Figure 5: NAFL/IMIQ preferably recruits pDCs.

(a) Chemokine production at the inoculation site. The levels of indicated chemokines were measured in BALB/c mice after varying times of laser treatment by quantitative real-time PCR (qPCR), normalized to GAPDH and expressed as fold increases relative to time zero. Each square represents the mean value of four mice and colour indicates a fold increase from low (blue) to high (red). (b) Recruitment of pDCs into the inoculation site. pDCs were identified by B220⁺ CD11b⁻PCDA-1⁺ Ly6C⁺ cells by flow cytometry as described in Supplementary Fig. 6 and the percentages of pDCs in total skin cells were determined 6 and 24 h after vaccination with influenza vaccine alone or in the presence of indicated adjuvants. n=4. (c) The inoculation sites were also stained with siglec H-specific antibody (upper panel) or isotype control antibody (lower panel) 24 h after NAFL/IMIQ treatment. Note: Siglec H staining (red) was concentrated around MTZ (white dash lines). Representative results of six similar samples in two separate experiments. Blue, 4',6-diamidino-2-phenylindole staining of cell nuclei. Scale bar, 100 μm. Insets in c show a MTZ (white dash line), part of which is outlined by a white square and enlarged in c. (d) Cytokine expression at the inoculation sites 6 h after immunization. The expression levels of indicated cytokines were measured by qPCR and expressed as fold increases relative to those in the mice receiving no adjuvant. Each square represents a mean of 4 mice. (e,f) Increases in the number of mature DCs in dLNs in mice receiving influenza vaccine and NAFL/IMIQ. dLNs were collected at indicated times after immunization and mature DCs were identified as CD86⁺ CD11c⁺ cells (e) or CD40⁺ CD11c⁺ cells (f). n=6, except for the NAFL/IMIQ group (n=8). (g) Effects of pDC depletion on NAFL/IMIQ-mediated adjuvanticity. Mice were intraperitoneally injected with 400 μg anti-mPDCA-1 antibody or control antibodies (control) at 24 and 0 h before immunization. One week later cellular immune responses were measured (middle and lower panel), and humoral immune responses were measured two weeks later (upper panel). 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.