55P Transcriptional Profiles of Engineered T Cells Stimulated with Different Receptor Structures and Co-Stimulatory Domains

T cells engineered with chimeric antigen receptor (CAR-T) have shown success in treating hematological malignancies. However, the limited efficacy and potential side effects hinder the use of CAR-T in solid tumors. Research on this heterogeneous cell therapeutics at single-cell level can advance the current understanding and strategy of CAR-T therapy. We used single-cell transcriptome analysis to characterize the transcriptional profile of stimulated CAR-T. T cells engineered with second-generation CAR constructs (with 4-1BB or CD28 co-stimulation domains) were stimulated with anti-CD3/CD28-coated beads and Nalm6 and subjected to microwell-based single-cell transcriptome analysis. A broad spectrum of transcripts was differentially expressed in T cells under various conditions. The sequence derived from the 3’-untranslated region of the CAR transcript distinguished CAR-T from non-transduced T cells within the analyzed cell population. CAR-T stimulation, mediated by either CD3/CD28 or CAR, significantly changed the gene-expression landscape compared to unstimulated CAR-Ts. Notably, CAR stimulation triggered higher expression of immune-related genes compared to CD3/CD28 stimulation. Additionally, comparing 4-1BB and CD28 co-stimulatory domains showed distinct transcriptional profiles contingent upon each co-stimulation signal. This study revealed characteristic features of CAR-T signals affected by receptor structure (CD3/CD28 vs. CAR) and co-stimulation domain (4-1BB vs. CD28). These findings provide insights into a deeper understanding of CAR-T therapy, ultimately leading to the development of more effective CAR-T therapy.