Luo Siwei, Notaro Amber, Lin Lan
Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Nat Commun. 2025 Jan 2;16(1):216. doi: 10.1038/s41467-024-54675-3.
Discovering antigen-reactive T cell receptors (TCRs) is central to developing effective engineered T cell immunotherapies. However, the conventional technologies for isolating antigen-reactive TCRs (i.e., major histocompatibility complex (MHC) multimer staining) focus on high-affinity interactions between the TCR and MHC-antigen complex, and may fail to identify TCRs with high efficacy for activating T cells. Here, we develop a microfluidic single-cell screening method for antigen-reactive T cells named ATLAS-seq (Aptamer-based T Lymphocyte Activity Screening and SEQuencing). This technology isolates and characterizes activated T cells via an aptamer-based fluorescent molecular sensor, which monitors the cytotoxic cytokine IFNγ secretion from single T cells upon antigen stimulation, followed by single-cell RNA and single-cell TCR sequencing. We use ATLAS-seq to screen TCRs reactive to cytomegalovirus (CMV) or prostate specific antigen (PSA) from peripheral blood mononuclear cells (PBMCs). ATLAS-seq identifies distinct TCR clonotype populations with higher T cell activation levels compared to TCRs recovered by MHC multimer staining. Select TCR clonotypes from ATLAS-seq are more efficient in target cell killing than those from MHC multimer staining. Collectively, ATLAS-seq provides an efficient and broadly applicable technology to screen antigen-reactive TCRs for engineered T cell immunotherapy.
发现抗原反应性T细胞受体（TCR）是开发有效的工程化T细胞免疫疗法的核心。然而，用于分离抗原反应性TCR的传统技术（即主要组织相容性复合体（MHC）多聚体染色）侧重于TCR与MHC-抗原复合物之间的高亲和力相互作用，可能无法识别激活T细胞效率高的TCR。在此，我们开发了一种用于抗原反应性T细胞的微流控单细胞筛选方法，称为ATLAS-seq（基于适体的T淋巴细胞活性筛选和测序）。该技术通过基于适体的荧光分子传感器分离并表征活化的T细胞，该传感器监测抗原刺激后单个T细胞分泌的细胞毒性细胞因子IFNγ，随后进行单细胞RNA和单细胞TCR测序。我们使用ATLAS-seq从外周血单核细胞（PBMC）中筛选对巨细胞病毒（CMV）或前列腺特异性抗原（PSA）有反应的TCR。与通过MHC多聚体染色回收的TCR相比，ATLAS-seq识别出具有更高T细胞活化水平的不同TCR克隆型群体。从ATLAS-seq中选择的TCR克隆型在杀伤靶细胞方面比从MHC多聚体染色中选择的更有效。总的来说，ATLAS-seq提供了一种高效且广泛适用的技术，用于筛选用于工程化T细胞免疫疗法的抗原反应性TCR。
