Ucoidan can promote antigen-presentation or cross presentation by DCs. Mice had been injected with PBS, OVA or OVA + fucoidan for 24 hrs, after which measured for expression of MHC class I and II on spleen Lineage2CD11c+ cDCs. As shown Figure 5A, spleen CD11c+ cDCs drastically up-regulated surface expression of MHC class I and II molecules HSP90 Inhibitor Formulation immediately 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 had been transferred into CD45.1 congenic mice and 24 hrs later, the mice received injection of PBS, OVA or OVA + fucoidan. Soon after three days, the proliferation of OT-I and OTII cells was determined by CFSE dilution assay. OT-I and OT-II T cells proliferation was robustly elevated in mice immunizedFucoidan Functions as an Adjuvant In VivoFigure 1. In vivo administration of fucoidan induces spleen cDC maturation. C57BL/6 mice have been treated with 10 mg/kg fucoidan for 24 hrs. (A) Flow cytometric analysis of CD40, CD80, CD86 and MHC class II expression around 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 had been 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 (suitable panel) and MHC class II (left panel) on CD8a+ and CD8a2 cDCs was shown. All data are representative of or the average of analyses of six independent samples (2 mice per experiment, total 3 independent experiments). doi:ten.1371/journal.pone.0099396.gwith OVA + fucoidan compared to those in mice immunized with OVA alone (Figure five 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 ErbB3/HER3 Inhibitor Storage & Stability B16-OVA tumor cellsBased on the observation that fucoidan functioned as an adjuvant to activate OVA-specific CD4 and CD8 T cells, wefurther investigated regardless of whether this response can guard mice grafted with OVA-expressing B16 tumor cells. C57BL/6 mice had been immunized i.p. with PBS, OVA, fucoidan or OVA + fucoidan on days 0, 15 and 30 and had been inoculated s.c. with B16-OVA tumor cells on day 35. Mice immunized with OVA + fucoidan had been pretty much absolutely protected from B16-OVA tumor challenge (Figure 6A, B and C), and furthermore, they didn’t develop tumor just after 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 One | plosone.orgFucoidan Functions as an Adjuvant In VivoFigure 2. Fucoidan promotes production of pro-inflammation cytokines in cDCs. Expression levels of IL-6, IL-12p40, IL-23p19 and TNF-a mRNA in spleens have been measured 3 hrs just 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 were isolated by cell sorter two hrs right after fucoidan injection. Isolated cDCs were incubated in culture medium for 4 hrs, after which analyzed for IL-6, IL-12p70, IL-23 and TNF-a levels in the culture supernatants were measured by ELISA. (D) mRNA leve.