Th150 - Single-Cell Analysis of TCR and Gene Expression Diversity in NY-ESO-1-Specific HLA-DRB3*02:02-Restricted CD4+ T Cells from Healthy Donors

Abstract Text: Antigen-specific natural transgenic T-cell receptors (tTCRs) have been successfully isolated against NY-ESO-1 119−143, a cancer/testis antigen with minimal expression in healthy tissues but broad presence in tumors, making it a promising immunotherapy target. Unlike CAR T-cell therapies, which recognize only extracellular proteins, tTCRs enable the recognition of peptides from both intracellular and extracellular sources, expanding their therapeutic potential. However, most TCR-based therapies target HLA class I alleles, which are susceptible to downregulation, allowing tumors to evade immune detection. To overcome this, we focused on the HLA-DRB3*02:02 allele, expressed in ~50% of the Caucasian population.
CD4+ T cells were pre-sensitized and expanded in vitro using NY-ESO-1 119−143-pulsed monocytes. CD4+ CD154+ T cells were sorted and expanded through multiple rounds to enhance specificity. After 30 days, a highly specific CD4+ CD25− T-cell population targeting NY-ESO-1 119−143 on HLA-DRB3*02:02 was obtained, maintaining 97% CD4 purity, with specificity significantly improving after the second sorting round.
Single-cell RNA sequencing (scRNAseq) and TCR sequencing (scTCRseq) were performed to analyze TCR repertoires, gene expression, and clonal expansion. This analysis identified potential TCR candidates for therapeutic development. These findings demonstrate the feasibility of isolating highly specific CD4+ TCRs against NY-ESO-1 119−143 in the context of HLA-DRB3*02:02, supporting the potential of this strategy as an effective immunotherapy for NY-ESO-1-expressing tumors.