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.

single cell transcriptomics, single T cell receptor repertoire analysis, cDNA, combined transcriptome, and T cell receptor repertoire assay, low cell number,
Frontiers in Immunology
Department of Internal Medicine

Litjens, N.H.R, Langerak, A.W, van der List, A.C.J., Klepper, M, de Bie, M, Azmani, Z., … van IJcken, W.F.J. (2020). Validation of a Combined Transcriptome and T Cell Receptor Alpha/Beta (TRA/TRB) Repertoire Assay at the Single Cell Level for Paucicellular Samples. Frontiers in Immunology, 11. doi:10.3389/fimmu.2020.01999