Fig. 3. Optimization of vaccination routes for inducing efficacious overall and HPV-specific CD8⁺ T cell immune responses.

a C57BL/6J mice (n = 5) were vaccinated subcutaneously or intravenously (10 μg/100 μl) on days 0, 5, and 10 with mRNA-LNPs. Whole blood, spleen, and inguinal lymph nodes (LNs) were collected on day 16 to measure the frequency of overall and dextramer⁺ CD8⁺ T cells. b T-SNE maps showing cluster distribution of CD8⁺ T cells using flowcytometry data (top) and t-SNE heatmaps for each marker applied on CD8⁺ T cell events (bottom). c Expression of different immune markers on CD8⁺ T cells in spleen, blood, and inguinal LNs in (Error bar = mean ± SEM, n = 5). Statistics were assessed by two-way ANOVA with Tukey’s multiple comparison tests. *P < 0.05, **P < 0.01 and ***P < 0.001. d Histograms showing the differential expression of phenotypic markers expressed by dextramer⁺ or dextramer- populations after LNP-IV (top) or LNP-SC (bottom) assessed using flow cytometry (concatenated, n = 15). Data are representative of three independent experiments. Naive unvaccinated, IV intravenous, SC subcutaneous.