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目的探讨乳腺癌患者外周血单个核细胞中CD4~+CD25~+CD127~(dim/-)Tregs细胞TCRB链CDR3的基因,氨基酸序列及初步结构。方法采用免疫磁珠细胞分选技术,分选出10例乳腺癌患者及5例正常患者外周血中的CD4~+CD25~+CD127~(dim/-)Tregs细胞,流式细胞术检测分选纯度;采用毛细吸管电泳法分析TCRB链CDR3优势利用情况,对单克隆增生的家族进行基因测序。结果 5例正常CD4~+CD25~+CD127~(dim/-)Tregs细胞TCR BV CDR3长度和多态性表达频率不一致,大部分呈高斯分布;10例乳腺癌患者CD4~+CD25~+CD127~(dim/-)Tregs细胞TCR BV CDR3家族长度和多态性表达频率不一致,乳腺癌患者外周血CD4~+CD25~+CD127~(dim/-)Tregs细胞TCR BV CDR3区谱型异常率明显大于正常人(χ~2=32.345,P<0.01),TCRBV22、TCRBV15、TCRBV16 3个家族被多例患者限制性取用,出现优势利用,单克隆增生的家族进行基因测序发现,病例1 TCR BV11和病例2 TCR BV4存在共同基序“ETQYF”;病例2 TCR BV13.1和病例7 TCR BV15有共同基序“EQFF”;病例2 TCR BV13.2和病例5 TCR BV22存在共同基序“TGELFF”。结论乳腺癌患者外周血CD4~+CD25~+CD127~(dim/-)Tregs细胞存在不同程度的克隆增生,部分TRBV家族被多例乳腺癌患者限制性取用并在免疫应答中发挥重要作用。多数TCR BV家族CDR3谱系存在单峰谱型,且部分单峰谱型的T细胞具有不同的CDR3序列,提示多个抗原表位参与了T细胞免疫应答。多数病例单峰谱型不同家族间存在共同基序,推测调节性T细胞可能识别抗原肽的相同部分。
Objective To investigate the gene, amino acid sequence and primary structure of TCRB chain CDR3 in CD4 ~ + CD25 ~ + CD127 ~ (dim / ~) Tregs from peripheral blood mononuclear cells from patients with breast cancer. Methods The CD4 ~ + CD25 ~ + CD127 ~ (dim / -) Tregs in peripheral blood from 10 patients with breast cancer and 5 normal controls were sorted by immunomagnetic bead sorting. Flow cytometry Purity; Capillary electrophoresis analysis of TCRB chain CDR3 advantages of utilization, the family of monoclonal proliferation gene sequencing. Results The frequency and frequency of TCR BV CDR3 in 5 normal CD4 ~ + CD25 ~ + CD127 ~ (dim / -) Tregs cells were not consistent, most of which were Gaussian distribution. The frequencies of CD4 ~ + CD25 ~ + CD127 ~ The frequency of TCR BV CDR3 family members in peripheral blood of patients with dim (- / -) Tregs was not consistent with the frequency of TCR BV CDR3 family members. The frequency of TCR BV CDR3 pattern abnormalities in CD4 ~ + CD25 ~ + CD127 ~ (dim / The three families of TCRBV22, TCRBV15 and TCRBV16 were restrictedly used in a number of patients, and the families with superior utilization and monoclonal hyperplasia were found to be sequenced. The cases 1 TCR BV11 and Case 2 TCR BV4 has the common motif “ETQYF”; Case 2 TCR BV13.1 and Case 7 TCR BV15 have a common motif “EQFF”; Case 2 TCR BV13.2 and Case 5 TCR BV22 have a common motif “TGELFF ”. Conclusion There are varying degrees of clonal proliferation in CD4 ~ + CD25 ~ + CD127 ~ (dim / -) Tregs cells in peripheral blood of patients with breast cancer. Some TRBV families are restricted access to many breast cancer patients and play an important role in immune response. Most TCR BV family CDR3 lineages exist unimodal patterns, and some unimodal T-lines have different CDR3 sequences, suggesting that multiple epitopes are involved in the T-cell immune response. In most cases, there is a common motif between different families in the unimodal spectrum, suggesting that regulatory T cells may recognize the same part of the antigenic peptide.