Abstract 4066: High throughput single-cell TCR sequencing method identifies cancer-specific TCR clonotypes

Abstract Cancer cells produce tumor-specific antigens and neoantigens that can be recognized by T cells and induce their clearance. Single cell profiling of T cell receptors (TCRs) and gene expression enables the identification and characterization of tumor infiltrating T cells that have antigen recognition and killing capabilities. Existing microfluidic-based single cell sequencing methods have limited cell and sample throughput making it hard to perform longitudinal studies to capture changes in TCR repertoire dynamics during tumor progression and therapeutic interventions. Understanding these dynamics will be key in designing the next generation of immunotherapies. To overcome these limitations, we developed a combinatorial barcoding approach to simultaneously characterize the TCRs alongside the full transcriptomes of up to 1 million fixed T cells from up to 96 unique samples in a single experiment. To demonstrate this approach, we profiled 1 million human T cells stimulated with a variety of antigens including the tumor antigens MART-1, NY-ESO-1, HER2/Neu, MAGE-A10, WT-1 and Tyrosinase. Using our technology, we recovered paired TCR alpha and beta clonotype information in 82% of all T cells. We estimated the clonotype diversity and found that the tumor antigen-stimulated samples had lower diversity than the controls. Specifically, when looking at the top three most expanded clonotypes for each sample we show that they account for 8-99% of all detected clonotypes. Next, we searched publicly available databases and showed our method not only detects previously annotated disease-specific CDR3 sequences but also uncovers previously unknown ones. Finally, we ran multiple sequence alignments and identified CDR3 sequence motifs unique to each antigen-stimulated population. In summary, we present a highly flexible and scalable method to single cell TCR profiling to allow researchers to profile up to 1 million T cells and up to 96 samples in a single experiment. We envision our approach to be invaluable in investigating T cell functional responses during infection, cancer, autoimmunity, or therapeutic interventions and pave the way for the development of new immune-based therapies. Citation Format: Efthymia Papalexi, Crina Curca, Bryan Hariadi, Alexa Suyama, Simone Marrujo, Gary Geiss, Charles Roco, Alexander Rosenberg. High throughput single-cell TCR sequencing method identifies cancer-specific TCR clonotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4066.