ATLAS-seq: a microfluidic single-cell TCR screen for antigen-reactive TCRs

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. Briefly, we used the microfluidic chip to generate droplets co-encapsulating aptamer decorated single CD8+ T cell and antigen-loaded aAPCs. Generated droplets were collected and incubated for 2 days to allow T-cell activation by antigen-loaded aAPCs. An activated T-cell containing cognate TCR for the target antigen pMHC may release IFNγ molecules into the same droplet which, in turn, bind to the IFNγ specific aptamer beacons on the same T-cell surface and emit Cy3 fluorescence signal. The droplet emulsions were then broken to release aptamer labelled CD8+ T cells for fluorescent sorting using flow cytometry. CD8+Cy3high T cells were then collected and subject to single cell TCR and RNA sequencing (scTCR-seq and scRNA-seq) prepared by 10X Genomics single-cell sequencing system.