TCR repertoire sequencing analysis using RNA on fibrotic livers
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE208635 | GEO | 2022/09/15
REPOSITORIES: GEO
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Project description:We developed a modular, high-throughput discovery platform to simultaneously test whether Mut PIK3Ca is immunogenic and to retrieve paired a/b TCR gene sequences that confer specificity to this NeoAg. This method, termed Stimulation Induced Functional TCR sequencing (SIFT-seq), combines single-cell (sc) TCR V(D)J and transcriptome sequencing. Here, microwell cultures of in vitro stimulated T cells with confirmed neoantigen-specific recognition are selected for SIFT-seq. Matched aliquots of selected wells are acutely stimulated with autologous antigen-presenting cells presenting WT or Mut PI3Ka and the transcriptomic profile of individual clonotypes is assessed to identify neoantigen-specific T cells and retrieve their TCR gene sequences.
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Project description:Diversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and β chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3’-biased single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3’ scRNA-seq is not available. Here we present an analysis pipeline to process TCR variable regions enriched from 3’ scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells (PBMCs) from a healthy donor and detect TCR sequences in a high proportion of single T-cells. Detection of TCR sequences is negligible in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T-cells than other T-cell types, indicating an association between T-cell clonotypes and differentiation states.
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Project description:The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. Here, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCR-alpha-beta clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. Together, these findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.
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Project description:To investigated global changes caused by pressure overload-induced heart failure in ADAM12 knockout mice using a microarray analysis. A disintegrin and metalloproteinase (ADAM) 12 is has long been considered to promote cardiac dysfunction based on according to the finding report that a small molecule ADAM12 inhibitor, KB-R7785 ameliorated cardiac function in a transverse aortic constriction (TAC) model by inhibiting the through inhibition of proteolytic activation of HB-EGF signaling. HoweverOn the other hand, this compound has poor selectivity for ADAM12, and the role of ADAM12 inon cardiac dysfunction has not yet been investigated usingby genetic loss-of-function mice. Anoperation. ADAM12 deficiency resulted in significantly more expanded cardiac fibrosis accompanied byaccompanying with increased collagen-related gene expression in their failing hearts. The results of a genome-wide transcriptional analysis suggested a strongly enhanced highly elevated focal adhesion- and fibrosis-related signaling pathway in ADAM12 knockout hearts. The present results study revealed that the loss of ADAM12 enhanced focal adhesion and canonical TGF-β signaling by regulating the abundance of the integrin β1 and TGF-β receptors.
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