Fig. 3: In vivo assessments of antigen-specific immune activation by three different mRNA LiNx formulations.

a–h C57BL/6 mice were administered three different LiNx formulations loaded with mOVA via s.c. injection (30 μg per mouse). Mice were sacrificed two weeks after the injection. a, b The number of OVA-specific CD8 T cells within the LiNx (a) and dLNs (b) was analysed by flow cytometry. c–h Their splenocytes were isolated and restimulated in vitro with OVA and SIINFEKL peptides (100 μg mL⁻¹ OVA and 2 μg mL⁻¹ SIINFEKL) for 12 h and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of CD8⁺IFN-γ⁺ cells (c), CD8⁺TNFα⁺ cells (d), CD8⁺ Granzyme B⁺ cells (e), CD4⁺IFN-γ⁺ cells (f), CD4⁺TNFα⁺ cells (g), and CD4⁺IL-4⁺ cells (h). i Frequency of IFN-γ-producing cells among restimulated splenocytes isolated from vaccinated mice on day 30 post-vaccination, assessed via ELISPOT. j Titre of OVA-specific IgG antibodies in blood serum on day 30, determined by ELISA. k–o C57BL/6 mice were administered with three different LNP/mRNA LiNx formulations loaded with mOVA via s.c. injection (30 μg per mouse). Mice were sacrificed three months after the injection, and their splenocytes were isolated for analysis. The percentages of OVA-specific CD8 T cells (CD3⁺ CD8⁺ OVA⁺ cells) (k), OVA-specific effector CD8 T cells (CD3⁺CD8⁺OVA⁺CD44⁺CD62L^– cells) (l), and OVA-specific central memory CD8 T cells (CD3⁺CD8⁺OVA⁺CD44⁺CD62L⁺ cells) (m) were determined. Splenocytes were restimulated in vitro with OVA and SIINFEKL peptide (100 μg mL⁻¹ OVA and 2 μg mL⁻¹ SIINFEKL) for 12 h and assessed via flow cytometry and intracellular cytokine staining to determine the percentages of CD3⁺CD8⁺TNFα⁺ cells (n) and CD3⁺CD8⁺IFN-γ⁺ cells (o). p Titre of OVA-specific IgG antibodies in blood serum on day 90, determined by ELISA. Data represent the mean ± s.e.m. (n = 7 mice (a, b), n = 4 mice (c–j), n = 8 mice (k–p)). Data were analysed using one-way ANOVA and Tukey’s multiple comparisons test for (a–p). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.