| Chapter 1 Identification and functional analysis of early and advanced tumor-specific CD8~+T cell subsets in tumor-draining lymph nodesObjective:To identify the heterogeneity of cell subsets,discover new cell types with clinical guiding value,and contribute to the development of innovative therapeutic strategies in tumor immunity targeting the main anti-cancer main force,CD8~+T cells,through the data analysis of single-cell sequencing of tumor antigen-specific CD8~+T cells in non-metastatic tumor-draining lymph nodes(TDLN)of mice with lung adenocarcinoma.Methods:(1)Single-cell sequencing data of tumor antigen-specific CD8~+T cells from 2 samples(1 early TDLN,1 advanced TDLN)from KP-NINJA lung adenocarcinoma tumor-bearing mice were downloaded from the Gene Expression Omnibus(GEO)database.Principal Component Analysis(PCA)and t-Distributed Stochastic Neighbor Embedding(t-SNE)cluster analysis and differential gene expression analysis were used to identify the heterogeneity of gene profiles in each cell group.(2)The gene sets of human pan-cancer CD8~+T cell subsets were downloaded.Based on the perspective of cell function,the 6 cell subsets through Gene Set Enrichment Analysis(GSEA)functional enrichment analysis were manually annotated,and the differential gene bubble chart was used to show the marker genes where the cells of each subset were in the gene set.(3)Based on the characteristics of exhausted T cells in development and differentiation,the gene sets of cells at different stages of differentiation were download,6 cell subsets through GSEA functional enrichment analysis were manually annotated,and the differential gene bubble chart was used to show the marker genes where the cells of each subset were in the gene set.(4)t-SNE clustering was performed on tumor antigen-specific CD8~+T cell subsets in TDLN of early-stage and late-stage mice,respectively,and pie charts were drawn to analyze the distribution of each cell subset in TDLN in early-stage and late-stage tumors,respectively.The ratio of each cell subset number in early and advanced tumor stage TDLN was analyzed by histogram.Resu Lts:(1)Tumor antigen-specific CD8~+T cells for all samples was clustered into 6 subgroups(0-5 clusters)in t-SNE,and the gene heat map expression profiles of the 6 cell subgroups did not have similar expression,which cou Ld be considered as cells from 6 subpopu Lation.(2)Based on the perspective of cell function,6 cell subsets of TDLN tumor antigen-specific CD8~+T cells were annotated as 6 types of cells,including:1)Cluster 0-TCF-7~+TEX,representing stem-like depleted CD8~+T cells;2)Cluster 1-TN;3)Cluster 2-ZNF683~+CXCR6~+TRM,representing T cells with tissue-resident memory signature genes;4)Cluster 3-KIR~+EOMES~+NKT;5)Cluster 4-IL17 TM;6)Cluster 5-NME1~+T.(3)Based on the development and differentiation of exhausted T cells,6 cell subsets of TDLN tumor antigen-specific CD8~+T cells were annotated as 4 types of cells,including:1)Cluster 0 was annotated as TEXPROG1,representing the first stage restricted TCF-1~+depleted T cells in tissue and in a quiescent state;2)Cluster 4 and Cluster 5 are annotated as TEXPROG2;3)Cluster 1 and Cluster 3 are annotated as TEXINT;4)Cluster 2 is annotated as TEXTERM,representing the fourth stage terminally exhausted T cells.(4)The number of CD8~+T cell subsets characterized by Cluster 0-Cluster 2 significantly increased in advanced tumor TDLN,suggesting that tumor antigen-specific CD8~+T in advanced tumor TDLN have TCF7-TEX and TRM phenotypes,as well as TEXPROG1 and TEXTERM differentiation status respectively.Conclusion:By analyzing the differences of tumor-specific CD8~+T cell subsets in early-advanced TDLN in the characteristics and distribution,it was found that the CD8~+T cell subset with the characteristics of stem TCF7-TEX and tissue-resident memory TRM increased in advanced stage TDLN.At the same time,the cells in this group also had the characteristics of TEXPROG1 and TEXTERM differentiation,which might indicate the value of evaluating the malignant progression of tumors.Chapter 2 Gene characteristics and variation analysis of tumor-specific and non-specific CD8~+T cells in the draining lymph nodes of head and neck squamous cell carcinomaObjective:To study the functional characteristics of tumor antigen-specific CD8~+T cell transcriptome genes in non-metastatic TDLN of patients with head and neck squamous cell carcinoma,and further validate the resu Lts at the single cell level.Methods:(1)Flow sorting was used to obtain TDLN tumor antigen-specific(PD-1~+)and non-specific(PD-1~-)CD8~+T cells from patients with head and neck squamous cell carcinoma,and PCA principal components and gene differential expression analysis based on ordinary transcriptome sequencing were used.(2)The Tx-E cell and Tx-NE cell marker gene sets were download,GSEA functional enrichment on the PD-1~+CD8~+T and PD-1~-CD8~+T cell gene lists was performed,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed.(3)The effector cell(TERMINAL,GZMK+TEM,TCF7+TEX,OXPHOS-TEX,TEMRA,GZMK-TEX,and TC17)phenotype marker gene sets were download,and PD-1~+CD8~+T and PD-1~-CD8~+T cell gene list was used for GSEA functional enrichment.(4)The CLUSTER0 and CLUSTER2 marker gene sets were downloaded,and GSEA functional enrichment on the PD-1~+CD8~+T and PD-1~-CD8~+T cell gene lists was performed.(5)The TRM and non-TRM cell marker gene sets were downloaded,and GSEA functional enrichment for the PD-1~+CD8~+T and PD-1~-CD8~+T cell gene lists was performed.Resu Lts:(1)PCA principal component analysis,differential gene heat map and volcano plot showed that the gene expression of PD-1~+CD8~+T and PD-1~-CD8~+T cells were heterogeneous.Most of the differential genes in PD-1~+CD8~+T cells were significantly up-regu Lated.(2)GSEA resu Lts showed that PD-1~+CD8~+T cells had a positive correlation in gene function with tumor-infiltrating Tx-E cells in response to immunotherapy.GO analysis showed that PD-1~+CD8~+T cells were mainly enriched for cell growth and differentiation Function.KEGG analysis showed that PD-1~+CD8~+T cells were mainly enriched in virus-related immune pathways,suggesting that PD-1~+CD8~+T cells have stronger potential for differentiation and proliferation.(3)GSEA resu Lts showed that PD-1~+CD8~+T cells were significantly positively correlated and enriched in each effector cell phenotype pathway,suggesting the characteristics of stronger effector cell and stemness.(4)PD-1~+CD8~+T cells were significantly positively enriched in CLUSTER0 and CLUSTER2 subset cell phenotype pathways,suggesting that PD-1~+CD8~+T cells in head and neck squamous cell carcinoma draining lymph nodes have the characteristics of CD8~+T cells in advanced malignant tumor.(5)PD-1~+CD8~+T cells were significantly positively correlated and enriched on the TRM cell phenotype pathway,while PD-1~-CD8~+T cells were significantly positively correlated and enriched on the non-TRM cell phenotype pathway.PD-1~+CD8~+T cells had the genetic signature of tissue-resident memory TRM cells,whereas PD-1~-CD8~+T cells did not have this phenotypic signature.Conclusion:Through the analysis of the gene difference between tumor-specific and non-specific CD8~+T cells in the draining lymph nodes of head and neck squamous cell carcinoma,it was found that PD-1~+CD8~+T cells had the characteristics of stemness and TRM gene of CD8~+T cell in advanced malignant tumors.Chapter 3 CD8~+T cell subsets with stem like and TRM dual phenotypes in draining lymph nodes of head and neck squamous cell carcinomaObjective:To detect the infiltration ratios of stem like and TRM phenotype PD-1~+CD8~+T cells in peripheral blood,non-metastatic TDLN,metastatic TDLN and tumor specimens of patients with head and neck squamous cell carcinoma.Methods:(1)Flow cytometry was used to detect the expression ratios of TRM phenotype markers(CD69 and CD103)in peripheral blood,non-metastatic TDLN,metastatic TDLN and tumor specimens of patients with head and neck squamous cell carcinoma.(2)Flow cytometry was used to detect the expression of CD8~+T cell stemness phenotype marker(TCF-1)and PD-1 in peripheral blood,non-metastatic TDLN,metastatic TDLN and tumor specimens of patients with head and neck squamous cell carcinoma.(3)Flow cytometry was used to detect the co-expression of TCF-1 and CD69 in PD-1~+CD8~+T cells in non-metastatic TDLN,metastatic TDLN and tumor specimens of patients with head and neck squamous cell carcinoma.Resu Lts:(1)Flow cytometry analysis showed that CD69~+CD8~+T cells,CD103~+CD8~+T cells and CD69~+CD103~+CD8~+T cells were mainly present in non-metastatic TDLN,metastatic TDLN and tumor tissues in patients with head and neck squamous cell carcinoma.The highest proportion was in tumor tissue,and they were almost absent in peripheral blood,which was consistent with the TRM phenotype CD8~+T cell tissue resident characteristic.(2)Flow analysis showed that there were TCF-1~+CD8~+T cells,PD-1~+CD8~+T cells and TCF-1~+PD-1~+CD8~+T cells in the peripheral blood,non-metastatic TDLN,metastatic TDLN and tumor specimens of patients with head and neck squamous cell carcinoma.The highest proportion of PD-1 expression was in tumor tissue.The highest proportion of TCF-1 expression was in non-metastatic TDLN,while its expression in tumor tissue was relatively less,suggesting that the infiltration of cancer cells wou Ld make the phenotype of TCF-1stemness of CD8~+T cells gradually subside.At the same time,it was found that the majority of CD8~+T cells in non-metastatic TDLN expressed stemness marker TCF-1.(3)The resu Lts of flow cytometry analysis showed that TCF-1~+CD69~+PD-1~+CD8~+T cells mainly existed in solid tissue TDLN,and they were almost absent in tumor tissue,suggesting that there were PD-1~+CD8~+T cells with dual phenotypes of stemness and TRM in TDLN.Conclusion:Flow cytometry analysis showed that PD-1~+CD8~+T cells in the draining lymph nodes of head and neck squamous cell carcinoma had dual phenotypes of stemness and resident.Chapter 4 The clinical value of stem like and TRM cells in CD8~+T cell subsets in the draining lymph nodes of head and neck squamous cell carcinomaObjective:To investigate the relationship between the infiltration of tumor antigen-specific CD8~+T cell with stemness and TRM dual phenotypes in non-metastatic TDLN of patients with head and neck squamous cell carcinoma and clinical characteristics,and to clarify the significance of the existence of this subset of cells on the survival and prognosis of patients with head and neck squamous cell carcinoma.Methods:(1)Flow cytometry was used to analyze the co-expression of CD103 and CD69 in CD8~+T cells of TDLN specimens from patients with laryngeal cancer and oral cancer.(2)Flow cytometry was used to analyze the different infiltration ratios of this subset of cells in proximal(region II/III)TDLN and distal(region IV)TDLN.(3)Flow cytometry was used to analyze the infiltration of this subset of cells in the proximal TDLN located on the ipsilateral and contralateral sides of the in-situ tumor.(4)Correlation analysis was used to clarify the relationship between the number of this cell subset in TDLN and the size of the primary tumor.Resu Lts:(1)The resu Lts of flow cytometry analysis showed that the proportion of CD69~+CD103~+subset cells in both laryngeal cancer(P<0.001)and oral cancer(P<0.0001)tissues was significantly higher than that in peripheral blood.There was almost no CD69~+in peripheral blood.The presence of CD103~+CD8~+T cells was consistent with the expression trend of the comprehensive analysis of head and neck squamous cell carcinoma tumor types.(2)Flow analysis showed that the proportion of CD69~+CD8~+,CD103~+CD8~+T cells and CD69~+CD103~+CD8~+T cells in proximal TDLN was significantly higher than that in distal TDLN,suggesting that CD8~+T cells with the characteristics of stem like and TRM were enriched in the proximal TDLN of head and neck squamous cell carcinoma.(3)Flow cytometry analysis showed that there was no significant difference in the proportion of CD69~+,CD103~+single expression and CD69~+CD103~+co-expression between the CD8~+T cells of the TDLN located on the ipsilateral side of the tumor and those of the TDLN located on the contralateral side of the tumor,suggesting that the enrichment effect of the CD8~+T cell subsets with the TRM phenotype in the proximal draining lymph nodes of head and neck squamous cell carcinoma was independent of tumor side.(4)Correlation analysis showed that the percentage of CD69~+cell subsets in CD8~+T cells in TDLN was significantly positively correlated with tumor size(r=0.7078,P=0.02).Similarly,the percentage of CD103~+cell subsets in CD8~+T cells also showed a very significant and highly positive correlation(r=0.8974,P=0.0004)with tumor size,suggesting that the increased resident phenotypic characteristics of CD8~+T cells with dual-phenotype of stemness and TRM have an important role in evaluating the late progression of malignant tumors.Conclusion:The presence of PD-1~+CD8~+T cells with dual phenotypes of stemness and resident in the draining lymph nodes of head and neck squamous cell carcinoma cou Ld indicate the degree of tumor progression.Chapter 5 Radiation-induced remove the resident property of stem like CD8~+T cell subsets in head and neck squamous cell carcinoma draining lymph nodesObjective:To observe the effect of radiotherapy on the tumor antigen-specific CD8~+T cell subsets with dual-phenotype of stemness and TRM in head and neck squamous cell carcinoma TDLN by in vivo experiments.Methods:(1)Subcutaneous injection of head and neck squamous cell carcinoma cell line Meer was used to construct C57 mouse xenograft animal model.During the period,4Gy local radiotherapy was administered to the experimental group for two consecutive days to interfere with the tumor site,and the tumor growth in the control group and the experimental group was observed and recorded.The tumor volume growth curve was drawn,and the difference in tumor weight between the two groups was calcu Lated.The infiltration and functional changes of effector T cells in tumor tissues of the two groups of mice were compared by flow cytometry.(2)Flow cytometry was used to detect and compare the infiltration ratio of the tumor antigen-specific CD8~+T cell subsets with dual-phenotype of stemness and TRM in bilateral TDLN of two groups of mice.Resu Lts:(1)The tumor volume growth(P<0.05)and tumor dry weight(P<0.0001)of the mice in the radiotherapy group were significantly lower than those in the control group,suggesting that radiotherapy had a significant inhibitory effect on the growth of the transplanted tumors of the head and neck squamous cell carcinoma mice.The number of total CD8~+T cells in the transplanted tumor of the mice in the radiotherapy group was significantly higher than that in the control group(P<0.05),and the number of PD-1~+CD8~+T cells in the tumor tissue of the radiotherapy group was significantly higher than that in the control group(P<0.05).However,there were no significant differences in the numbers of PD-1~-CD8~+T cells,GZMB~+CD8~+T cells,TNF-α~+CD8~+T cells and IFN-γ~+CD8~+T cells between the two groups,suggesting that radiotherapy induces C57 to be smaller.The infiltration of PD-1~+CD8~+T cells in the primary foci of mouse head and neck squamous cell carcinoma increased,but the effector function did not change significantly.(2)Flow cytometry showed that the proportion of TCF-1~+cell subsets in the PD-1~+CD8~+T cells in the ipsilateral draining lymph nodes of the tumor in the control group and the radiotherapy group were 99.6%and 99.0%,respectively.The proportions in the lateral draining lymph nodes were 98.5%and 98.3%,respectively.The resu Lts showed that the majority of PD-1~+CD8~+T cells in the contralateral and ipsilateral TDLNs of the head and neck squamous cell carcinoma of C57mice were stem cells.There was no effect on the stemness phenotype of PD-1~+CD8~+T cells in the bilateral TDLN.The proportions of CD103~+cell subsets in the PD-1~+CD8~+T cells in the ipsilateral draining lymph nodes in the control and radiotherapy groups were 58.5%and 31.95%,respectively,and the proportions in the contralateral draining lymph nodes were 49.11%and 26.14%,respectively.This indicated that the resident of PD-1~+CD8~+T cells in the contralateral and ipsilateral TDLN of head and neck squamous cell carcinoma of C57 mice was weakened by radiotherapy.The number of TCF-1~+CD103~+PD-1~+CD8~+T cells in the TDLN on both the ipsilateral(P<0.05)and contralateral(P<0.05)sides of the tumor in the radiotherapy group was significantly lower than that in the control group,suggesting that radiotherapy cou Ld eliminate the resident of PD-1~+CD8~+T cells in head and neck squamous cell carcinoma TDLN in C57 mice.Conclusion:Radiotherapy cou Ld eliminate the resident property of stem like and TRM dual phenotypes CD8~+T cells in the draining lymph nodes of head and neck squamous cell carcinoma. |
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