Fig. 1

PGA/Alum recovers low DC migration, activation, and DC-mediated CD8⁺ T cell activation in aged mice. A Aged mice (n = 3 per group) were s.c. injected into the right footpad with IR800-OVA alone or mixed with γ-PGA, alum, or PGA/Alum. IR800-OVA-young mice (n = 3) were used for comparison. At 1, 3, 6, 12, and 24 h, in vivo NIR fluorescence signals were acquired using in vivo imaging system. Fluorescent intensities of LN of interest (dotted circle) were quantitatively measured using ImageJ. B and C Aged mice (n = 3 per group) were intramuscularly (i.m.) administered Fluor-OVA alone or mixed with γ-PGA, alum, or PGA/Alum. Fluor-OVA-young mice (n = 3) were used for comparison. At 3 h post-immunization, the number of DCs (gated as CD11c⁺CD3⁻) and Fluor-OVA⁺ DCs were analyzed in injected muscle region (B) and dLNs (C) via flow cytometry. D–G Splenic DCs from aged mice were stimulated with γ-PGA, alum, or PGA/Alum. (D and E) After 30 h, the DCs were stained with a fluorescent dye-conjugated anti-CD11c, CD40, and CD86 Abs. Expression of the costimulatory molecules was analyzed in CD11c⁺ DCs via flow cytometry. (F and G) After 6 h, the DCs were washed and co-cultured with CFSE-labeled OT-I CD8⁺ T cells for 5 days (F) or OT-I CD8⁺ T cells in the presence of monensin for 6 h  (G). Statistical significance was analyzed using two-way ANOVA/Bonferroni (A) and one-way ANOVA/Bonferroni (B–G); *P < 0.05, **P < 0.01, and ***P < 0.001