N1-Methylpseudouridine-Modified mRNA-Transfected Dendritic Cells Enable Efficient Generation of Functional Tumor Antigen-Specific Cytotoxic T Lymphocytes

Efficient generation of tumor-specific cytotoxic T lymphocytes (CTLs) remains challenging. Although mRNA-based approaches enable the delivery and expression of diverse tumor-specific antigens, unmodified mRNA can trigger innate immune responses that suppress translation. Here, we investigated whether 1-methyl-pseudouridine (m¹Ψ)-modified mRNA enhances antigen expression in human dendritic cells (DCs) and promotes the induction of antigen-specific CTLs, using survivin and MART-1 as model tumor-associated antigens. Remarkably, m¹Ψ modification increased protein production in DCs by up to 20-fold compared with unmodified mRNA. DCs transfected with m¹Ψ-modified mRNAs efficiently primed survivin- and MART-1-specific CD8⁺ T cells from autologous peripheral blood mononuclear cells of healthy donors, as demonstrated by HLA class I peptide-MHC dextramer staining. Expanded CTL lines exhibited potent effector functions, including IFN-γ production, CD107a mobilization, and robust cytotoxicity against peptide-pulsed T2 cells and tumor cell lines expressing survivin or MART-1. Translation of the engineered mRNA variants in human HeLa cells and DCs is strongly dependent on the poly(A) tail. Together, these findings demonstrate that m¹Ψ modification substantially enhances antigen expression and supports the efficient generation of functional, antigen-specific CTLs, providing a promising platform for next-generation mRNA-based cancer vaccines and adoptive T-cell therapies.