Characteristic Analysis Of Peripheral Blood TCR α CDR3 And TCR β CDR3 Repertoire And Screening Of Immune-related Genes In Patients With Active And Inactive SLE

Objective:1.Comparative analysis of the characteristics of the peripheral blood TCR α CDR3 and TCR β CDR3 repertoires of Systemic Lupus Erythematosus(SLE)patients in active,SLE patients in inactive,and healthy volunteers.2.Comparative analysis of the peripheral blood transcriptome gene expression of patients with active SLE,patients with inactive of SLE and healthy volunteers,and preliminary screening of candidate genes related to immunity.Methods:1.From outpatient and inpatients of the Department of Nephrology and Rheumatology of the Affiliated Hospital of Zunyi Medical University,according to the SLE diagnostic criteria of the American Academy of Rheumatology and the Systemic Lupus Erythematosus Disease Activity Index(SLEDAI)scoring criteria,follow the patient’s knowledge Under the principle of consent,4 patients with active SLE and 4 patients with SLE in inactive were screened.2.Collect 10 m L of peripheral blood from 4 active SLE patients,4 SLE patients in inactive,and 4 healthy volunteers.Isolating PBMCs and extracting total RNA.Each sample named:SLE inactive period: Inactive＿1,Inactive＿2,Inactive＿3,Inactive＿4;SLE activity period:Active＿1,Active＿2,Active＿3,Active＿4;healthy volunteers: HC＿1,HC＿2,HC＿3,HC＿4.3.Human T cell TCR α CDR3,TCR β CDR3 repertoire construction principle and process:Reverse transcription of the total RNA of each sample into c DNA,using c DNA end rapid amplification technology-5’RACE technology to build the repertoire,using the Illumina Nova Seq platform determine the gene sequence of the TCR α CDR3 and TCR β CDR3 repertoire of each sample(the repertoire construction and sequencing were completed by 6Hangzhou Aimun Biotech Co.,Ltd.).4.Human peripheral blood m RNA has a reference transcriptome sequencing process:m RNA is isolated from the total RNA of each sample,reverse transcribed into c DNA,double-stranded DNA is synthesized,a database is constructed by PCR,and the Illumina Nova Seq 6000 platform is used to determine the transcription of each sample group gene expression level(database construction and sequencing were completed by Lianchuan Biotechnology Co.,Ltd.).5.Analysis of sequencing results:(1)Analysis of the composition and characteristics of T cell TCR α CDR3 and TCR β CDR3 libraries: The sequence of each sample obtained after sequencing is converted into FASTA format and submitted to the IMGT database,using IMGT/High V-QUEST online software analyzes the output IMGT summary file,analyzes the diversity of T cell TCR α CDR3,TCR β CDR3 repertoire,the usage and pairing of V and J gene families,and the overlap of CDR3 regions.Sequence analysis and storage are in completed in Excel and graphed by Graphpad Prism 8 software.(2)Comparative analysis of differentially expressed genes in peripheral blood of patients with SLE in active phase,patients in inactive phase and healthy volunteers,and screening of immune-related genes.Results:1.Diversity of CDR3 repertoire: The diversity of TCR α CDR3 and TCR β CDR3 repertoire of Inactive and Active groups is less than that of HC group.And the diversity of the Inactive group is slightly lower than that of the Active group.2.Usaging and pairing of V and J gene families: Compared with the HC group,the Inactive group had significantly more usage to TRAV1-1,TRAV26-2,TRAV35,TRAV5,TRAV8-6,and TRBV5-4 genes(P<0.05);the TRAJ24 gene usage of the Active group was also higher than that of the HC group(P<0.05);the TRAV23,TRAV25 and TRAV8-1 genes of the Active group were significantly higher than the Inactive group(P<0.05);while the Inactive group the TRBV11-2,TRBV6-4 and TRBJ2-5 genes were used more than Active group(P<0.05).3.The length,amino acid usage,insertion and trimmed of the CDR3 repertoire: The CDR3 length of the TCR α CDR3 repertoire of SLE patients is normally distributed with11 and 12 amino acids,while the TCR β CDR3 repertoire is 11,12 and With 13 amino acids as the main normal distribution,there is no obvious difference between the Inactive group and the Active group;in the amino acid usage,there is a difference in Tryptophan between the Inactive group and the Active group(P<0.05),and the others have no significant difference;In the TCR α CDR3 repertoire there is a difference in the ratio of the number of 4 nucleotides inserted at 3’V between the Inactive group and the Active group(P<0.05),insert 5 nucleotides at 5’J,delete 3 and 8 There is a difference in the ratio of the number of nucleotides(P<0.05);in the TCR β CDR3 repertoire,most of the differences between the groups are in the insertion of longer nucleotides(>15)(P<0.05).4.CDR3 overlap: In the overlap sequence analysis of the Inactive group and the Active group,it was found that their overlap rate was higher than that of the HC group,and there was statistical significance between the TCR α CDR3 repertoire of the Inactive group and the HC group(P<0.05).At the same time,the overlap rate of the TCR α CDR3 repertoire in the Inactive group was higher than that of the Active group,while the overlap rate of the TCR β CDR3 repertoire was slightly lower than that of the Active group.5.CDR3 high-frequency sequence: In the first 10 sequences of the high-frequency cloning of the TCR α CDR3 repertoire of SLE patients,conservative motifs such as"SYQLT","ANNLF","GNQFY","GNKLV","YDKVI","GNKLV" ","SQGNLI",etc.are expressed in multiple SLE patients;among the first 10 sequences of high-frequency cloning of the TCR β CDR3 repertoire of SLE patients,"CASMDGNTYEQYF","SDEQY","NTGELIF" and other sequences are present in many patients Expression,the sequence CATSPAGNEQFF,is expressed in 7 samples of the first 10 high-frequency CDR3(SLE and healthy control group)of 12 samples,and the other 5 samples are all low-frequency expressions.6.Transcriptome results: In the analysis of differentially expressed genes,1746 genes were significantly up-regulated and 1757 genes were significantly down-regulated in Active group compared with HC group,1427 genes were significantly up-regulated and1020 genes were significantly down-regulated in Inactive group compared with HC group Compared with Inactive group,281 genes were significantly up-regulated and 457 genes were significantly down-regulated in Active group.Between active SLE and remission patients,TNFSF13 B and ATP6V1G2-DDX39 B genes in active SLE patients have significantly higher expressions.And CCR1,CLEC7 A,THBD,RNASE2,PTX3,CEACAM1 and other genes are closely related to SLE.There are 46 SLE-related genes in Active group and HC group,Inactive group and HC group,Active group and Inactive group and immune-related genes.Conclusion:1.The diversity of the TCR α CDR3 and TCR β CDR3 repertoire of peripheral blood of SLE patients is significantly lower than that of the control group.2.There is a certain degree of bias in the use of V and J genes in the peripheral T cell CDR3 library between the SLE inactive group and the active group;at the same time,the T cell CDR3 repertoire has a large difference in the number of inserted nucleotides.3.The CDR3 sequence that overlaps in the inactive group and the active group of SLE and has significant high-frequency expression may be related to the mechanism of SLE self-response T cells.4.There are multiple immunological-related genes differentially expressed in the transcriptome of the active and inactive group of SLE and Treg cell differentiation,basophil chemotaxis,etc.