Ucoidan can promote antigen-presentation or cross presentation by DCs. Mice have been injected with PBS, OVA or OVA + fucoidan for 24 hrs, and after that measured for expression of MHC class I and II on spleen Lineage2CD11c+ cDCs. As shown Figure 5A, spleen CD11c+ cDCs considerably up-regulated surface expression of MHC class I and II molecules after therapy with OVA + fucoidan, whereas those treated with OVA alone did not. Subsequent, we performed an adoptive transfer experiment to detect OVA particular OT-I and OT-II T cell proliferation. CFSE-labeled OT-I CD 8 T cells or OT-II CD4 T cells have been transferred into CD45.1 congenic mice and 24 hrs later, the mice received injection of PBS, OVA or OVA + fucoidan. Right after three days, the proliferation of OT-I and OTII cells was HSP90 Antagonist manufacturer determined by CFSE dilution assay. OT-I and OT-II T cells proliferation was robustly improved in mice immunizedFucoidan Functions as an Adjuvant In VivoFigure 1. In vivo administration of fucoidan induces spleen cDC maturation. C57BL/6 mice had been treated with ten mg/kg fucoidan for 24 hrs. (A) Flow cytometric analysis of CD40, CD80, CD86 and MHC class II expression on the gated lineage2CD11c+ cDCs in splenocytes (upper panels). Lineage markers integrated CD3, Thy1.1, B220, Gr1, CD49b and TER-119. (B) Imply fluorescence intensity (MFI) of CD40, CD80, CD86 (left panel) and MHC class II (ideal panel) was shown. (C) Lineage2CD11c+ cDCs were additional divided into CD8a+ and CD8a2 cDCs. Expression of CD40, CD80, CD86 and MHC class II was shown by histogram. (D) MFI of CD40, CD80, CD86 (appropriate panel) and MHC class II (left panel) on CD8a+ and CD8a2 cDCs was shown. All information are representative of or the average of analyses of six independent samples (two mice per experiment, total 3 independent experiments). doi:ten.1371/journal.pone.0099396.gwith OVA + fucoidan in comparison to those in mice immunized with OVA alone (Figure 5 B). These data demonstrated that fucoidan functions as an adjuvant to improve antigen presentation and antigen-specific CD4 and CD8 T cell activation.OVA-immunization with fucoidan as an adjuvant protects mice from a challenge with B16-OVA tumor cellsBased on the observation that fucoidan functioned as an adjuvant to activate OVA-specific CD4 and CD8 T cells, wefurther investigated whether this response can protect mice grafted with OVA-expressing B16 tumor cells. C57BL/6 mice have been immunized i.p. with PBS, OVA, fucoidan or OVA + fucoidan on days 0, 15 and 30 and were inoculated s.c. with B16-OVA tumor cells on day 35. Mice immunized with OVA + fucoidan were nearly totally protected from B16-OVA tumor challenge (Figure 6A, B and C), and furthermore, they did not create tumor following a second tumor challenge, indicative of formation of longterm memory (information not shown). We also investigated the functional activity of CTL in an in vivo cytotoxicity assay. OnPLOS A single | plosone.orgFucoidan Functions as an Adjuvant In VivoFigure two. Fucoidan promotes production of pro-inflammation cytokines in cDCs. Expression levels of IL-6, IL-12p40, IL-23p19 and TNF-a mRNA in HDAC7 Inhibitor MedChemExpress spleens were measured three hrs soon after fucoidan injection. (A) mRNA levels of IL-6, IL-12p40, IL-23p19 and TNF-a in spleens. (B) IL-6, IL-12p70, IL23 and TNF-a concentration in serum. (C) Lineage2CD11c+ cDCs had been isolated by cell sorter two hrs immediately after fucoidan injection. Isolated cDCs had been incubated in culture medium for 4 hrs, and then analyzed for IL-6, IL-12p70, IL-23 and TNF-a levels inside the culture supernatants had been measured by ELISA. (D) mRNA leve.