Dataset Information

Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion.

ABSTRACT: To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate 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.

SUBMITTER: Huang H

PROVIDER: S-EPMC6500157 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Select sequencing of clonally expanded CD8<sup>+</sup> T cells reveals limits to clonal expansion.

Huang Huang H Sikora Michael J MJ Islam Saiful S Chowdhury Roshni Roy RR Chien Yueh-Hsiu YH Scriba Thomas J TJ Davis Mark M MM Steinmetz Lars M LM

Proceedings of the National Academy of Sciences of the United States of America 20190416 18

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of periphe ...[more]

PMID: 30992377

Similar Datasets

Project description:Background & aimsHBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.MethodsDNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.ResultsHBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.ConclusionsTargeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.Lay summaryFresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Dataset Information

Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion.

Publications

Select sequencing of clonally expanded CD8<sup>+</sup> T cells reveals limits to clonal expansion.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets