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.

T 细胞受体 (TCR) 通过 V/D/J 片段的体细胞重组产生多达 10 ¹⁵独特的序列。需要高度敏感和特异性的技术来分离和鉴定对感兴趣的抗原有反应的稀有 TCR 序列。在这里，我们描述了通过交联剂调节的细胞内表型 (CLInt-Seq) 使用 mRNA 测序，以有效恢复细胞中的抗原特异性 TCR，这些细胞内蛋白（如细胞因子或转录因子）的组合被染色。这种方法可以对任何抗原特异性的低频 TCR 进行高通量识别和分离。作为原理证明，TNFα 和 IFNγ 的细胞内染色鉴定了巨细胞病毒 (CMV) 和 Epstein-Barr 病毒 (EBV) 反应性 TCR，其效率类似于最先进的肽-MHC 多聚体方法。在一个单独的实验中，调节性 T 细胞基于细胞内 FOXP3 染色进行了分析，证明了基于转录因子检查表型的能力。我们进一步优化了细胞内染色条件，以使用与当前单细胞测序技术兼容的化学可裂解伯胺交联剂。TNFα 和 IFNγ 的 CLInt-Seq 与 EBV 反应性 TCR 的多聚体染色分离的表现相似。我们预计 CLInt-Seq 将能够从任何需要通过细胞内标记物分离少量群体的组织中进行基于液滴的单细胞 mRNA 分析。TNFα 和 IFNγ 的 CLInt-Seq 与 EBV 反应性 TCR 的多聚体染色分离的表现相似。我们预计 CLInt-Seq 将能够从任何需要通过细胞内标记物分离少量群体的组织中进行基于液滴的单细胞 mRNA 分析。TNFα 和 IFNγ 的 CLInt-Seq 与 EBV 反应性 TCR 的多聚体染色分离的表现相似。我们预计 CLInt-Seq 将能够从任何需要通过细胞内标记物分离少量群体的组织中进行基于液滴的单细胞 mRNA 分析。