Distribution And Clinical Significance Of T Cell Receptor ? Chain Repertoire In Healthy Dornors And Patients With NSCLC

Background and objective:T cell repertoire describes lymphocytes characterized by TCR expression,which plays a critical role in antigen recognition.Long read sequencing allows determination of TCR genes including V,D,J and C regions.In recent years,advances in high-throughput sequencing technologies known as next-generation sequencing have rapidly progressed and enabled large-scale analysis of sequence data.The analysis of the human T-cell repertoire with age ranges was limited in these studies.The frequency,characteristics,and significance of the CDR3 repertoire with respect to TCR? chain V with J rearrangement are not well-understood in healthy Chinese individuals.In our study,PBMCs samples from 154 healthy individuals were collected(age range:6-70)and each age group of about 20 individuals(male:female=1:1).A newly developed next-generation sequencing-based TCR repertoire analysis was performed.The study enabled us to reveal the longitudinal behavior of individuals T-cell clones in healthy Chinese individuals with ages.Lung cancer was the first malignant tumor incidence and the death toll in the global,and non-small cell lung cancer(NSCLC)accounts more than 85%of patients.Surgery,chemotherapy,radiaotherapy and targeted therapy were the major therapeutic methods,but the five-year overall survival of patients with advanced stage was poor,in addition,specific prognostic marker for NSCLC was limited.Thus,it is an important research direction to explore novel therapeutic strategies and markers for improving clinical efficacy and predicting prognosis.In recent years,the immune system was found to play an important role in the process of tumor occurrence and recession.At the same time,adoptive T cell therapy showed great potential in cancerimmunotherapy,and the study confirmed that antigen specific T cells in infiltrating tumor site actually played the anti-tumor role.Cancer testis antigen families were restrictive expressed in tumor tissues and germ cells and considered as tumor specific antigen.MAGE-A3 as a member of the family,has been confirmed to express in various cancers and link to poor prognosis.Previous studies have confirmed the MAGE-A3 was highly expressed in NSCLC tissue,the frequency of positive expression was associated with poor survival in patients.And its effective present peptides restrict by HLA-I also have been identified.The functional antigen specific CD8-T cells in peripheral blood were closely related with the prognosis in cancer patients.However,the function of MAGE-A3 specific CD8+ T cells and its clinical significance were not reported in patients with NSCLC.T cells recognized and killed cancer cells were through T cell receptor integrated MHC antigen peptide compounds to activate T cells proliferation.Thus exploring the TCR repertoire expression of antigen specific CD8+ T cells is critical to reveal the importance of T cells in cancer patients.Meanwhile,there was not reported that whether TCR repertoires persistant differences between reactive antigen specific CD8+T cells and non-responsive T cells.Therefore,this study firstly revealed the distribution TCR tepertoire diversity in healthy dornors,which provided the foundation to compare TCR tepertoire diversity in the different ages and genders.Furthermore,the significances of MAGE-A3 specific CD8+ T cells in patients with lung cancer were studied.The results showed that the proportion of reactive MAGE-A3 specific CDIA T cells was negatively correlated with poor prognostic clinical parameters in patients with NSCLC.In addition,TCR was the center of the T cells to identify cancer cells.TCR-v? repetoires between reactive and non-responsive MAGE-A3 specific CD8+ T cells existed obvious differences,and the differences also existed individual variation.The expression of TCR-v?9 was positively accociated with prognostic in patients with NSCLC.Part 1 The repertoire feature of T cell receptor ?-chain at different age and gender groups in the healthy Chinese individualsMethods:1)The whole blood of 154 healthy volunteers were collected,using density gradient centrifugation to obtain peripheral blood mononuclear cells(PBMC).2)The genomic DNA from PBMC was extracted and placed in liquid nitrogen.3)High-throughput sequencing of the CDR3 region.RNA was extracted,reverse transcribed into cDNA,cDNA was used as a template,multiple PCR amplification of VDJ region,access to the antigen receptor gene region.4)The amplified PCR product was purified,building a library.5)High-throughput sequencing.6)bioinformatics analysis to assess the diversity of the immune repertoire.Results:1)We demonstrated that the extent of TCR diversity was significantly different between age groups but not gender groups.2)The number of the TCR-CDR3 amino acid sequences was larger at the age of 21-30 years.3)We found the combination of TRBV with TRBJ genes was highest at the age of 21-30 years.4)TRBD was not significantly diverse in different age groups.5)We found that TRBV6-1 and TRBV27 were highly expressed throughout the whole healthy individuals.6)We identified TRBJ2-1 and TRBV7-2 with higher expression at the age of 6-10 and 11-20 years than other age groups.SummaryThese results showed that the different feature of TCR p-chain repertoire was in different age groups in the healthy Chinese individuals.In conclusion,it was expected that these TCR repertoires could serve as a useful tool for investigating the role of immune profiling in the healthy Chinese individuals.Part 2 Clinical significance of the frequency and function and MAGE-A3 specific CD8+ T cells in patients with NSCLCMethods:1)Selected fifty-three HLA-A2 and MAGE-A3 double positive patients with NSCLC and cultured MAGE-A3 specific CD8+T cells in vitro.MAGE-A3 specific CD8+ T cells were detected using MAGE-A3 specific tetramer by flow cytometry.2)The specific response to MAGE-A3 specific CD8+T cells was detected by flow cytomtery and ELISPOT assay.3)Evaluated the presence of functionally reactive MAGE-A3 specific CD8+ T cells and the correlation with clinical parameters of patients with NSCLC.Results:1)In the patients with NSCLC,MAGE-A3 tetramer positive CD8+ T cells were not be observed in the early stage after obtaining the leukapheresis product(detection threshold<0.01%),however,after stimulations twice with DC loaded peptide,the tetramer positive T cells were at frequencies of>0.2%..2)The frequency of tetramer positive CD8+ T cells was higher in patients with elder age but lower in patients with lymph node metastasis and late tumor stage(P<0.05).The lower CD 107a expression level of CD8+ T cells responding to MAGE-A3 peptide was significantly associated with poor outcome parameters,including advanced stage and lymph node metastasis(P<0.05).The expression level of CD107a+/IFN-y+ specific CD8+T was negatively correlated with lymph node metastasis.3)In addition,functional T cells responding to MAGE-A3 peptide,according to CD 107a high expression,not the frequency of MAGE-A3 specific CD8+T cells,could be used as an independent prognostic factor in Cox regression analysis.SummaryOur results indicated that MAGE-A3 specific CD8+ T cells could be induced with MAGR-A3 peptide.The presence of functional MAGE-A3 specific CD8+ T cells had strong prognostic impact on survival in NSCLC.In addition,our findings provided a rationale for pursuing MAGE-A3 based vaccination and T-cell adoptive transfer strategy.Part 3 T cell receptor variable P gene repertoire in reactive MAGE-A3 CD8+ T cells from patients with NSCLCMethods1)The responsive CD 107a+/IFN-?+and non-responsive CD107a-IFN-?-MAGE-A3 antigen specific CD8?T cells were sorted by flow cytometry.2)To synthetize the cDNA by one step method to characterize the repertoire of T cell receptor variable(TCR-vp)by real time PCR in CD107a+/IFN-y+and CD107a-/IFN-?'MAGE-A3 antigen specific CD8+T cells.3)Compared the differential TCR-v? between responsive and non-responsive MAGE-A3 antigen specific T cells.4)Then the relationships of top five dominating TCR-v?in patients with NSCLC with clinical characteristics were evaluated.Results1)TCR-v? repertoires of MAGE-A3-specific CD8+ T cells were distinct in different individuals.2)The expression levels of TCR-v? repertoires were distinct in responsive and non-responsive MAGE-A3-specific CD8+T cells.3)The top five differential TCR-v? genes in responsive antigen specific CD8+ T cells were different in NSCLC patients.The frequencies of top five differential TCR-v? were existing distinct.4)The frequencies of differential TCR-v? genes were associated with certain clinical parameters.SummaryTCR-vP repertoires of MAGE-A3-specific CD8+ T cells were distinct in different individuals.The expression levels of TCR-v? repertoires were distinct in responsive and non-responsive MAGE-A3-specific CD8+ T cells.The frequencies of differential TCR-vp genes were associated with certain clinical parameters.Conclusions:(1)This study revealed the diversity of TCR P-chain repertoire in the healthy Chinese individuals,which provides based data for comparing TCR ?-chain repertoire in different diseases to healthy dornors.(2)The frequency of reactive MAGE-A3 specific CD8+ T cells was posieively better prognostic parameters and survial.(3)The expression levels of TCR-v? repertoires were distinct in responsive and non-responsive MAGE-A3-specific CD8+ T cells in patients with NSCLC.In addition,different patiens existed differences and high expression of TCR-v?9 was positively associated with survival of NSCLC patients.