Paired TCR and transcriptome analysis of single T cells characterizes clonally expanded human conventional and unconventional CD8+ T cells

Purpose: Single cell RNAseq has revolutionized our understanding of the heterogeneity of immune system. The goal of this study is to characterize functions of Mycobacterium tuberculosis (Mtb) specific CD8+ T cell clones using single cell RNAseq Methods: PBMCs from Mtb exposed patients were stimulated ex vivo with Mtb lysate for 12 hours. Activated CD8+ T cells were selected and single cell sorted based on the upregulation of activation markers, including CD154 and CD137. Results: Using an optimized Smart-seq2 protocol, we could successfully combine TCR sequencing with transcriptome sequencing on single T cell. TCR sequence analysis allows us to preferentially identify and select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. In summary, we analyzed 921 single cells with 2,500-3000 detected genes for each cell. The iNKT and MAIT cells have a nearly superimposable transcriptional pattern, indicating that they are variations of the same cell type, and differ considerably from conventional CD8+ T cells. While there are no distinct gene expression differences between clonally expanded versus unique iNKT or MAIT cells, highly expanded conventional CD8+ T cells downregulate the interleukin-2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire. Conclusions: we combine targeted TCR sequencing with a sensitive RNA-seq protocol that yields both paired TCR sequences and extensive transcriptional information on single T cells. This enables the selection of rare, clonal and specific T cell subtypes, enabling analysis of their transcriptomes. Our targeted approach allowed us to efficiently and economically select clonal cells with a sensitive clone size dynamic range. And by using a short ex vivo stimulation (12~24 hours) to identify pathogen-responsive T cells, we could circumvent long-term ex vivo culture artifacts. By leveraging single cell analysis of clonal T cells in additional contexts we may improve our understanding of immune system responses to diseases such as cancer, additional infections, as well as aid in vaccine development. Parellel single cell TCR and RNA-seq to study the transcriptome of clonally expanded conventional and unconventional cells responding to Mycobacterium tuberculosis