| Objective:In order to explore the effects of age on the TCRβCDR3 repertoires of naive CD4~+T cell in spleen,T cells in thymus and spleen and the homogeneity and heterogeneity of T cells TCRβCDR3 repertoires in different tissues of mice were analyzed by high throughput sequencing.Methods:1.Genomic genes were extracted from spleen and thymus tissues of 3,12 and20-month-old female BALB/c mice;naive CD4~+T cell were selected by magnetic bead sorting,and then genomic genes were extracted and DNA purity was identified by flow cytometry.There are 9 groups including 5 samples in each group,2.PCR was used for the amplification of these above genomic genes to prepare the TCRβCDR3 library,and the PCR purified product was sent to BGI Company for sequencing by high-throughput sequencing.3.We have Converted the results of the sequencing from BGI Company into fasta format and uploaded it to the IMGT basic database.The files were downloaded from the IMGT database for analysis.Results:1.With the age increasing,the cortex and medulla boundaries of the mice thymus disappeared,the medulla increased,the cortex decreased,and the secretory vacuole formed at aged 20 months;the structure of spleen white pulp became more and more anomalous.2.With the age increasing,the diversity of TCRβCDR3 groups in mice spleen naive CD4~+T cells and thymus T cells changed,the diversity decreased,while the diversity of the spleen T cella TCRβCDR3 group did not change with age-related increasing.3.All 45 samples used the serine,threonine,glycine,alanine and tyrosine at high frequency,and the usage of TRBJ was similar.4.The TCRβCDR3 repertoires of 45 samples showed Gaussian distribution dominated by 12 amino acids,but with the increase of month age,the Gaussian distribution becomes more and more irregular.5.The common clone overlapping ratio of TCRβCDR3 repertoires of thymus and spleen T cell as well as the spleen CD4~+naive T cell increased with the increase of month age.6.When the mice was at 3 months,the diversity ofTCRβCDR3 repertoires among different tissues was different.7.High or low frequency usage of TRBV and TRBJ gene families and TRBV-TRBJ pairing in different tissues at the same month:(1)At 3 months age,TRBV1,TRBV13-2,and TRBV2 were accessed for a high frequency in the TCR of three groups,and TRBV12-2,TRBV20,TRBV23,and TRBV24 usage were for a low frequency;however,naive CD4~+T cells TCRβ CDR3 repertoires in spleen were also used TRBV13-1 and TRBV19 for a high frequency,the spleen T cell used TRBV19 and TRBV4 for a high frequency,and TRBV3,TRBV4 and TRBV26 were used by the thymus T cell at high frequency.The two TCR groups in the spleen used TRBV26 at low frequency,while the thymuscellusedTRBV30foralowfrequency.TRBV1-TRBJ1-2,TRBV1-TRBJ1-3,TRBV1-TRBJ2-1 and TRBV1-TRBJ2-5 were used for a high frequency in all 3 samples,and TRBV24-TRBJ1-4,TRBV24-TRBJ1-5 and TRBV24-TRBJ2-1 were used for a low frequency.Comparing these samples between two groups,it was found that there were 20 different types of TRBV and TRBJ gene families in the mice spleen T cells and naive CD4~+T cells.There were 73 different types between mice spleen CD4~+T cells and thymus T cells,and there were 132 different types of TRBV and TRBJ pairing usage between thymic T cells and spleen T cells.At the same time.(2)At 12 months age,TRBV1,TRBV2,and TRBV4 were used for a high frequency in the TCR among three groups,TRBV12-2,TRBV20,TRBV23,and TRBV24 were used for a low frequency;but spleen cells and naive CD4~+T cells used the TRBV13-1 and TRBV14 for a high frequency,and used TRBV26 for a low frequency;and thymus T cells used TRBV30 for a low frequency,but used the TRBV14 and TRBV3 for a high frequency.the three groups of samples used TRBV1-TRBJ1-1,TRBV1-TRBJ1-2,TRBV1-TRBJ2-1 and TRBV1-TRBJ2-5 for a high frequency,and used TRBV24-TRBJ1-5 and TRBV24-TRBJ2-1 for a low frequency,but there were many differences in the pairing usage of TRBV and TRBJ gene families among three groups.There are 16 different pairs between spleen T cells and naive CD4~+T cells,and there were 96 different pairing usage between thymus T cells and spleen naive CD4~+T cells,and 83 different pairs between thymus T cells and spleen T cells.(3)At 20 months age,the TCRβCDR3 repertoires among three groups used TRBV1,TRBV2,TRBV13-2,and TRBV4 for a high frequency,and used TRBV12-2,TRBV20,TRBV23,and TRBV24 for a low frequency;spleen T cells and naive CD4~+T cells used TRBV14 for a high frequency,and used TRBV26 for a low frequency;the thymus T cell bank used TRBV3 for a high frequency but a low frequencyforusingTRBV30;TRBV1-TRBJ1-2,TRBV1-TRBJ2-1and TRBV1-TRBJ2-5 were used for a high frequency,and TRBV24-TRBJ1-1,TRBV24-TRBJ1-2,TRBV24-TRBJ1-4,TRBV24-TRBJ1-5 and TRBV24-TRBJ2-5 were used for a low frequency.Gene family pairing usage were used approximately similar,spleen T cells and naive CD4~+T cells are different in TRBV1-TRBJ1-5 usage.Compared with thymic T cell,there were 5 and 6 different pairing usage of the TRBV and TRBJ gene family pairing usage in the spleen naive CD4~+T cells and T cells,respectively.Conclusion:1.The diversity of the TCRβCDR3 repertoires of naive CD4~+T cells in spleen and T cells in thymus in mice decreased with age increasing,and the greater the age,the larger the clonal expansion will occur,and the clonal amplification has preference.Large-scale clonal expansion of TCRβCDR3 repertoires in the spleen T cell will not occur until 12 months.While the diversity of T cells TCRβCDR3 in spleen was not affected by the age.2.The distribution of CDR3 length of naive CD4~+T cell in spleen and T cells in spleen and thymus of the mice changed with age increasing,and the Gaussian distribution became more and more irregular with age increasing.3.The TRBV gene family usage of na?ve CD4~+T cells TCRβCDR3 repertoires in spleen,T cells in spleen and thymus were different at different ages,and the TRBJ gene family were similarly used. |
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