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Background While Chimeric Antigen Receptor (CAR) T-cell therapy has revolutionized the treatment of heme malignancies, multiple challenges must be overcome in order to apply this therapeutic modality to solid tumors. In particular, robust tools to facilitate a comprehensive elucidation of CAR expression, activation, and signaling dynamics through simultaneous muti-omics profiling are lacking. Traditional single-cell RNA sequencing (scRNA-seq) provides only a partial view, necessitating assays that integrate proteomic and post-translational modification (PTM) data. Here, we applied InTraSeq™, a novel multimodal assay for CAR-T research that enables concurrent single-cell measurement of mRNA, surface, cytoplasmic, and nuclear proteins, including PTMs. 1 Methods Our experimental setup involved profiling four distinct conditions: Jurkat cells cultured alone, Jurkat cells co-cultured with Raji cells, anti-CD20 CAR-Jurkat cells cultured alone, and anti-CD20 CAR-Jurkat cells co-cultured with CD20 positive Raji cells. Each condition was analyzed at two timepoints, 2 and 24 hours, resulting in eight unique samples. All samples were uniquely hashed using CST’s Histone H3 InTraSeq conjugates, then pooled and processed with 10x Genomics 3’ kits. An anti-Whitlow/218 linker monoclonal antibody InTraSeq conjugate was specifically included to label the anti-CD20 CAR-Jurkat cells.Results Our data reveal successful de-multiplexing of all eight samples based on the unique Histone H3 barcodes. The anti-Whitlow/218 InTraSeq conjugate accurately confirmed CAR expression, enabling precise identification of engineered CAR-Jurkat cells. Notably, unique RNA clusters and intricate single-cell protein signaling events were observed exclusively in the activated CAR-Jurkat/Raji co-cultures at both timepoints, highlighting novel aspects of CAR biology.Conclusions This pioneering single-cell RNA and protein signaling study provides comprehensive insights into mechanisms of CAR by simultaneously examining transcriptomic and proteomic profiles within individual cells. InTraSeq™ offers an invaluable tool for dissecting the complex interplay of gene expression and protein activity during CAR-T cell activation.Acknowledgements The Jurkat cells engineered to stably express an scFv-based Anti-CD20 (Leu16) CAR containing a Whitlow/218 linker was kindly provided by the Lohmueller Lab, University of Pittsburgh.Reference Ariss, M. M., Huang, L., Ding, X., Sheth, S., Levy, T., Fisher, J., ...,Beausoleil, S. A. (2024). InTraSeq: A Multimodal Assay that Uncovers New Single-Cell Biology and Regulatory Mechanisms. bioRxiv, 2024; 09.