Dissecting the role of CAR signaling architectures on T cell activation and persistence using pooled screening and single-cell sequencing

Chimeric antigen receptor (CAR) T cells represent a promising approach for cancer treatment, yet challenges remain such as limited efficacy due to a lack of T cell persistence. Given its critical role in promoting and modulating T cell responses, it is crucial to understand how alterations in the CAR signaling architecture influence T cell function. Here, we designed a combinatorial CAR signaling domain library and performed repeated antigen stimulation assays, pooled screening and single-cell sequencing to investigate T-cell responses triggered by different CAR architectures. Parallel comparisons of CAR variants, at early, middle and late timepoints during chronic antigen stimulation systematically assessed the impact of modifying signaling domains on T cell activation and persistence. Our data reveal the predominant influence of membrane-proximal domains in driving T cell phenotype. Additionally, we highlight the critical role of CD40 costimulation in promoting potent and persistent T cell responses, followed by CTLA4, which induces a long-term cytotoxic phenotype. This work deepens the understanding of CAR T cell biology and may be used to guide the future engineering of CAR T cell therapies. Library CAR T cells derived from two healthy donors were subjected to a repeated antigen stimulation assay (co-cultured at 1:1 E:T ratio with SKBR3-GFP cells every 3 days). At days 0, 6 and 12 of the RAS assay, CAR library T cells were co-cultured with SKBR3-GFP cells for 6h in 30 IU/mL of IL-2. Following this time, the co-cultures were stained using DRAQ7, and the live GFP- negative population was sorted using a FACS. Cells were then stained using 20 Totalseq B antibodies and sequenced.