Analysis Of CD8~+T Cell Epitope Profile Of Hepatocellular Carcinoma Associated Antigens And Application Of The Epitope Peptides Cocktail

BackgroundHepatocellular carcinoma is one of the most common malignant tumors with high morbidity and mortality.Most patients are diagnosed in the middle or late stage,with limited optional treatments and very poor prognosis.The emergence of novel immunotherapies,such as immune checkpoint inhibitors,has brought new hope to patients with HCC.The main mechanism of immunotherapies is to induce and amplify tumor-specific T cells,especially CD8~+T cells,to enhance their reactivity or prevent them from functional exhaustion and apoptosis,so as to enhance the specific tumoricidal efficiency.Strong tumor antigen-specific CD8~+T cell response can inhibit HCC recurrence,with its strength being positively correlated with progression-free survival and even overall survival.Therefore,the dynamic monitoring of the reactivity of tumor antigen specific T cells can immediately and directly display the effect of immunotherapy,which is beneficial to guide the follow-up treatment plan and predict the prognosis.However,the current study on the T cell epitopes profile of HCC-associated tumor antigens is still limited,and the number of T cell epitopes that have been functionally identified and reported is very small.For example,there are only 22 CD8~+T cell epitopes from AFP,16 from h TERT,11 from GPC3,and even fewer from other antigens.Moreover,these validated epitopes are mainly restricted to a few HLA-A molecules(HLA-A0201,A2402 or A1101),which cannot cover the HLA molecular polymorphism of a broad population in an indicated geographical region,let alone be consistent with HLA polymorphism in the Chinese population.Therefore,the universal detection of tumor antigen-specific T cells for broad HCC patients is greatly limited,which seriously hinders the immediate and direct evaluation of the efficiency of immunotherapy,as well as the early prediction of prognosis and recurrence after traditional therapies.In-depth analysis of the T cell epitopes profile of HCC-associated tumor antigens is of great significance,which will provide molecular basis and targets for the detection of tumor antigen-specific T cell,the development of immunotherapy based on T cell immunity,and the understanding of interaction mechanism between immune and tumor in HCC patients.ObjectiveThere are 13 dominant HLA-A molecules with each gene frequency more than 1%in the Chinese population,whose total gene frequency is over 95%.There are many types of HCC associated tumor antigens,among which the positive expression rates of AFP,GPC3 and GP73 are 50%-80%,65%-80%and 63%-72%,respectively.This project intends to identify the dominant T cell epitopes in AFP,GPC3 and GP73 and presented by the 13 dominant HLA-A molecules.A broad-spectrum library of T cell epitope peptides was constructed,and combined with ELISPOT technique to establish a universal detection system of specific T cell reactivity for HCC patients.Peptide cocktail vaccine was prepared to investigate its ability to induce tumor antigen specific CD8~+T cell response and anti-tumor efficiency in HLA-A transgenic mice.Methods1.Prediction and screening of candidate T cell epitopes:The amino acid sequences of AFP,GPC3 and GP73 were collected from Uniprot database;using several T cell epitope prediction databases,CD8~+T cell epitope peptides restricted by the 13 predominant HLA-A molecules in the three HCC associated antigens were systematically predicted,and the epitope peptides that met the criteria of at least two prediction algorithms were selected as candidate epitope peptides and synthesized followed by further verification via a series of experiments.2.Immunogenicity verification of candidate epitope peptides:The PBMCs from HCC patients were co-cultured with candidate epitope peptides,and the immunogenicity of candidate epitope peptides was validated by IFN-γELISPOT assay.The candidate epitopes that can significantly stimulate the activation and IFN-γsecretion of memory T cells in PBMCs are identified as positive epitope peptides,indicating that they are immunogenic epitope peptides.The PBMCs from HLA-A matched HCC patients were co-incubated with each positive epitope peptide for 7 days in vitro.IFN-γintracellular staining and flow cytometry assay were used to detect the ability of positive epitope peptides to stimulate activation of memory CD8~+T cells.The positive epitope peptides were co-cultured with DC and PBL from HLA-A matched HCC patients for 14 days(DC-peptide-PBL co-stimulation assay)to detect the ability of the positive epitope peptides to stimulate the activation of naive CD8~+T cells.3.HLA cross-restriction analysis of positive epitope peptides:Using HMy2.CIR cell lines expressing the indicated HLA-A molecule,fluorescently labeled reference peptide,and these positive epitope peptides,the peptide competitive binding experiment of HLA-A molecule was performed by which the cross-binding ability of each positive epitope peptide with the relevant HLA-A molecules was analyzed.In parallel,peptide affinity experiments were conducted using T2 cell line and HLA-A0201 restricted positive epitope peptides to further verify the binding ability between the positive epitope peptides and HLA-A0201molecule.4.Detection of specific T cell reactivity:IFN-γELISPOT detection system was developed using the epitope peptides cocktail which was composed of the positive epitope peptides presented by a series of HLA-A molecules of AFP,GPC3 and GP73 proteins.Then,the reactivity of HCC-associated tumor antigen-specific T cells in PBMCs of HCC patients and healthy donors was detected using this detection system.5.Peptide cocktail vaccine to induce epitope-specific CD8~+T cell response:The positive epitope peptides of AFP,GPC3 and GP73 and presented by HLA-A0201 molecule were mixed with the adjuvant poly(I:C)to prepare a peptide cocktail/poly(I:C)vaccine.Then,HLA-A0201/DR1 transgenic mice were inoculated subcutaneously for three times.IFN-γELISPOT and intracellular cytokine staining methods were used to detect the frequency of epitope-specific CD8~+T cells in splenocyte of immunized mice.Cytotoxicity assay with7-AAD staining was used to detect the ex vivo specific killing ability of splenocyte from immunized mice against Hep G2.Flow cytometry was used to detect the proportion of T cell subsets and the expression level of PD-1 molecule on the splenocyte of immunized mice.H&E staining was used to evaluate the pathological changes of various organs after immunization.6.In vivo anti-tumor efficiency of peptide cocktail vaccine:Hep G2 cell line and NSG mice were used to get NSG mice xenograft model.The tumor-bearing NSG mice received the adoptive therapy of splenocyte from peptide cocktail/poly(I:C)immunized HLA-A2/DR1transgenic mice and the treatment of anti-PD-1 monoclonal antibody.At the same time,control group with adoptive cellular therapy of poly(I:C)vaccined splenocytes and anti-PD-1m Ab treatment,and the control group without anti-PD-1 m Ab were set up.The growth curve of the tumor was generated.The infiltration of CD8~+T cells in tumor was detected via immunohistochemistry.After coculturing the isolated tumor infiltrating lymphocytes and mixed peptides in 96-well plates for 20 hours,the concentration of IFN-γin the supernatant was quantitatively detected by ELISA.Results1.The amino acid sequences of AFP,GPC3 and GP73 were confirmed by Uniprot database.A total of 93 candidate CD8~+T cell epitopes restricted by the 13 dominant HLA-A molecules were predicted and selected,including 30 AFP epitopes,31 GPC3 epitopes and 32GP73 epitopes.2.A total of 35 candidate epitopes were confirmed to be immunogenic by IFN-γELISPOT assay,with 10,12 and 13 epitopes derived from AFP,GPC3 and GP73,respectively.After 7-days co-culture(HLA-A-matched HCC patient’s PBMCs and each positive epitope peptide)and intracellular IFN-γstaining,it is confirmed that all 23 tested positive epitope peptides can induce the activation of specific CD8~+T cells of the PBMCs from HCC patients.DC was induced from the PBMCs of HLA-A-matched healthy donor.After 14-days DC-peptide-PBL co-culture and intracellular IFN-γstaining,it confirmed that all 30 tested positive epitopes can induce epitope-specific CD8~+T cell activation in the PBL from healthy donors.3.The cross-binding ability of 35 positive epitope peptides with relevant HLA-A molecules,namely HLA cross restriction,was further analyzed through T2 cell binding assay,and peptides competitive binding assay of HLA-A molecules transfected HMy2.CIR cell lines.4.A total of 112 peptides from AFP、GPC3和GP73 were used to form wide-coverage peptide pools.The IFN-γELISPOT assay system was established to detect the antigen-specific T cells in PBMCs of 58 HCC patients and 11 healthy donors.The average number of spots in HCC patients was significantly higher than that in healthy donors.The AFP epitope-specific T cells were detectable in most HCC patients’PMBMCs,while the intensity of AFP specific T cell response was also stronger than that of GPC3 and GP73.5.HLA-A2/DR1 transgenic mice were immunized by a peptide cocktail-based vaccine,which was prepared by mixing the 14 positive epitopes presented by HLA-A0201 with poly(I:C).IFN-γELISPOT and IFN-γintracellular staining showed that the peptide cocktail/poly(I:C)vaccine induced a vigorous epitope-specific CD8~+T cell response in mice.The number of SFUs and the frequency of IFN-γ~+/CD8~+T cells were 5.6-11 times and 3.9-29.7times of that of the control group,respectively.Cytotoxicity assay showed that the splenocyte of mice immunized with peptide cocktail/poly(I:C)possessed specific killing efficiency on Hep G2 in vitro.However,there was no obvious cytolysis to the empty T2 cells and the T2cells loaded with irrelevant peptide.Flow cytometry showed that CD8~+T cell subsets and the ratio of CD8~+/CD4~+T cell were significantly increased in the splenocyte of mice immunized with peptide cocktail/poly(I:C).Meanwhile,the expression level of PD-1 on CD8~+T cells was higher than that of control group.H&E staining showed no obvious pathological damage in organs of the mice after peptide cocktail vaccine immunization.6.The best therapeutic effect with minimum tumor volume was achieved in the tumor-bearing NSG mice that received the adoptive infusion of the splenocyte from HLA-A2/DR1 transgenic mice immunized by peptides cocktail vaccine and also received anti-PD-1 m Ab treatment.There were more infiltrating CD8~+T cells in the tumor tissue of the combination therapy group,and the level of IFN-γsecreted by the infiltrating lymphocytes in ex vivo co-culture with peptides cocktail was also the highest.ConclusionsThis study successfully validated 35 novel CD8~+T cell epitopes of HCC associated tumor antigens and restricted by the dominant HLA-A molecules in the Chinese population,thus expanding the library of T cell epitopes of HCC associated antigens.The feasibility of using a broad-spectrum epitope peptide pool to detect antigen-specific T cells for broad HCC patients was verified.Peptides cocktail vaccine was prepared,and its ability to induce specific CD8~+T cell response and antitumor efficiency were confirmed in HLA-A transgenic mice.