![]() ![]() This background expression could make it difficult to specifically identify low-frequency responses. ![]() These surface activation markers are expressed on up to 0.5% of CD8 + T cells in absence of TCR stimulation. Cell surface markers like CD137 are used as proxies for the intracellular effector molecules that respond to T cell activation and can return a false positive signal. Once cells are isolated, single-cell sequencing can produce TCR alpha/beta pair identity of thousands of cells ( 16– 18). The activation marker CD137 has been used for isolation of novel tumor-associated, as well as neoantigen-reactive TCRs ( 8, 10). These activation marker techniques permit isolation of TCRs reactive with previously undefined epitopes ( 8, 10). More recently, surface activation markers such as CD137 and CD107a/b have been used to isolate live T cells that have been activated in vitro, allowing for isolation and sequencing of antigen-specific TCRs without multimer staining ( 5, 8). 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. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. ![]() 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. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. 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. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 10 15 unique sequences. ![]()
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