Figure 3

Multi-site vaccination increased the magnitude of transgene-specific CD8⁺ T cells in spleens and lymph nodes. Tumor-free female C57BL/6 mice were vaccinated intramuscularly with 1 × 10⁸ pfu of recombinant human serotype 5 adenovirus expressing the melanoma-associated antigen dopachrome tautomerase, with injections spread across one (semitendinosus; left hind limb), two (semitendinosus of both hind limbs) or four (semitendinosus of both hind limbs and the triceps brachii of both forelimbs) sites. (A,B) Spleen and (C,D) lymph node-derived CD8⁺ T cells specific for the immunodominant self-epitope DCT_180–188 were quantified fourteen days post-vaccination by flow cytometric assessment of intracellular cytokine staining after ex vivo re-stimulation with peptides. (A,B) One-way analysis of variance with Tukey’s multiple comparison test (upper panels) and Pearson correlation analysis (lower panels) was performed for the (A) frequency and (B) total number of antigen-specific splenic CD8⁺ T cells (n = 4/treatment). (C–D) Two-way analysis of variance with Tukey’s multiple comparison test (upper panels) and Pearson correlation analysis (lower panels) was performed for the (C) frequency and (D) total number of antigen-specific lymph node-derived CD8⁺ T cells (n = 4/treatment; L = left node, R = right node).