Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill"...

Area of Science:

• Immunology
• Genomics
• Biotechnology

Background:

• Synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells offer improved safety and efficacy for glioblastoma treatment.
• Tracking and profiling these engineered T cells *in vivo* is crucial for understanding their therapeutic potential but remains challenging.

Purpose of the Study:

• To develop a method for simultaneous detection and transcriptomic profiling of synNotch-CAR-T cells *in vivo*.
• To characterize the dynamics and tissue-specific behavior of engineered T cells using single-cell RNA sequencing.

Main Methods:

• Development of a single-cell RNA-sequencing (scRNA-seq) workflow incorporating custom spike-in probes targeting synNotch-CAR transcripts.
• Machine-learning-assisted classification for detecting engineered cells based on probe integration.
• Application of the workflow in *in vitro* assays and a xenograft model.

Main Results:

• The scRNA-seq workflow achieved high specificity and sensitivity in detecting engineered T cells *in vitro*.
• SynNotch-positive cells were detected *in vivo* across multiple organs, with highest frequency and activation in the brain.
• Single-cell transcriptomic analysis revealed tissue-specific differentiation, including cytotoxicity, proliferation, and memory phenotype acquisition.

Conclusions:

• The spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics.
• This platform provides a broadly applicable tool for monitoring engineered immune cells in various clinical settings.
• Understanding tissue-specific T cell behavior is key to optimizing engineered cell therapies.