| Objective:Using Illumina Solexa high-throughput sequencing to analysis the homogeneity and heterogeneity of CD4+CD25+ regulatory T cell(Treg)receptor beta chain complementarity determining region 3(TCR ? CDR3)repertoire in primary breast tumor tissues and spleens from spontaneous mammary tumor of C3H/HeJNju mice.To further explore the differences and associations between tumor tissues and systemic immune circulation of CD4+CD25+Treg,to provide a basis for further study of the mechanism and application of Treg in breast cancer.Methods:1.To screen three mice with spontaneous mammary tumor model of the age of 10 months,were labeled M1,M2 and M3 respectively.2.The primary breast tumor tissues(T)and spleens(S)were collected from M1.M2.M3 mice,respectively.The magnetic bead separation technology was used to select the CD4+CD25+Treg cells in the collected tissues,and the cells purity was identified by FCM.3.The genomic DNA of CD4+CD25+ Treg obtained from each sorting was extracted,and the serial number of each sample was named:MIT:M1 mouse breast tumor tissue sample;MIS:M1 mouse spleen sample;M2T:M2 mouse breast tumor tissue sample;M2S:M2 mouse spleen sample;M3T:M3 mouse breast tumor tissue sample;M3S:M3 mouse spleen sample.4.The six genomic DNA samples,sent to the Adaptive Biotechnologies Immuno SEQ corporation(The University of Washington School of Medicine)to detect,and the results showed that the quality of all samples was fulfill the requirements of construct CDR3 library,each sample CDR3 library was established according to TCR ? CDR3 receptoire the principle of mouse building library,then Illumina high-throughput technology was used to sequence the TCR CDR3 repertoire of each sample.5.We downloaded the original sequences of each sample from the original database of the Immuno SEQ company.The original sequence names of the six samples are:DFFM1T-Treg?DFFM1S-Treg?DFFM2T-Treg?DFFM2S-Treg?DFFM3T-Treg?DFFM3S-Treg,respectively.Immuno SEQ and IMGT/High V-QUEST Systems(http://www.imgt.org)were used to analyze the composition and characteristics of CD4+CD25+Treg TCR ? CDR3 repertoire in breast tumor tissues and spleens of three model mice:(1)refer to the inverse Simpson's diversity index(1/DS)to analyze CDR3 diversity.(2)The distribution of AA within the CDR3 repertoire of high frequency(greater than 0.8%),intermediate frequency(0.4-0.8%),low frequency(less than 0.4%),the clone value-added ratio,composition and characteristics of the top twenty highest-frequency sequences in CDR3 repertoire.(3)The usage and gene paring of TRBV-TRBJ.(4)The length and usage of CDR3 AA sequences of CDR3 repertoire.(5)The number and rate of overlap among CDR3 sequences was analyzed using Venn diagram software.Results:1.The tumor growth was observed in C3H/HeJNju mice,which was screened by the spontaneous mammary tumor model.The original tumor tissues were confirmed by pathological HE staining(Nanjing Biomedical Reseach institute of Nanjing University).Three model mice were different types of breast cancer,and M1 mouse were considered as invasive breast cancer with medullary characteristics.M2 mouse were considered as breast fibroadenocarcinoma.M3 mouse were considered as breast papillary carcinoma.2.The CD4 + CD25 + Treg cells sorted by magnetic bead sorting technology of primary breast tumor tissues and spleens of three model mice were identified by FCM and the purity reached more than 85%,the total DNA of each sample was extracted,and a clear band appeared at the predicted position on the 0.7%agarose gel.3.The ratio of the total number of Productive CDR3/Unique CDR3 sequences of CD4+CD25 +Treg TCR ? CDR3 sequences in the six samples:MIT:488/239,MIS:29568/9232,M2T:5173/2597,M2S:33963/14429,M3T:1545/347,M3S:37227/14338.4.The cloning prolife distribution of CD4 + CD25 + Treg TCR ? CDR3 repertoire in the six samples.(1)The 1/DS values of six samples respectively:MIT:120.27,MIS:841.58;M2T:144.63 M2S:1269.92;M3T:69.08,M3S:553.35.(2)In the three model mice,the percentage and the distribution of high-frequency(greater than 0.8%)and intermediate-frequency(0.4-0.8%):breast tumor tissues were higher than spleens,there are many clone sequences in the middle and high frequency of tumor tissues.On the contrary,the percentage and the distribution of low frequency:spleens were higher than breast tumor tissues,and was dominated by low-frequency proliferative clones.(3)There were three or more identical CDR3 sequences between the same mouse breast tumor tissue and spleen in the top 20 sequences of high-frequency cloning amplification.Among them,there are three identical CDR3 sequences of M1 mouse tumor tissue and spleen,which are CASSQDRVGNTLYF,CASVDWGTLYF and CASGPQGSAETLYF,respectively.There are six identical CDR3 sequences of M2 mouse tumor tissue and spleen,which are CASSHGLGGYAEQFF.CASSIPGYSYEQYF?CASSPGTDNQAPLF?CASNPQGWGSDYTF?CASTWGEDEQFF and CASSRDGKSSYEQYF,respectively.There are three identical CDR3 sequences of M3 mouse tumor tissue and spleen,which are CASSQDWIEQYF?CASSDAGGAGDTQYF and CASSGWTGEQAPLF,respectively.5.The usage and gene paring of TRBV/TRBJ of CD4+ CD25+ Treg TCR ? CDR3 repertoire in the six samples:(1)The shared TRBV gene family of M1,M2,M3 mice primary breast tumors and spleens predominance(the top five CDR3 sequences)include TRBV19-1.(2)The shared TRBJgene family of M1,M2,M3 mice primary breast tumors and spleens predominance(the top five CDR3 sequences)include TRBJl-3,TRBJ 2-1,TRBJ 2-7.(3)The common TRBV-TRBJ gene family dominant pairing(great than 2%)in M1,M2,M3 mice are involved in efficient gene rearrangements in primary breast tumor tissues and spleens,which are TRBV05-O1-TRBJ02-07,TRBV19-01-TRBJ02-07.Breast tumor tissues of different mice showed a dominant pairing of more than 3%of TRBV-TRBJgenes,but low-frequency expression in the spleen or not found.The TRBV-TRBJ gene family dominant pairing of breast tumor tissues include the following:M1 mouse tumor tissue predominantly paired with TRBV13-03-TRBJO1-03(6.15%),TRBV13-02-TRBJ02-04(3.69%),TRBV13-03-TRBJ02-05(3.69%).M2 mouse tumor tissue predominantly paired with TRBV02-01-TRBJ02-01(8.77%),TRBV19-01-TRBJ01-03(3.39%).M3 mouse tumor tissue predominantly paired with TRBV13-02-TRBJ02-02(4.47%),TRBV01-01-TRBJ01-03(4.4%),TRBV17-01-TCRBJ01-05(3.56%).(4)The paired TRBV-TRBJ gene family between the tumor tissues and the spleens was statistically analyzed and there were statistically significant pairs of paired gene family including TRB V03-01-TRBJ01-02,TRBV03-01-TRBJ01-06,TRBV13-01-TRBJOI-01,TRBV16-01-TRBJ01-03,TRBV16-01-TRBJ02-04,TRBV19-01-TRBJ02-05,TRBV21-01-TRBJ02-01,TRBV21-01-TRBJ02-05,TRBV21-01-TRBJ02-07,TRBV22-01-TRBJ01-06,TRBV22-01-TRBJ02-03,TRBV22-01-TRBJ02-04,TRBV22-01-TRBJ02-07,TRBV23-01-TRBJ02-07,TRBV24-01-TRBJ02-01,TRBV24-01-TRBJ02-03,TRBV29-01-TRBJ01-06,TRBV31-01-TRBJ01-03.6.The usage and length distribution of amino acid of CD4 + CD25 + Treg TCR ?CDR3 repertoire in the six samples:(1)The high frequency of aminoacid of CD4 + CD25 + Treg TCR ? CDR3 repertoire in M1,M2,M3 mice primary breast tumors and spleens was hydrophilic amino acids represented by serine(S).(2)The length of CD4 + CD25 + Treg TCR ? CDR3 repertoire in M1,M2,M3 mice breast tumors and spleens were mainly 12 AA dominated Gauss distribution.7.The clonal overlap rate of CD4 + CD25 + Treg TCR ? CDR3 repertoire in the six samples:MIT&M1S=37.24%,MIS&M1T=0.96%;M2T&M2S=35.7%,M2S&M2T=6.43%;M3T&M3S=68.01%,M3S&M3T=1.65%;8.The unique amino acid overlapping sequences of CD4 + CD25 + Treg TCR ? CDR3 repertoire in the six samples(1)The unique amino acid overlaping sequences of CD4 + CD25 + Treg TCR ? CDR3 in M1,M2,M3 mice primary breast tumor and spleen samples were 89,927 and 236,respectively.There is very few overlaping sequences between the three mices breast tumor tissues,and there are many identical amino acid overlapping sequences among the spleen tissues,as shown below:There was an amino acid overlap sequence between M1 and M2 mouse breast tumor tissues.There were four amino acid overlapping sequences between M2 and M3 mouse breast tumor tissues.There was no amino acid overlap sequence between M1 and M3 mouse breast tumor tissues.There were 535 amino acid overlapping sequences between M1 and M2 mouse spleen.There were 531 amino acid overlapping sequences between M1 and M3 mouse spleen.There were 726 amino acid overlapping sequences between M2 and M3 mouse spleen.(2)Three mice breast tumor tissues with unique amino acid sequence of overlapping between the spleens,high-frequency expression in breast tumor tissues of top 20 CDR3 sequences,most of the low frequency expression in the spleens.Conclusion:1.There may be more high-frequency clonal proliferation of Treg cells in the tumor tissues of spontaneous mammary tumor mice,it may be in the tumor inhibition effect anti-tumor immune response of T cells,thereby promoting tumor growth and metastasis.2.There are differences in the TRBV-TRBJ gene family dominant pairing between tumor tissues and spleens,as well as the number and characteristics of overlapping CDR3 sequences in spontaneous mammary tumor mice,suggesting that there are different treg in the local and circulating immune system of tumor.To further explore the origin,characteristics and functional of Treg in tumor microenvironment. |
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