| Objective: In the article,we take healthy monozygotic twins(MZ),dizygotic twins(DZ)and non twins(NT)aged 20-50 years as the research objects,and analyze the homogeneity and heterogeneity of TCR ? chain pseudogene CDR3,out of frame rearrangement CDR3,productive CDR3 repertoires in the peripheral blood among MZ,DZ and NT groups,as well as the dynamic changes of the TCR ? CDR3 repertoires among MZ,DZ and NT before and after 5 years.A preliminary study of the influence of individual genetic factors on the bias of V(D)J recombination and the selection effect MHC-self peptide tolerance of TCR provides a basis for further research on the mechanism and application of TCR rearrangement and selection.Methods: 1.Collecting respectively 10 ml of peripheral blood from 6 pairs of healthy twin volunteers(D,e,F,G,h,i),isolating PBMCs and extracting total DNA;designing and synthesizing 30 human TRBV gene family-specific upstream primers,14 human TRBJ gene familyspecific downstream primers,using the total DNA as a template to multiplex PCR to amplify the TCR ? CDR3 repertoires,and recovering the PCR products and sending them to BGI(Beijing Genomics institution).The TCR ? CDR3 repertoires of each sample are sequenced by Illumina Solexa high-throughput technology after quality inspection and quality control are completed.2.Collecting respectively 10 ml of peripheral blood from 3 pairs of healthy twin volunteers(A,B,c)in 2012 and 2017(completed by the research team),isolating PBMCs and extracting total DNA,total DNA is sent to Adaptive Biotechnologies immune SEQ companies(Washington Hospital,USA)and BGI,constructing the TCR ? CDR3 repertoires by multiplex PCR technology and sequencing by Illumina Solexa highthroughput technology.3.The DNA of 9 pairs of twin samples is sent to Beijing Boao Jingdian Biotechnology Co.Ltd.,and the HLA-A/B/C/DRB1 locus is tested by PCR-SBT method.4.Comparative analysis of TCR ? CDR3 repertoires characteristics:(1)Comparative analysis for the homogeneity and heterogeneity of pseudogene CDR3,out of frame rearrangement CDR3 and productive CDR3 sequences among MZ(D1/D2,F1/F2,G1/G2),DZ(e1/e2,h1/h2,i1/i2),and NT(D1/e1,D1/F1,D1/G1,D1/h1,D1/i1)groups;(2)Comparative analysis for dynamic changes of TCR ? CDR3 repertoires among MZ(A1/A2,B1/ B2),DZ(c1/c2)and NT(A1/B1,A1/c1,A1/c2,B1/c1,B1/c2)before and after 5 years.Results: 1.HLA typing results of twins show that A,B,D,F,and G are monozygotic twins,and c,e,h,and i are dizygotic twins.2.TCR ? CDR3 repertoires diversity analysis:(1)There is no statistical difference in the diversity among MZ,DZ and NT groups.(2)There is no statistical difference in the diversity of the productive CDR3 repertoires before and after 5 years.3.TCR ? CDR3 repertoires TRBV/TRBJ gene usage:(1)In the pseudogene CDR3 sequences,TRBV/TRBJ gene usage of MZ is more similar,the difference among MZ,DZ and NT three groups are significantly different;the dynamic changes between MZ is more consistent before and after 5 years.(2)In the out of frame rearrangement CDR3 sequences,TRBV/TRBJ gene usage of MZ and DZ are similar,the dynamic changes have no significant difference among MZ,DZ and NT groups before and after 5 years.(3)In the productive CDR3 sequences: compared with the pseudogene CDR3 sequences,the similarity for the TRBV and TRBJ gene usage of MZ is reduced,and the difference among MZ,DZ and NT groups is reduced;the dynamic changes in TRBV gene usage is no significant difference among MZ,DZ and NT groups.(4)The similarity in TRBV/TRBJ gene usage of the MZ in the pseudogene CDR3 sequence is significantly higher than the similarity of the out of frame CDR3 sequences and the productive CDR3 sequences.4.TCR ? CDR3 repertoires V-J pairing:(1)In the pseudogene CDR3 sequences,the V-J pairing distribution of MZ is the most similar,and the dynamic changes between MZ are more consistent before and after 5 years.(2)In the out of frame rearrangement CDR3 sequences,the V-J pairing partly of MZ is similar,and the dynamic changes between MZ are more consistent before and after 5 years.(3)In the productive CDR3 sequences,the V-J pairing distribution among MZ,DZ and NT groups is random,and there is no significant difference in the dynamic changes among MZ,DZ and NT groups before and after 5 years.5.TCR ? CDR3 repertoires AA usage and length distribution: There is no significant difference in AA usage and length distribution among MZ,DZ and NT groups and there is no significant difference in the dynamic changes among MZ,DZ and NT groups before and after 5 years.6.Overlap of TCR ? CDR3 repertoires AA sequences:(1)There is no statistical difference in the overlap among MZ,DZ and NT groups and the overlap ratio of some MZ and DZ is higher than that of unrelated individuals in productive high-frequency segmented AA sequences.(2)There is no significant differences in the dynamic changes among MZ,DZ and NT groups before and after 5 years.7.TCR ? CDR3 repertoires nucleotide insertion and deletion:(1)In the pseudogene CDR3 sequences,the similarity of different nucleotide numbers inserted and deleted proportion: MZ> DZ> NT,the dynamic changes between MZ are more consistent.(2)In the out of frame rearrangement CDR3 sequences,the proportion of different nucleotide numbers inserted and deleted is similar among MZ,DZ and NT groups;the dynamic changes between MZ are more consistent.(3)In the productive CDR3 sequences,the proportion of different nucleotide numbers inserted and deleted is similar among MZ,DZ and NT groups;there is no significant differences in the dynamic changes among MZ,DZ and NT groups before and after 5 years.Conclusions: 1.(1)In the TCR ?pseudogene CDR3 sequences,MZ(D1/D2,F1/F2,G1/G2)and DZ(e1/ e2,h1/h2,i1/i2)show more homogeneity in V/J gene usage,V-J pairing,nucleotide insertion and deletion compared with NT(D1/e1,D1/F1,D1/G1,D1/h1,D1/i1)and the overlap ratio of some MZ and DZ is higher than that of unrelated individuals in productive high-frequency segmented AA sequences.(2)There is no significant difference in AA usage,AA length distribution of TCR ? CDR3 among MZ,DZ and NT groups.It suggests that individual genetic factors are biased towards V(D)J initial recombination and MHC-autopeptide tolerance selection in the thymus,and influence the composition characteristics of the peripheral adaptive immune response total T cells ? CDR3 repertoires.The experiment can provide a basis for further research on the effects and mechanisms of individual genetic factors on V(D)J recombination bias,tolerance selectivity,and peripheral adaptive responses.2.(1)In the TCR ? pseudogene CDR3 sequences and TCR ? out of frame rearrangement CDR3 sequences,the dynamic changes between MZ(A1/A2,B1/B2)are more consistent in V,V-J pairing,nucleotide insertion and deletion of the TCR ? CDR3 repertoires before and after 5 years,and DZ(c1/c2)and NT(A1/B1,A1/c1,A1/c2,B1/c1,B1/c2)are inconsistent.(2)There is no significant difference among MZ,DZ and NT groups in J gene usage,AA usage,AA length distribution of TCR ? CDR3 and overlap of TCR ? CDR3 repertoires before and after 5 years.It further suggested that genetic factors may have an effect on the bias of the CDR3 repertoires of total T cells in peripheral blood,and also suggested that adaptive immune response of the individual's experience needs to be increased to assess the degree of impact on the results in the study. |
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