Litjens Nicolle H R, Langerak Anton W, van der List Amy C J, Klepper Mariska, de Bie Maaike, Azmani Zakia, den Dekker Alexander T, Brouwer Rutger W W, Betjes Michiel G H, Van IJcken Wilfred F J
Department of Internal Medicine Section Nephrology and Transplantation, Erasmus MC, University Medical Center, Rotterdam, Netherlands.
Laboratory Medical Immunology, Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, Netherlands.
Front Immunol. 2020 Aug 28;11:1999. doi: 10.3389/fimmu.2020.01999. eCollection 2020.
Transcriptomics can be combined with TRA and TRB clonotype analysis at the single cell level. The aim of this study was to validate this approach on the ICELL8 Single-Cell system and to evaluate its usefulness to analyse clinical paucicellular samples. For this purpose, we carefully selected T cell lines with defined TRA/TRB clonotypes as well as clinical samples enriched for CD3 T cells that possess a complex TCR repertoire. Low cell numbers of the different samples were dispensed in a chip on the ICELL8 Single-Cell System. Two sequencing libraries were generated from each single cell cDNA preparation, one for the TRA/TRB repertoire and one for the 5' ends of transcripts, and subsequently sequenced. Transcriptome analysis revealed that the cell lines on average express 2,268 unique genes/cell and T cells of clinical samples 770 unique genes/cell. The expected combined TRA/TRB clonotype was determined for on average 71% of the cells of the cell lines. In the clinical samples the TRA/TRB repertoire was more complex than those of the cell lines. Furthermore, the TRB clonotype distribution of the clinical samples was positively correlated to frequencies of TCRVβ families in CD3 T cells obtained by a flow cytometry-based approach (Spearman's Rho correlation coefficient 0.81, = 6.49 10). Combined analyses showed that transcriptome-based cell type-specific clusters in clinical samples corresponded to clinical features such as CMV status. In conclusion, we showed that the ICELL8 Single-Cell System enabled combined interrogation of both TRA/TRB repertoire and transcriptome of paucicellular clinical samples. This opens the way to study the response of single T cells within heterogeneous samples for both their transcriptome and TRA/TRB clonotypes in disease or upon treatment.
转录组学可在单细胞水平上与TRA和TRB克隆型分析相结合。本研究的目的是在ICELL8单细胞系统上验证这种方法，并评估其对分析临床少细胞样本的有用性。为此，我们精心挑选了具有明确TRA/TRB克隆型的T细胞系以及富含具有复杂TCR库的CD3 T细胞的临床样本。将不同样本的少量细胞分配到ICELL8单细胞系统的芯片上。从每个单细胞cDNA制备物中生成两个测序文库，一个用于TRA/TRB库，一个用于转录本的5'端，随后进行测序。转录组分析显示，细胞系平均每个细胞表达2268个独特基因，临床样本中的T细胞平均每个细胞表达770个独特基因。对于细胞系中平均71%的细胞，确定了预期的联合TRA/TRB克隆型。在临床样本中，TRA/TRB库比细胞系的更复杂。此外，临床样本的TRB克隆型分布与通过基于流式细胞术的方法获得的CD3 T细胞中TCRVβ家族的频率呈正相关（斯皮尔曼等级相关系数0.81，P = 6.49×10⁻¹⁰）。联合分析表明，临床样本中基于转录组的细胞类型特异性簇与CMV状态等临床特征相对应。总之，我们表明ICELL8单细胞系统能够对少细胞临床样本的TRA/TRB库和转录组进行联合检测。这为研究异质样本中单个T细胞在疾病或治疗时的转录组和TRA/TRB克隆型反应开辟了道路。
