Fig. 1: Integrated glyco-nanovaccine (iGN) provokes antigen-specific cytotoxic immune responses.

a Schematic diagram of nanoparticles ESIINFEKL–1V209–αMan–GNPs (short peptide GNPs) and SGLEQLESIINFEKL–1V209–αMan–GNPs (long peptide GNPs). Schematic diagrams of components conjugated to each nanoparticle are shown in the gray box. b Experimental schedule for evaluating immune response induced by nanoparticles. Splenocytes were analyzed by flow cytometry at 7 days after vaccination with each type of nanoparticle (0.4 nmol 1V209/injection). c Expression of interferon-gamma (IFNγ) in CD8⁺ T cells derived from splenocytes stimulated with SIINFEKL peptide (10 μg/ml) was analyzed by flow cytometry 7 days after vaccination in mice immunized with ESIINFEKL–1V209–αMan–GNPs (short peptide GNPs) or SGLEQLESIINFEKL–1V209–αMan–GNPs (long peptide GNPs). Representative scatterplots of the gated CD8⁺ T cells are shown in the right panel. Data are presented as means ± SEM; n = 6 mice for the short peptide GNPs group, n = 7 mice for the PBS or long peptide GNPs group. ***p < 0.001, One-way ANOVA test followed by Tukey’s multiple comparison test. d Schematic diagram of (HHHHHHSGLEQLESIINFEKL)Ni-NTA–1V209–αMan–GNPs (long peptide Ni-GNPs). e Expression of IFNγ in CD8⁺ T cells derived from splenocytes stimulated with SIINFEKL peptide (10 μg/ml) was analyzed by flow cytometry 7 days after vaccination in mice immunized with (HHHHHHSGLEQLESIINFEKL)Ni-NTA–1V209–αMan–GNPs (long peptide Ni-GNPs). The percentage of IFNγ⁺CD8⁺ T cells in PBS or SGLEQLESIINFEKL–1V209–αMan–GNPs (long peptide GNPs) in Fig. 1c is shown in light gray or light pink, respectively, for reference. Data are presented as means ± SEM; n = 7 mice for each group. ***p < 0.001, One-way ANOVA test followed by Tukey’s multiple comparison test. f Schematic diagram of nanoparticles: αMan–GNPs, SGLEQLESIINFEKL–αMan–GNPs, and 1V209–αMan–GNPs. g Expression of IFNγ in CD8⁺ T cells derived from splenocytes stimulated with SIINFEKL peptide (10 μg/ml) was analyzed by flow cytometry at 7 days post vaccination in mice immunized with αMan–GNPs, SGLEQLESIINFEKL–αMan–GNPs, 1V209–αMan–GNPs, or SGLEQLESIINFEKL–1V209–αMan–GNPs (iGN; integrated glyco-nanovaccine). Data are presented as means ± SEM; n = 6 mice for each group. ***p < 0.001, One-way ANOVA test followed by Tukey’s multiple comparison test. h Mice were immunized with PBS or iGN (0.4 nmol 1V209/injection) as described in Fig. 1c and were then subjected to flow cytometry to analyze CD44 and CD62L expression at 7 days after vaccination. Representative scatterplots of the gated CD8⁺ T cells are shown in the right panel. Data are presented as means ± SEM; n = 7 mice for each group. **p < 0.01, unpaired t test. i Schematic representation of in vivo cytotoxicity assay using carboxyfluorescein succinimidyl ester (CFSE) labeling is shown in the gray box. Splenocytes pulsed with SIINFEKL peptide or irrelevant peptide were labeled with low or high concentration of CFSE, respectively. The two types of CFSE-labeled splenocytes were mixed at a 1: 1 ratio, followed by injection into mice immunized with PBS or iGN (0.4 nmol 1V209/injection). Five hours after the injection, splenocytes from immunized mice were analyzed to measure the ratio of CFSE-low and -high by flow cytometry. Antigen-specific killing activity was calculated and is shown at the right. Data are presented as means ± SEM; n = 5 mice for PBS group, n = 10 mice for iGN group. ***p < 0.001, unpaired t test.