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

Abstract

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.