Characterization Of The Immunodominant Antigen-specific CD4~+T Cell Responses Of Helicobacter Pylori And Influenza A Virus

BackgroundInfectious diseases caused by pathogenic microorganisms were still the major killers tohuman. Host immune responses against the pathogens determine the outcomes of thediseases. Adaptive immunity is the key point of the host immune responses againstpathogens during the re-infection and it is also the fundamental basis of vaccine effect.CD4+T cell responses work as a central role in the adaptive immunity. They promote Bcells differentiation and antibody producing, help the antibody types switching; theypromote CD8+T cell differentiation and memory formation; they also secret multiplecytokines to interfere virus replication directly and recruit other immune cells to theinfection site to clear the pathogens indirectly; they even can kill those virus infected cellsdirectly, just like CD8+T cells.CD4+T cell responses were antigen specific, HLA restricted and characterized byimmunodominance hierarchies. Antigen specificity means the CD4+T cell responses canjust recognize epitopes consisted by13-20amino acids. They can not recognize the wholepathogen or protein antigens. This will cause the responses specific to different epitopeseven induced by the same pathogen or protein. HLA restriction means different HLAmolecules could mediate different responses through presenting different epitopes to T cells.This will cause the responses specific to different epitopes between subjects with differentHLA alleles. Immunodominance refer to the phenomenon that the CD4+T cell responsestend to focus on a very limited number of epitopes even during immune responses tocomplex microorganisms. Up to six different HLA class II alleles could be co-expressed inthe same subject and they may all have the ability to present epitopes. However, theresponses with different specificity will compete each other, some stronger responses willbecome immunodominant. Immunodominant CD4+T cells are not only more numerical but also provide better protection than subdominant ones. But the pathogens can mutate theanchor residues within the immunodominant epitopes and thus evade killing. Therefore,studies on the antigen specificity, HLA restriction and immunodominance of CD4+T cellresponses will help us understanding the mechanisms of the protective effect mediated byantigen specific CD4+T cells during infectious diseases, and help us designing moreeffective vaccines.Helicobacter pylori (H. pylori) is an important pathogenic microorganism colonized inthe human gastric mucosa. H. pylori infection is causally linked to chronic gastritis, pepticulcer, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma.Gastric cancer causes200thousands death each year in China. Influenza A virus (IAV) isone kind of RNA virus which causes influenza. IAV infection causes about0.5million deathworldwide annually. CD4+T cell responses were proved to be very important in theprotective immune effect against H. pylori and IAV infection. However, it was still not clearabout their antigen specificity, HLA restriction and immunodominance characteristics.Objectives1. To establish H. pylori and IAV antigen specific CD4+T cell lines by using thememory cells in the peripheral blood of H. pylori and IAV infected subjects. To observetheir effector productions and explore their potential effect in the anti-infection immunity;2. To identify the immunodominant antigens and epitopes recognized by H. pylori andIAV antigen specific CD4+T cells, align their sequence conservatism and evaluate theirpotential roles in vaccine designing.3. To determine the HLA restrictions of the immunodominant CD4+T cell responsesand characterize their immunodomiance hierarchies between individuals with the same ordifferent HLA alleles;4. To detect the frequencies of immunodominant responses between different subjectsand analyze their relationship with gastritis, peptic ulcer and gastric cancer for revealingtheir effect and mechanisms in the development of various H. pylori caused gastric diseases;Methods1. Characterization of the immunodominant H. pylori adhesion A subunit (HpaA)specific CD4+T cell responsesH. pylori infection was diagnosed by13C breath test, serum anti-H. pylori antibody ELISA and Urease dipstick. HpaA specific CD4+T cell lines were expanded in vitro thoughrecombinant HpaA protein stimulation. Effectors produced by HpaA specific CD4+T cellswere detected by intercellular cytokine staining. Overlapping synthetic peptides were usedto identify the immunodominant epitopes. HLA genotyping was performed by PCR-SBT.Antibody blocking and BLCLs presenting tests were used to determine the HLA restrictions.In vitro induced dentritic cells and BLCLs were used to characterize their natural processingand presenting property. MACS and ELISPOT were used to analyze their memoryphenotypes and ex vivo frequencies of epitope specific CD4+T cells. Theimmunodominance hierarchies were compared between multiple HLA-DRB1*1501positive subjects. And the frequencies of immunodominant CD4+T cells were furthercompared between groups with gastritis, peptic ulcers and gastric cancers to analyze theireffect on the development of different diseases caused by H. pylori infection.2. Characterization of the immunodominant IAV specific CD4+T cell responsesLysates of IAV infected P815cells were used to expand the IAV specific CD4+T celllines in vitro. Individual IAV protein antigens produced by recombinant vaccinia viruseswere used to identify the immunodominant protein antigens. Mapping of immunodominantepitopes was performed by overlapping synthetic peptides. HLA restrictions weredetermined by PCR-SBT, antibody blocking and BLCLs presenting tests. Intercellularcytokine staining was used to detect the effectors produced by IAV specific CD4+T cells.Multiple sequence alignment was used to evaluate the sequence conservatism of epitopes.Results1. Characterization of the immunodominant HpaA specific CD4+T cell responses1.1Bulk culture method of HpaA specific CD4+T cell lines were successfullyestablished. By using this method, HpaA specific CD4+T cell lines can be specificallyexpanded from H. pylori infected subjects, but can not be expanded from H. pyloriuninfected subjects.1.2IFN-γ was the major production of HpaA specific CD4+T cells, none of IL-4,IL-10or IL-17A was detected, so they belonged to type I CD4+T cell responses.1.3Three H. pylori antigen derived functional immunodominant CD4+T cell epitopes:HpaA192-204, HpaA88-100and HpaA200-212were identified here at the first time.1.4HpaA192-204, HpaA88-100and HpaA200-212specific CD4+T cell responses were restricted to DRB1*0406, DRB1*1501and DQB1*0301respectively.1.5These epitopes specific CD4+T cells had memory cell phenotypes. And the in vitroexpansion would not change the immunodominance hierarchies.1.6All of these epitopes could be natural processed and presented by antigenpresenting cells. They had conservative sequences and could be used as candidate epitopefor designing H. pylori epitope vaccines.1.759H. pylori infected HLA-DRB1*1501positive subjects were screened from1115patients with gastric diseases. HpaA88-100specific CD4+T cell response was found to bedominant in these H. pylori infected HLA-DRB1*1501positive subjects.1.8At the first time, we demonstrated that the frequency of HpaA88-100specificCD4+T cell response was negatively associated with the severity of the gastric diseasescaused by H. pylori infection, such as gastritis, peptic ulcer and gastric cancer, which meantthe protective effect of HLA-DRB1*1501against gastric diseases was mediated by theimmunodominant CD4+T cell responses, which also meant the epitope HpaA88-100was aprotective epitope specific to H. pylori.2. Characterization of the immunodominant IAV specific CD4+T cell responses2.1Soluble IAV whole cell antigens were successfully prepared through lysing IAVinfected P815cells. By using this lysate, bulk culture method of IAV specific CD4+T celllines were successfully established.2.2IAV specific CD4+T cells expressed IFN-γ and TNF-α in major, did not expressIL-2, however, their CD107expression was up-regulated, indicating that they exert theiranti-IAV protective immunity through interfering the IAV replication by IFN-γ and TNF-αand through direct cytotoxicity.2.3Soluble individual IAV antigens were successfully produced. By using them, wefound M1and NP were the most dominant targets of IAV specific CD4+T cell responses.2.4M1129-141, M1209-221, NP404-416and NP463-475specific CD4+T cellresponses were identified as immunodominant ones. M143-55, M194-106, M1103-115,M1105-117, NP102-114and NP115-127specific CD4+T cell responses were identified assubdominant ones.2.5M1129-141, M1209-221, NP404-416and NP463-475specific immunodominantCD4+T cell responses were restricted to DRB1*0101, DPB1*0301, DRB1*0404and DRB1*0901respectively; M143-55, M194-106, M1103-115, M1105-117, NP102-114and NP115-127specific subdominant CD4+T cell responses were restricted to DRB1*0701,DRB1*1302, DRB1*0404, DRB1*0901, DPB1*0101and DPB1*0601respectively.2.6Epitope variants were wildly observed in the newly pandemic IAV strains. And themutation frequency was significant higher in the immunodominant epitopes thansubdominant ones.Significance1. A bulk culture method of HpaA specific CD4+T cell lines was established in thisstudy by using the PBMCs of H. pylori naturally infected subjects. We identified the firstHLA restricted and immunogenic H. pylori CD4+T cell epitope. The immunodominancehierarchy was the first time analyzed systematically here. Most importantly, wedemonstrated that HLA-DRB1*1501mediated HpaA88-100specific immunodominantCD4+T cell responses were protective against H. pylori caused gastritis, peptic ulcer andgastric cancer. These results provide us a new method to identify immunodominant epitopesof H. pylori protective antigens, help us understanding the immunodominancecharacteristics and the protective mechanisms of antigen specific CD4+T cells in H. pyloriinfection, and also provide us new candidate antigens for H. pylori vaccine design.2. Our study established a bulk culture method of IAV polyclonal antigen specificCD4+T cell lines by using a soluble IAV whole cell antigen produced by IAV infected P815cells. M1and NP were confirmed as the most dominant antigen targets at a whole geneprotein level. We identified several novel dominant and subdominant CD4+T cell epitopesderived from M1and NP. The sequence conservatism of dominant and subdominantepitopes was systematically compared. The immunodominance was correlated with anincreased frequency of mutations, reflecting that these prominent epitopes are under greaterselective pressure. These data provide a new insight into the immunodominancecharacteristics of antigen specific CD4+T cells in IAV infection, and also provide usconsiderations for choosing candidate antigens in designing novel IAV vaccine based on Tcell response.