The Diversity Of The TCR β Chain CDR3 Repertoire In Renal Cell Carcinoma

Background:In recent years,the incidence of renal cell carcinoma in China has been on the rise.The occurrence and development of renal cell carcinoma is a process of continuous interaction of multiple factors,and its exact etiology is not clear.At present,surgical resection is still the main means of renal cell carcinoma treatment,and surgical treatment can significantly improve the survival rate of patients.Although the prognosis of renal cell carcinoma has improved significantly with the improvement of diagnosis and treatment,there are still some patients with tumor recurrence or even progression and metastasis.Previous studies have shown that the occurrence of malignant tumors is associated with immune dysfunction.Renal cell carcinoma is a highly immunogenic cancer.In recent years,immunotherapy has become a new treatment for advanced renal cell carcinoma.Although it has shown some therapeutic effect,it cannot completely control and cure the tumor due to the occurrence of drug resistance.Therefore,it is necessary to further study the immune mechanism of renal cell carcinoma and analyze the tumor immune microenvironment.In the tumor microenvironment,the immune system plays an important role.The tumor microenvironment regulated by the immune system is called the tumor immune microenvironment.Tumor infiltrating lymphocytes(TIL)play a key role in the tumor immune microenvironment.One of the tumor infiltrating lymphocytes that play an important role in tumor killing is T lymphocytes.T lymphocytes recognize the antigen presented by antigen presenting cells through TCR is an important step to start its biological effect.Therefore,the immune system plays an important role in the tumor microenvironment.Studying the functional characteristics and immunological mechanisms of T cell receptors is of great significance for understanding tumor immunity.TCR in human body is mainlyαβTCR,in which β chain is the main peptide chain in TCR repertoire research,because β chain(the main component of D gene fragment in VDJ gene fragment)has higher combination potential than a chain,and its diversity is more complex.TCR a chain and β chain are composed of extracellular domain and intracellular domain,respectively.The extracellular domain has two structural domains,and the amino acid sequence at the amino end changes greatly,which is called the variable region,which contains the complementary determining region,and there are three complementary determining regions,namely CDR1,CDR2 and CDR3.Among the three complementary determinants,CDR3 had the largest variation,and TCR mainly relied on it for antigen-specific recognition.After antigen stimulation,T cells proliferate and activate into a group of T lymphocyte clones,and the TCRs of these T lymphocytes are exactly the same.When T lymphocytes contact with different antigens,specific receptors in TCR repertoire combine with them.When antigen persists,T lymphocytes with specific TCR proliferate continuously,that is,clonal proliferation driven by antigen occurs.Therefore,from the diversity of TCR repertoire can understand the human immune response and a variety of immune response,which is very important for the study of tumor immune molecular mechanism.Studies have shown that TCR repertoire plays an important role in the diagnosis,treatment and prognosis of tumors,autoimmune diseases,infectious diseases and other diseases.Studying the characteristics of the TCR repertoire of the disease can understand the immune response in patients.The study of T cell receptor diversity can have a more in-depth analysis of the immune function of the human body,and screen out the gene fragments and amino acid sequences that have a specific relationship with the disease.It can help to select the best treatment for the disease-free state,judge the therapeutic effect and estimate the prognosis.It can also identify disease-specific T cell clones and use immunotherapy.Previous studies on TCR repertoire of renal cell carcinoma mainly focused on the phenotype and functional characteristics of CD4+/CD8+tumor infiltrating T lymphocytes,as well as the comparison of immune repertoire of cancer tissue and peripheral blood mononuclear cells.There are few studies on TCR immune repertoire of renal cell carcinoma tissue and adjacent normal renal tissue.The understanding of the characteristics of TCR CDR3 in cancer tissues and adjacent non-tumor tissues is still insufficient,so it is necessary to further study the characteristics of TCR CDR3 in cancer tissues and adjacent non-tumor tissues.Objective:The purpose of this study is to analyze the diversity and clone expression of TCR in renal cell carcinoma and adjacent non-tumor tissues,and to analyze the diversity and clone sequence of T cell receptor in renal cell carcinoma and adjacent non-tumor tissues by high-throughput sequencing technology.Methods:1.Collect renal cell carcinoma tissues and adjacent non-tumor tissues,and extract tissue RNA;2.TCR CDR3 was amplified by 5’ RACE and nested PCR;3.Construction of sequencing library;4.High-throughput sequencing5.To analyze the diversity of Vβ,Jβ gene fragments,V-D-J gene combinations and TCR CDR3 in cancer tissues and adjacent non-tumor tissues.Results:1.Analysis of the usage patterns of Vβ and Jβ gene fragments in TCR repertoire:Thirteen Jβ gene fragments were identified in all samples of cancer tissues and adjacent non-tumor tissues.The 62 Vβ gene fragments belonged to 29 Vβ gene subfamilies,and the 13 Jβ gene fragments belonged to 2 Jβ gene families,respectively.There was no significant difference in the number of Vβ gene types between cancer tissues and adjacent non-tumor tissues by comparing the difference in the use of Vβ gene types between cancer tissues and paracancerous tissues.The use patterns of Vβ and Jβ genes in cancer tissues and adjacent non-tumor tissues were generally similar.There are some high-frequency Vβ gene fragments and Jβ gene fragments in some samples.Most of the genes used in each sample were less than 5%.The frequencies of four Vβ gene fragments in cancer tissues and adjacent non-tumor tissues were statistically different,which were TRBV5-5,TRBV7-7,TRBV9 and TRBV25-1,respectively.Only the frequency of TRBJ2-5 gene fragments was different.2.Analysis of V-D-J Gene Combination in Cancer Tissue and adjacent non-tumor tissuesThe average Simpson index and NSDE values of adjacent non-tumor tissues tissues were higher than those of cancer tissues,indicating that the diversity of V-D-J gene combination mode in adjacent non-tumor tissues was higher than that in cancer tissues,but there was no significant statistical difference between the two.The average MHSI of cancer tissues and adjacent non-tumor tissues of each paired sample was 0.28,and the MHSI value of most paired samples was lower than 0.3.The similarity of V-D-J gene combination mode between cancer tissues and adjacent non-tumor tissues was low.There was statistical difference in the number of V-D-J gene combinations between cancer tissues and adjace nt non-tumor tissues.3.Diversity analysis of TRB CDR3 in cancer tissues and adjacent non-tumor tissues.(1)Quantity difference of clonal TRB CDR3 amino acid(CDR3 aa)between cancer tissues and adjacent non-tumor tissuesThe median of CDR3 amino acid clone in cancer tissues was 3804[3110-7459],and that in adjacent non-tumor tissues was 2038[1177-3710].The number of CDR3 amino acid clone in cancer tissues was larger than that in adjacent non-tumor tissues,but there was no significant statistical difference between the two.(2)Distribution characteristics of amino acid length of TRBCDR3 in cancer tissues and adjacent non-tumor tissuesAnalysis of TRB CDR3 amino acid clone length distribution(Figure 3-8),most samples of CDR3 amino acid sequence length of 5-30 amino acids,each sample used more amino acid length range of 11-18.The sum of frequency of top 5 amino acid clones in cancer tissues was higher than that in adjacent tissues.There may be clonal proliferation of CDR3 amino acids in cancerous or adj acent non-tumor tissues.(3)high expended cloning Analysis of Cancer Tissues and adjacent non-tumor tissuesThe average number of HEC in cancer tissues and adjacent non-tumor tissues was 120 and 152,respectively.There was no significant statistical difference in the overall number of HEC between cancer tissues and adjacent non-tumor tissues(4)Comparison of TRB CDR3 amino acid polymorphism between cancer tissues and adjacent non-tumor tissuesSimpson index and NSDE were lower in cancer tissues than those in adjacent non-tumor tissues,and CDR3 amino acid diversity in cancer tissues was lower than that in adjacent non-tumor tissues,but there was no significant statistical difference.(5)Cloning and similarity analysis of TRB CDR3 amino acids in cancer tissues and adjacent non-tumor tissuesIn all samples of cancer tissues or adjacent non-tumor tissues,with the increase in the number of samples,the proportion of shared CDR3 amino acid clones decreased significantly,and the shared cloning was very low.The co-clones of each pair of cancer tissues and paired adjacent tissues were also low,indicating that the co-clones between different samples in cancer tissues or aadjacent non-tumor tissues were very low.(6)Similarity of TCR CDR3 amino acid usage frequency in cancer tissues and adjacent non-tumor tissues.The median MHSI value of each pair of cancer tissues and adjacent tissues was 0.035,and most samples were less than 0.03.It indicated that the frequency of TCR CDR3 amino acids in cancer tissues and adjacent non-tumor tissues was low.(7)Principal component analysis of TCR CDR3 amino acid usage in cancer tissues and adjacent non-tumor tissuesThe frequency of CDR3 amino acids was calculated by principal component analysis(standardized by samples),and the first and second principal component values were plotted.Cancer tissue and adjacent tissue can not be completely divided into two groups,the principal component values of each sample are different.(8)Cloning analysis of tumor-associated CDR3 amino acidFor each CDR3 amino acid clonotype,the number of CDR3 amino acid clonotypes in cancer tissues and adjacent non-tumor tissues was calculated respectively.When the number of samples in cancer tissues was higher than that in adjacent non-tumor tissues,and exact test showed statistically significant differences,it was considered that the CDR3 amino acid clonotype might be tumor-related CDR3 amino acid clonotype.A total of 17 tumor-specific CDR3 amino acid clonotypes were identified,and it was speculated that it might be related to tumor antigens.(9)The relationship between TCR CDR3 diversity and clinical prognosis in cancer tissues and adjacent tissuesThe NSDE values of different clinical stages of cancer tissues were compared for clinical prognosis analysis.Grouped by clinical stages,the NSDE of stage II patients was slightly higher than that of stage I and stage III patients,and there was no significant statistical difference among the three groups.The diversity of TCR CDR3 in cancer tissue was not significantly correlated with the clinical prognosis.Conclusion:1.The pattern of utilization of Vβ and Jβ genes in renal cell carcinoma tissues and adjacent non-tumor tissues are generally similar,and there are Vβ and Jβ gene fragments with statistical differences.2.The diversity of TCR CDR3 amino acids in cancer tissues was lower than that in adjacent non-tumor tissues,and the similarity of TCR CDR3 amino acids between cancer tissues and adjacent non-tumor tissues was low.3.Renal cell carcinoma and adjacent non-tumor tissues have specific TCR CDR3 amino acid pattern of utilization.