两种方法分离免疫细胞用于高通量测序TCR组库分析的比较

Comparative study of the different methods used to isolate immune cells for high-throughput sequencing based TCR repertoire analysis

目的探讨两种方法分离免疫细胞进行高通量T细胞表面的T细胞变体(TCR),组库分析的区别及可行性。方法分别采用免疫磁珠分选纯化CD3阳性T细胞和Ficoll-Paque梯度离心法分离外周血淋巴细胞的方法制备TCR测序所需样品各1例,提取DNA后多重聚合酶链式反应(PCR)扩增CDR3区域,将扩增得到的PCR产物纯化后构建免疫组库,进行高通量测序及生物信息学分析。结果两种方法获得的免疫细胞提取DNA后均能够满足TCR免疫组库建库需要,高通量测序结果显示互补决定簇3(CDR3)区域长度呈高斯分布,来自健康志愿者的TCR具有丰富多样性,肿瘤患者TCR多样性呈寡克隆分布。结论纯化的T细胞或者直接分离得到的淋巴细胞均可用于免疫组库构建及后续TCR CDR3受体库高通量测序分析,TCR CDR3多样性可能直接反应机体免疫状态。

Objective To observe the differences between and feasibility of two methods of isolating immune cells for highthroughput sequencing based TCR repertoire analysis. Methods CD3 positive T cells,isolated by immuno-magnetic beads from cryopreserved PBMC,and lymphocyte,obtained from healthy donor peripheral blood using Ficoll-Paque gradient centrifugation,were each used to prepare a sample for TCR repertoire analysis. Total DNA was extracted from purified T cells and lymphocytes. Multiplex PCR was performed using Multiplex PCR Kit to amplify CDR3 region with specific primers using DNA as template. DNA library was prepared with purified PCR product. Paired-end sequencing of samples was carried out with a read length of 100 bp using the Illumina Hiseq2500 platform. Results Both of the samples were qualified for high-throughput sequencing based TCR repertoire analysis. CDR3 length distribution from two samples followed a Gaussian distribution. Difference on V-J paring distribution from two samples was significant. TCR repertoire from healthy donor indicated abundant diversity while TCR repertoire from patient with cancer followed an Oligoclonal distribution. Conclusion Both purified T cells and lymphocyte can be used for high-throughput sequencing based TCR repertoire analysis. TCR diversity may reflect the individual immune status.