AUTHOR=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. TITLE=Validation of a Combined Transcriptome and T Cell Receptor Alpha/Beta (TRA/TRB) Repertoire Assay at the Single Cell Level for Paucicellular Samples JOURNAL=Frontiers in Immunology VOLUME=11 YEAR=2020 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.01999 DOI=10.3389/fimmu.2020.01999 ISSN=1664-3224 ABSTRACT=

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, P = 6.49 * 10−7). 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.