Characterization Of Immunodominant Epitope-specific CD4+T Cell And CD8+T Cell Responses During Helicobacter Pylori Infection

BackgroundHelicobacter pylori(H.pylori)is gram-negative bacteria,which resides in the mucosa of stomachs.The infectious percentage of H.pylori in the world’s population is up to 50%.Persistent infection of H.pylori causes peptic ulcer,chronic gastritis,and gastric cancer.Thus,H.pylori is classified to be a class I carcinogen by WHO(The world Health Organization)in 1994.Current clinicians use at least two antibiotics and proton-pump inhibitor to treat H.pylori infection.Unfortunately,the reckless use of antibiotics leads to the increasing resistance in worldwide.Besides,the cost of current antibiotic treatment is expensive.Therefore,vaccination is considered to an effective solution against H.pylori infection.Currently,several types of vaccines aginst H.pylori are designed,for example,whole-pathogen vaccines,full-length protein vaccines,protein-subunit vaccines and vaccines based DNA sequences.Most vaccines induced significant humoral immune responses post the vaccination.However,the effects of humoral immune responses to eradicate H.pylori colonization in stomachs are not satisfactory.CD4+T cells are important during the infection of extracellular bacteria.Especially,antigen-specific CD4+T cells have been confirmed to be protective against H.pylori infection.Thus,it is important to develop vaccines inducing significant CD4+T cell responses.Post the infection of H.pylori,several types of CD4+T cell responses including Th1,Th2 and Th17 can be detected.However,it is still unclear about which subtypes of CD4+T cell responses can protect against H.pylori infection effectively.Urease secreted by H.pylori is an important antigen for bacteria colonization,survive and infection,which could induce obvious specific antibodies and T cell responses.Thus,many vaccines against H.pylori were designed based on Urease,especially the B subunit(Ure B).Post the vaccination of Ure B,the colonization of H.pylori in stomachs decreased and CD4+ T cell responses were detected.Thus,in this study,the protective antigen,Ure B,was used a model to immunize mice.Then,protective effects of antigen-specific CD4+T cell responses were analyzed.Furthermore,immunodominant epitopes of Ure B were screened and identified.And the protective mechanisms of immunodominant epitope-specific CD4+T cells were clarified.H.pylori is extracellular bacteria;however,it was reported that H.pylori could infect and proliferate in dendritic cells,gastric epithelial cells,macrophage,etc.,as well.Thus,CD8+ may also take part in the immune responses during H.pylori infection.In human patients with H.pylori infection,CD8+ T cell responses were detected.Moreover,using a pig model,significant proliferation of CD8+ T cells was detected post H.pylori infection.Unfortunately,few researches about CD8+ T cells were reported in a mouse model,and the functions of CD8+ T cells are still not clear during H.pylori infection.In this study,UreB were used as a model to immunize mice.Then,antigen-specific T cells were expanded in vitro.We assess the effects of Th1,Th1,Th17 and antibodies,and screened immunodominant epitopes.Then,the MHC restriction and the protective effects of the screened epitopes were confirmed.Furthermore,we characterized mucosal CD8+T cells post the infection of H.pylori.Objectives1.Clarify the protective mechanisms of immunodominant epitope-specific CD4+T cells post H.pylori infectionTo establish Ure B antigen-specific CD4+ T cell lines in vitro using splenic lymphocytes from Ure B immunized mice.To assess the effects of antigen-specific T cells against H.pylori.To screen the immunodominant T cell epitopes of Ure B,characterize the profile of the screened epitopes and confirm the protective effects.The screened protective epitopes will be used as candidates for the design of epitope-based vaccines.To investigate the mechanisms underlying the protective immune responses against H.pylori infection.2.Characterize mucosal CD8+ T cells during H.pylori infection in mice modelTo determine the characterization of mucosal bacteria specific CD8+ T cells and investigate the mechanisms underlying the persistent colonization post H.pylori infection.Methods1.Clarify the protective mechanisms of immunodominant epitope-specific CD4+T cells post H.pylori infectionSPF female BALB/c mice were immunized with protein emulsified in CFA subcutaneously(sc)in four limbs to establish mice model.3H-Td R assay and RT-PCR were used to detect the proliferation and cytokines of antigen-specific CD4+T cells post Ure B immunization ex vivo.FCM was used to determine the cytokines of expanded antigen-specific CD4+T cells in vitro.Immunodominant epitopes of Ure B were screened using overlapping peptides.MHC-restriction of the screened epitopes were analyzed through MHC antibodies blocking assay.BM-DCs pulsed UreB antigen were co-incubated with epitope-specific CD4+T cells.Then,the natural processing properties of epitopes were characterized.Immunodominant properties of screened epitopes were determined by comparing the responses of CD4+T cells to the screened epitopes and predicted epitopes.Mice were immunized with peptides in Cp G via the intranasal route four times with one-week intervals between vaccinations.One week after the final boost,mice were challenged four times with 2 × 108 colony-forming units of H.pylori by intubation.Then,the effects of epitopes were analyzed through gastric mucosal H.pylori colonization detection.Protective effects of epitope-specific CD4+ T cells were determined through adoptive transformation experiments and data from IL-17-/-mice.Protective mechanisms of immunodominant epitope-specific CD4+T cells were clarified through the determination of homing receptors and chemokine receptors on CD4+ T cell surface.2.Characterize mucosal CD8+ T cells during H.pylori infection in mice modelMice were challenged four times with 2 × 108 colony-forming units of H.pylori by intubation to establish infectious mice model.The stomachs of H.pylori infected mice were removed.Next,the stomachs were treated with HBSS(Hanks Balanced Salt Solution)containing EDTA,DTT and FCS for 50 min at 37°C.Supernatant contains the cells from the gastric mucosa.FCM,CCK8 and CFSE were used to detect the proliferation of CD8+T cells.Antigen-specific CD8+T cells were expanded in vitro through the direct stimulation of Ure B antigen,which indicated the effects of cross-presentation during the CD8+T cell responses to H.pylori.Immunodominant CTL epitopes of Ure B were screened using overlapping peptides.ICS and FCM were used to determine the cytokines,homing receptor and chemokine receptor of mucosal CD8+ T cells.Results1.Clarify the protective mechanisms of immunodominant epitope-specific CD4+T cells post H.pylori infection1.1 Two immunodominant Th epitopes from UreB,UreB317-329 and Ure B409-421 were screened.And,these two epitopes could induce strong Th1 and Th17 responses simultaneously.1.2 Ure B317-329 and UreB409-421 could be naturally processed and presented by antigen-presenting cells(APCs).UreB409-421 binds with surficial molecule MHC-II(I-A)of APCs,then,activates specific Th1 and Th17 responses.MHC restriction of Th1 epitope UreB317-329 is MHC-II(I-A);however,activation of UreB317-329 specific Th17 responses need the participation of both MHC-II(I-A)and MHC-II(I-E).1.3 The identified immunodominant epitopes,Ure B317-329 and UreB409-421 induced much stronger Th1 responses compared with previous published epitopes that predicted using a bioinformatics approach.And,these screened epitopes in this study can not be predicted precisely using software.1.4 The colonization of H.pylori and the inflammatory scores in stomachs decreased significantly post the immunization of UreB317-329 and Ure B409-421 respectively.1.5 TCRvb repertoires of Ure B317-329 specific CD4+ T cells were different from those of Ure B409-421 specific CD4+ T cells.No significant differences were detected of The TCRvb repertoires of Th1 and Th17 that response to the same epitopes.1.6 Homing and chemokine receptors,L-selectin and α4β7,take part in the protective immune responses against H.pylori infection.2.Characterize mucosal CD8+ T cells during H.pylori infection in mice model2.1 Gastric mucosal CD8+ T cells increased obviously post the infection of H.pylori.And the percentage increased of mucosal CD8+ T cells were much higher that that of mucosal CD4+ T cells.2.2 Gastric CD8+ T cells were classical TCRαβ+CD8αβ+,few CD8αα T cells and γδ T cells were detected in the gastric mucosa post H.pylori infection.2.3 Post H.pylori infection,gastric CD8+ T cells were induced which response to H.pylori bacteria and H.pylori supersonic supernatants specifically.2.4 Post H.pylori infection,gastric CD8+ T cells mainly secret IFN-γ and TNFa.Conclusion1.Using a systematic method,two novel immunodominant epitopes of Ure B antigen which could induce significant Th1 and Th17 responses were identified and the MHC restriction profiles were characterized.Furthermore,we compared the responses induced by these new epitopes and three epitopes predicted through software,and we found that the novel epitopes induced stronger responses.Then,we confirmed that the epitopes identified in this study could be naturally processed and presented by APCs.To further evaluate the effect of the immunodominant epitopes,mice were immunized with the epitopes and challenged with H.pylori.Finally,we showed that the two novel immunodominant epitopes could protect mice from H.pylori infection obviously.And the protective effect was independent of humoral immunity.Thus,these epitopes may serve as novel candidates for developing epitope-based vaccines.2.After H.pylori infection,mucosal CD8+ T cells were detected.The characterization of gastric CD8+ T cells was identified.And we found that classic CD8+ T cells proliferated and response to H.pylori specifically post the infection.Specific mucosal CD8+ T cells secreting IFN-γ and TNFa homed to stomachs and took part in the immune response post infection.The results obtained in this project contribute to the characterization of the relationship between immune responses and persistent infection of H.pylori.