Intramuscular plasmid DNA electroporation sequesters neoantigen-specific CD8⁺ T cells in treated muscle and limits...

DNA electroporation of neoantigen cancer vaccine is a well-established approach to induce high expression of neoantigens and likely enlarge the immune response that is shaped by the vaccination site influencing T cell trafficking. Using a DNA plasmid vaccine encoding MC38-derived neoantigens, we found that in presence of tumor, the vaccine delivery by muscle electroporation promotes extensive activation of immune-related genes and CD8⁺ T cell enrichment in muscle together with inflammatory markers, whereas tumors show minimal transcriptional changes compared with untreated controls. Flow cytometry analysis revealed accumulation of neoantigen-specific CD8⁺IFNγ⁺ T cells in electroporated muscles compared to spleen or tumor, consistent with preferential localization at the vaccination site. Vaccination in a second site with an unrelated plasmid DNA electroporation partially reproduced this effect, consistent with neoantigen-independent recruitment of activated CD8⁺ T cells. Adoptive T cells transfer further demonstrated preferential T cell homing to electroporated tissue. These findings identify electroporation-induced tissue remodeling as a major contributor associated with altered T cell localization and suggest that modulating local tissue responses may improve the systemic efficacy of DNA-based cancer vaccines.