Depletion Of Regulatory T Cells Enhances Helicobacter Pylori Vaccine Immune Effacacy: The Mechanism Study

Objective:To investigate the role of regulatory T cells (Treg) in H. pylori vaccine inducedprotection in mice and to explore the mechanism CpG-ODN-TLR9signal pathway inenhancing H. pylori vaccine efficacy.Methods:1、 The mechanism study of Treg to enhance immunoprotection of H.pylorivaccine:19Mice with deletion of Treg cells through injecting anti-CD25(n=5) andthose without deletion (n=7)and controls injecting PBS(n=7) were compared fortheir response to H.pylori challenge. The two groups were immuinized through mixedwith H.pylori whole cell sonicate(WCS) and complete Freund’s adjuvant otherwiseothan controls.And injecting anti-CD25were received antibody4days beforeimmunization. Within4weeks postimmunization, IgG and subtype IgG1,IgG2a fromblood of mice were evaluated by enzyme-linked immunosorbent assay. Hp bacterialload were accessed by quantitative culture in gastric specimen within4weeksH.pylori challenge and execution to mice. CD4+CD25+Treg,CD80+cells,CD19+cellsand CD8a+cells of Splenocytes were analyzed by Flow cytometry; IFN-γ,IL-4,IL-12and IL-17were measurement by ELISA for the suspension collected fromSplenocytes culture.2、The study of CpG-ODN adjuvant to enhance the immunoprotection of Hp vaccine:28Mice were compared for their response to CpG-ODN adjuvant enhance theimmunoprotection,which injecting ODN2088mixed with WCS(n=7)and ODN1826mixed with WCS (n=7)and only WCS without adjuvant(n=7)and PBS as control(n=7).Within4weeks postimmunization, IgG and subtype IgG1,IgG2a from blood of micewere evaluated by enzyme-linked immunosorbent assay. Hp bacterial load were accessed by quantitative culture in gastric specimen within4weeksH.pylori challenge and execution to mice. CD4+CD25+Treg,CD80+cells,CD19+cellsand CD8a+cells of Splenocytes were analyzed by Flow cytometry; IFN-γ,IL-4,IL-12and IL-17were measurement by ELISA for the suspension collected fromSplenocytes culture.Results：1. The mechanism study of Treg to enhance immunoprotection of H. pylori vaccine.1.1After last immunization, immunized mice had been induced anti-Hp IgG andsubtype IgG1,IgG2a antibodies;and the levels of anti-Hp IgG and subtype IgG1,IgG2aantibodies in WCS+anti-CD25group was obviously higher than WCS group（P＜0.01）. the levels of subtype IgG1,IgG2a antibodies were same as anti-Hp IgG.1.2CD4+CD25+Treg cells of splenocytes were analyzed by Flow cytometry，WCS+anti-CD25group lower WCS group（P＜0.01）；however CD8a+cells andCD80+cells，WCS+anti-CD25group higher than WCS group（P＜0.01）；CD19+cells,There was no signficant difference between WCS+anti-CD25group and WCS group（P>0.05）.1.3WCS+anti-CD25compared with WCS, H.pylori colonization density weresignificantly decreased (P＜0.01),1.4Compared with WCS group,significantly higher levels of IFN-γ，IL-4，IL-12andIL-17were detected in mice suspension of splenocytes immunized withWCS+anti-CD25group（P＜0.01）, Delpetion of Treg cells would enhanced Th1andTh2response.2. Research CpG-ODN adjuvants enhance the immunoprotection of Hp vaccines.2.1After last immunization, immunized mice had been induced anti-Hp IgG; and thelevels of anti-Hp IgG and subtype IgG1,IgG2a antibodies in WCS+ODN1826groupwas obviously higher than WCS group（P＜0.01）.2.2CD4+CD25+Treg cells of splenocytes were analyzed by Flow cytometry，WCS+ODN1826group lower WCS group（P＜0.01）；however CD8a+cells，WCS+ODN1826group higher than WCS group （P＜0.01） CD19+cells andCD80+cells, There was no signficant difference between WCS+ODN1826group and WCS group（P>0.05）.2.3WCS+ODN1826compared with WCS, H.pylori colonization density weresignificantly decreased (P＜0.01),.2.4Compared with WCS group, significantly higher levels of IFN-γ，IL-4，IL-12andIL-17were detected in mice suspension of splenocytes immunized withWCS+ODN1826group （P＜0.01）.Conclusions：1.Depletion of Treg specification could enhance both humoral (Th1)and cellular (Th2)immunity induced by H. pylori Vaccine, strengthen cytotoxicity of CD8+T cells andpresentation of antigen presenting cells, and reinforce immunoprotection ofvaccine against H. pylori infection ultimately.2. CpG-ODN through TLR9could reduce Tregs and possible reverse Treg byactivation of macrophages and Dendritic cells and secretion of IL-6, lead toTh1-related immunity respond, and reinforce immunoprotection of vaccine againstH.pylori infection ultimately.