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DNA sequencing for multiple rounds of the pMHC-yeast display selection for 2W, Yae and Fat TCR

ABSTRACT: A peptide library was displayed on Ab MHC class II allele expressed on yeast. TCRs of interest were used to select and enrich for cognate peptide-MHC.

ORGANISM(S): synthetic construct

PROVIDER: GSE151070 | GEO | 2020/06/15

REPOSITORIES: GEO

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DNA sequencing for multiple rounds of the pMHC-yeast display selection for TCR6, TCR7 and FluTCR [other]

Project description:A peptide library was displayed on Db MHC class I allele expressed on yeast. TCRs of interest were used to select and enrich for cognate peptide-MHC.

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Project description:We tested orphan TCR autoreactivity using the peptide MHC-TCR chimeric receptor (MCR) co-culture system. In this system, cognate antigen recognition leads to TCR specific NFAT activation in MCR reporter cells expressing a mouse I-Ab MHC class II extracellular domain covalently linked to candidate peptides and an intracellular TCR signaling domain. We used mixed autoimmune bone marrow chimera spleens and kidneys as sources of cDNA to generate a transcriptome-wide library of natural autoantigen peptides . We cloned this cDNA-derived peptide (CDP) autoantigen library into the MCR retroviral backbone and transduced NFAT reporter cells to make a murine autoantigen MCR reporter library (MCR-Lib). We then used this library to screen orphan TCRs identified by scTCR-seq for autoreactivity.

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TCR repertoire profiling of CMV specific and SARS-CoV2 Spike specific CD8+ T cells using single cell sequencing

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2022-01-01 | E-MTAB-11229 | biostudies-arrayexpress

DNA sequencing for multiple rounds of the pMHC-yeast display selection for 2W, Yae and Fat TCR

Project description:DNA sequencing for multiple rounds of the pMHC-yeast display selection for 2W, Yae and Fat TCR

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Altered thymic selection in Themis-deficient mice leads to expansion of an unusual virtual memory and bystander-competent CD8+ subpopulation bearing Va3.2 TCR

Project description:This study aimed to compare the TCRab repertoires from CD8 T cell subsets in a different T cell progression stage in Themis-sufficient and Themis-deficient mice. Analysis of the NGS-derived data revealed an overall reduction of the TCRa repertoire diversity in Themis's absence. In contrast, the CD8 compartment bearing the Va3.2 TCRs in the Themis-deficient model retains higher diversity than the repertoire from the Themis-sufficient counterpart. Furthermore, analysis of CDR3 peptide sequences of Va3.2+ and Va3.2- TCRs disclose the greater contribution of the hydrophobic residues in the former repertoires, indicating enhanced self-reactivity within this CD8+ T cell fraction.

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TCR-pMHC bond conformation controls TCR ligand discrimination.

Project description: Not available

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Droplet based mRNA sequencing of fixed and permeabilized cells by CLInt-Seq allows for antigen specific TCR cloning

Project description:T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via Crosslinker regulated Intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low frequency TCRs specific for any antigen. As a proof-of-principle, intracellular staining for TNFα and IFNγ identified Cytomegalovirus and Epstein-Barr virus reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically-cleavable primary amine crosslinker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ staining followed by single-cell sequencing performed similarly to isolation with multimer staining for Epstein-Barr virus reactive TCRs. We anticipate CLInt-Seq will enable droplet based single cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.

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Transcription profiling time series of thymocytes from HNT-TCR transgenic mice injected with HA peptide to induce thymic negative selection

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TCR/pMHC Optimized Protein crystallization Screen.

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Mtb HLA-E tetramer sorted CD8+ T-cells have a diverse TCR repertoire

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