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Learn MorePurpose: 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. SOURCE: Michael Sikora (mjsikora@stanford.edu) - Steinmetz Stanford University
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