cell proliferation and activation.Many studies have proved that DC/tumor cell vaccine can activate CD4~+T cells and CD8~+Tcells to anti-tumor effect,but clinical trials are not so ideal.It is suggested that there may be an immune mechanism for the destruction of these vaccines.So it is very necessary to explore the mechanism of immune suppression in the future to improve the DC/tumor hybrid cell vaccine for cancer treatment.The ultimate goal of the DC vaccine is to stimulate a strong and persistent CD4~+T cells and CD8~+Tcells response to clear tumor cells.However,we found that the DC/tumor fusion cells vaccine in vivo induce CD4~+T cells and CD8~+T cells which anti-tumor effect,in addition to a type of regulatory T cells.We found that the regulatory T cells is a novel subset of CD8??Tregs and derived from CD4~+T cells.In vivo CD4~+T cells transformed into the novel CD8??Tregs.CD8??Tregs originate from three positive thymocytes(CD4~+CD8??~+CD8??~+),after positive selection,transfer into a single positive thymocytes,migrate to the small intestine.CD8??Tregs by identifying MHC-Ib Qa-1,inhibit pathological CD4~+T cells in vivo,natural environment,it has not been reported that whether CD4~+T cells transfer into CD8??Tregs.Thus explored in vivo without any stimulation,CD4~+T cells can be transformed into CD8??Tregs and its possible mechanisms will provide reference to understand the process of T cells development and a new treatment for related antitumor immune and autoimmune diseases.Notch signaling pathway is an important signaling pathway regulating embryonic development and cell differentiation.After binging between Notch ligands and receptors,ICN is releasing activity by?-secretase cut fragments,translocation to the nucleus,and conjugate the transcription factor RBP-J(Recombination signal binding protein-J?,RBP-J),and activation of downstream target genes,multiple cells development and function.This study by using the gamma-secretase inhibitor DAPT and RBP-J gene conditional knockout mice and to verify that the Notch signaling pathway is mediated by CD4~+T cells transferred to CD8??Tregs.In this study,we further investigate the potential clinical significance of the novel CD8??Tregs to the anti-umor effect of tumor fusion cell vaccine and to the treatment of autoimmune diseases.The contents and results of this study are as follows:Part 1 CD4~+T cells transformed into a novel CD8??TregsObjective:To explore CD4~+T cells transformed into a novel CD8??Tregs.Methods:The frequency and surface marker expression of CD8??Tregs differentiation from CD4~+T cells were induced by fusion cells vaccine was detected by flow cytometry and the in vivo differentiation of CD4~+T cells in response to DC/B16 FCs.C57BL/6 background GFP transgenic mice CD4~+T cells were injected to the C57BL/6 mice,the percentage of CD8??Tregs were detected by flow cytometry in the spleen and lymph nodes of the mice.Results:FCs induced the differentiation of CD4~+T cells into CD8??Tregs.The results of flow cytometry showed that in vivo CD4~+T cells transformed into CD8??Tregs,and the percentages of CD8??Tregs in the lymph nodes and spleen were 90.8%and 82.5%.Flow cytometry results show that the conversion of CD8??Tregs expressed CD129,CD94,PD-1,CD44 molecules.Conclusions:The study found that DC/tumor fusion vaccine induced more CD4~+T cells into a novel CD8??Tregs.In vivo CD4~+T cells can transformed into the novel CD8??Tregs.Part 2 Study on the biological characteristics of the novel CD8??Tregs origination from CD4~+T cells.Objective:Study on the biological characteristics of the novel CD8??Tregs origination from CD4~+T cells.Methods:After treating CD4~+T cells with DC/B16 FCs,we detected cytokines secretion of differentiated CD8??Tregs by flow cytometry.We assessed the in vitro effects of CD8??Tregs on the proliferation and apoptosis of CD4~+T cells.We also assessed the in vitro effects of CD8??Tregs on the proliferation and apoptosis of CD4~+T cells.Results:Flow cytometry results show that the conversion of CD8??Tregs expressed CD129,CD94,PD-1,CD44 molecules,secreted IFN-?and IL-4,IL-10,IL-2 and TGF-?,inhibited the proliferation of activitated CD4~+T cells,and killed activitated CD4~+T cells.Conclusions:The study found that CD4~+T cells into a novel CD8??Tregs in vitro and in vivo.The novel CD8??Tregs play a role in immune suppression.Part 3 The mechanism of CD4~+T cells transformed into the novel CD8??TregsObjective:To expore the mechanism of CD4~+T cells transformed into the novel CD8??TregsMethods:After treating CD4~+T cells with DC/B16 FCs,we evaluated the effect of the Notch inhibitor DAPT in this process.The Notch signal pathway blocking agent DAPT and GFP~+CD4~+T cells were injected to the C57BL/6mice,the percentage of CD8??Tregs were detected by flow cytometry in the spleen and lymph nodes of the mice.GFP~+CD4~+T cells were injected to the RBP-J gene knockout mice,the percentage of CD8??Tregs were detected by flow cytometry in the spleen and lymph nodes of the mice.Results:FCs induced the differentiation of CD4~+T cells into CD8??Tregs,which was abrogated by DAPT treatment.The percentages of CD8??Tregs reduced in injected mice by DAPT and GFP~+CD4~+T cells.The percentages of CD8??Tregs reduced in injected RBP-J gene knockoutmice by GFP~+CD4~+T cells.Conclusions:CD4~+T cells can transform into CD8??Tregs dependenting on the Notch signaling pathway.Part 4 The novel CD8??tregs originated from CD4~+T cells affected on the antitumor effect of DC/tumor FCsObjective:To explore DC/tumor fusion cells vaccine induced CD4~+T cells differentiation into a novel CD8??regulatory T cells and further optimize the anti-tumor effect of DC/tumor fusion cells vaccine.Methods:We examined tumor growth and mouse survival of bearing tumor mice injected by the novel CD8??Tregs.Observe to DC/tumor fusion cells vaccine and DAPT against tumor function.After anti CD4 antibody blocking,the antitumor function of DC/tumor fusion cells vaccine and DAPT was observed.Results:In vivo,CD8??Tregs stimulated tumor growth and reduced mouse survival.The co-administration of DAPT and FCs significantly further boosted the anti-tumor properties of FCs,as well as increasing mean mouse survival.However,after anti CD4 antibody blocking,the co-administration of DAPT and FCs didn't significantly boost the anti-tumor properties of FCs.Conclusions:This novel CD8??Tregs group plays a role in immune suppression.Targeting this novel mechanism may further enhance the therapeutic benefits of FCs.Part 5 The novel CD8??Tregs differentiation from CD4~+T cells provides protection from EAEObjective:To determine the in vivo the novel CD8??tregs differentiation from CD4~+T cells provides protection from EAE.Methods:EAE was induced with MOG35-55.The novel CD8??Tregs were adoptively trasferred into EAE i.v.24h before the induction of EAE.Clinical symptoms were monitored daily and scored on a scale from 1 to 5.Results:Mice injected with the novel CD8??Tregs recover more rapidly from disease than those in the control group.Conclusions:Adoptive transfer of the novel CD8??Tregs protected from EAE.