| Objective:To analysis the composition and characterization of TCR ? chain CDR3 repertoires in Regulatory T ce LLs(Treg)in intestine and spleen of Germ-free(GF)CLass,Specific Pathogen-free(SPF),and Clean(CL)Class BALB/c Mice.To investigate the effects of differential composition of intestinal commensal microorganisms on the tolerability and response of CD4+CD25+Foxp3+Treg cells in BALB/c mice,which is the mechanism of Treg cells in GF,SPF and CL BALB/c mice.And applied research provides new ideas,monitoring techniques,and basic data for reference.Methods:1.A total of 9 BALB/c mice were purchased from Third Mi Litary Medica L University Fundamentals.9 BALB/c mice samples were named:GF1-SP(GF CLass 1 Spleen Tissue),GF1-In(GF CLass 1 Intestinal Tissue)GF2-SP(GF CLass 2 Spleen Tissue),GF2-In(GF CLass 2 Intestinal Tissue)GF3-SP(GF CLass 3 Spleen Tissue),GF3-In(GF CLass 3 Intestinal Tissue)SPF1-SP(SPF CLass 1 Spleen Tissue),SPF1-In(SPF CLass 1 Intestinal Tissue)SPF2-SP(SPF CLass 2 Spleen Tissue),SPF2-In(SPF CLass 2 Intestinal Tissu)SPF3-SP(SPF CLass 3 Spleen Tissue),SPF3-In(SPF CLass 3 Intestinal Tissu)CL1-SP(CL CLass 1 Spleen Tissue),CL1-In(CL CLass 1 Intestinal Tissue)CL2-SP(CL CLass 2 Spleen Tissue),CL2-In(CL CLass 2 Intestinal Tissue)CL3-SP(CL CLass 3 Spleen Tissue),CL3-In(CL CLass 3 Intestinal Tissue)2.Using MACS CD4+CD25+Regulatory T Cell Isolation Kit(mouse)to sorting the CD4+CD25+ Treg cells spleen and intestine of 9 BALB/c mice.3.Using Flow cytometry to identify more than 80% of the CD4+CD25+Foxp3+Treg ce LLs in each sample and to extract all samples genomic DNA.4.To collected fecal samples from 9 BALB/c mice and sent to Beijing Genomics Institute for genomic sequencing of fecal flora.5.To identified the each of DNA samples comply with a standard of sequencing requirements by the Adaptive Biotechnologies immuno SEQTM.The TCR ?-chain CDR3 repertoire of CD4+CD25+Foxp3+Treg cells in each sample was constructed and Illumina Solexa high-throughput sequencing was completed.6.After HTS,the repertoire of TCR ? CDR3 was analyzed by Immuno SEQ software,and the original document data(https://clients.adaptivebiotech.com)was download.7.To analysis the sequencing results from Beijing Genomics Institute.Results:1.The proportion of Total CDR3 sequences to the Unique CDR3 sequences in each samp Le were :GF1-In:100/129 GF1-SP:18452/25891 GF2-In:47/59GF2-SP:2616/3880 GF3-In:100/123 GF3-SP:19131/26071SPF1-In:59/77 SPF1-SP:19798/27897 SPF2-In:29/36SPF2-SP:41709/60573 SPF3-In:576/1116 SPF3-SP:(Building repertoire was unsuccessful)CL1-In:133/172 CL1-SP:12021/16528 CL2-In:63/80CL2-SP:19390/26116 CL3-In:17/20 CL3-SP:19368/25864.2.Distinct usage of the tota L TRBV-TRBJ genes(1)The high-frequency paired genomes TRBV-TRBJ usage of the intestine were:GF:TRBV19-01-TRBJ02-07,TRBV20-01-TRBJ02-07,TRBV13-02-TRBJ02-01;SPF:TRBV19-01-TRBJ02-07,TRBV13-01-TRBJ02-01;CL:TRBV19-01-TRBJ02-07.(2)The high-frequency paired genomes TRBV-TRBJ usage of the spleen were:GF: TRBV13-02-TRBJ02-07,TRBV05-01-TRBJ02-07,TRBV13-01-TRBJ02-07,TRBV19-01-TRBJ02-07,TRBV31-01-TRBJ02-07;SPF:TRBV05-01-TRBJ02-07,TRBV01-01-TRBJ02-07,TRBV02-01-TRBJ02-07,TRBV04-01-TRBJ02-07;CL:TRBV05-01-TRBJ02-07,TRBV19-01-TRBJ02-07,TRBV01-01-TRBJ02-07,TRBV02-01-TRBJ02-07.3.Distinct usage of the total TRBV?TRBJ genes(1)The advantage of access TRBJ genes usage of the intestine were:GF: TRBJ02-07?TRBJ02-01;SPF: TRBJ02-07?TRBJ01-04;CL: TRBJ02-07?TRBJ01-04.(2)The advantage of access TRBJ genes usage of the spleen were:GF: TRBJ02-07?TRBJ01-04;SPF: TRBJ02-07?TRBJ01-04;CL: TRBJ02-07?TRBJ01-04.(3)The advantage of access TRBV genes usage of the intestine were:GF: TRBV19-01?TRBV13-02;SPF: TRBV19-01?TRBV05-01;CL: TRBV19-01?TRBV05-01.(4)The advantage of access TRBV genes usage of the spleen were:GF: TRBV19-01?TRBV05-01;SPF: TRBV19-01?TRBV05-01;CL: TRBV19-01?TRBV05-01.4.Amino acid length and access The 17 samples are mainly serine and AA length distributions CDR3 sequences were normal distribution 12 AA5.Clonotype distribution and diversity of Treg TCR beta chain CDR3 repertoire:(1)The 1/DS values of 17 samples respectively:GF1-In:229 GF1-SP:19670 GF2-In:131GF2-SP:2359 GF3-In:277 GF3-SP:20622SPF1-In:127 SPF1-SP:9120 SPF2-In:52SPF2-SP:15141 SPF3-In:130CL1-In:229.7 CL1-SP:19670 CL2-In:64CL2-SP:18365 CL3-In:47 CL3-SP:13346(2)Expansive degree of total Treg TCR beta CDR3 clones from the 9 mices in spleen and intestine not the same.6.The stool flora sequencing in 9 BALB/c mice(1)Total sequence:GF1(MG-4):43874 GF2(MG-5):0 GF3(MG-6):0SPF1:45503 SPF2:44850 SPF3:45237CL1:44619 CL2:44400 CL3:44979(2)OUT abundance:GF1(MG-4):125SPF1:287 SPF2:294 SPF3:291CL1:327 CL2:305 CL3:2797.Spleen,intestine,mesenteric Lymph node tissue sections(1)Peripheral edge of the spleen white pulp narrowed in GF BALB/c mice.(2)Intestine villi become longer,central germinal center of mesenteric lymph node shrinks in GF BALB/c mice.Conclusion:1.Four-month-o Ld GF,SPF,and CL-grade BALB/c mice have different gut microflora composition and may be related to the development and composition of their different peripheral immune organs.The abundance and quantity of microbes in the feces are positively correlated with the changes in the microorganisms in the feeding environment.2.In the intestinal Treg TCR? CDR3 repertoire of GF,SPF and CL grade BALB/c mice at4 months of age,there was a significant difference in the availability of high frequency unique CDR3 AA sequences.And there is a greater heterogeneity in the access and pairing of TRBV and TRBJ gene families.The Treg TCR? CDR3 repertoire in the spleens of GF,SPF,and CL-grade BALB/c mice only showed differences in the access and pairing of some TRBV and TRBJ gene families.The composition of intestinal microbiota in GF,SPF,and CL-grade BALB/c mice may primarily affect the composition of the local Treg CDR3 repertoire and does not alter the overall properties of the circulating immune system Treg CDR3 repertoire. |
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