Figure 4: Accelerated migration of APCs into dLNs in the presence of NAFL/IMIQ.

MHC II-EGFP transgenic mice were ID inoculated in one ear with influenza vaccine adjuvantated with NAFL or NAFL/IMIQ. Accumulation of MHC II⁺ APCs around individual MTZs was visualized by intravital confocal microscopy at varying times after immunization and the one captured at 8 h post immunization was shown in a, left panel. A representative MTZ was tracked at 0, 3, 8 and 24 h after immunization of influenza vaccine with NAFL treatment (upper panel) or NAFL/IMIQ (lower panel) in (a), representative of 6 similar results in two separate experiments. White dash circles highlight MTZs. Scale bar, 200 μm. (b) Monitoring APCs within lymphatic vesseles. Ears were prepared and stained by whole-mount immunohistology 8 h post immunization. APCs (green), lymphatic vessels (blue and/or red) and blood vessels (red) were visualized by confocal microscopy, representative of six similar results in two separate experiments. Scale bar, 100 μm. Average number of APCs within each lymphatic vessel were determined by manual counting GFP⁺ cells inside the lymphatic vessels in 20 randomly selected fields with a total of more than 40 lymphatic vessels counted (c) during which three-D scanning was performed to confirm the intra-vessel localization of each APC (b). (d,e) Proportions of CD11⁺ DCs in the skin (d) and a total number of DCs in each dLN (e). CD11c⁺ DCs in the dorsal skin and dLNs were quantified by flow cytometry at indicated times after immunizing with the influenza vaccine alone or in the presence of indicated adjuvants as Fig. 1. n=6, except for NAFL/IMIQ group (n=8). Data are presented as mean±s.e.m. Statistical significance was analysed by ANOVA/Bonferroni. *P<0.05; **P<0.01 or ***P<0.001, respectively. All experiments were repeated twice with similar results.