气候变化领域集成服务门户(CCPortal): Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning

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DOI	10.1073/pnas.2021190118
	Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning
	Nesterenko P.A.; McLaughlin J.; Cheng D.; Bangayan N.J.; Sojo G.B.; Seet C.S.; Qin Y.; Mao Z.; Obusan M.B.; Phillips J.W.; Witte O.N.
发表日期	2021
ISSN	00278424
卷号	118 期号:3
英文摘要	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 cross-linker 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 (CMV)- and Epstein-Barr virus (EBV)-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 cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-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. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	TCR \| T cell receptor \| T cells \| single-cell sequencing \| mRNA sequencing
语种	英语
scopus关键词	amine; gamma interferon; major histocompatibility antigen; messenger RNA; T lymphocyte receptor; transcription factor FOXP3; tumor necrosis factor; antigen detection; antigen purification; antigen specificity; Article; cell level; cell permeabilization; controlled study; cross linking; Cytomegalovirus; Epstein Barr virus; human; human cell; molecular cloning; nonhuman; phenotype; priority journal; process optimization; proof of concept; regulatory T lymphocyte; RNA analysis; single cell RNA seq; virus reactivation
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180956
作者单位	Molecular Biology Institute, University of California, Los Angeles, CA 90095, United States; Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, United States; Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, United States; Division of Hematology–Oncology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, United States; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095, United States; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095, United States; Immune Education Department, Agenus, Lexington, MA 02421, United States
推荐引用方式 GB/T 7714	Nesterenko P.A.,McLaughlin J.,Cheng D.,et al. Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning[J],2021,118(3).
APA	Nesterenko P.A..,McLaughlin J..,Cheng D..,Bangayan N.J..,Sojo G.B..,...&Witte O.N..(2021).Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning.Proceedings of the National Academy of Sciences of the United States of America,118(3).
MLA	Nesterenko P.A.,et al."Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning".Proceedings of the National Academy of Sciences of the United States of America 118.3(2021).