Single-cell RNA-sequencing and paired TCR-sequencing of CD4⁺ and CD8⁺ T-cell differentiation in a mouse DNA vaccination model with or without CD4⁺ T-cell help

Briefly, we employed a DNA vaccination model in C57BL/6 mice based on epidermal “tattoo” delivery of plasmid DNA encoding the HPV E7 (E7₄₈–₅₇) MHC-I epitope either alone or linked to three MHC-II helper epitopes (P30, PADRE, NEF).

Vaccination on days 0, 3, and 6 induces antigen expression in keratinocytes and delivery to the draining lymph node (dLN) via migratory conventional dendritic cells and passive drainage. This model allows parallel tracking of antigen-specific CD8⁺ T cells (H-2Dᵇ/E7 tetramer) and CD4⁺ T cells (PADRE MHC-II tetramer), enabling controlled comparison of CD8⁺ T-cell responses in the presence or absence of CD4⁺ T-cell help, as well as mapping the differentiation trajectories of polyclonal CD4⁺ T cells.