Antigen coupled with Lewis-x trisaccharides elicits potent immune responses in mice Shih-Chang Hsu, BS, Tsung-Hsien Tsai, MD, Hirokazu Kawasaki, PhD,

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Antigen coupled with Lewis-x trisaccharides elicits potent immune responses in mice Shih-Chang Hsu, BS, Tsung-Hsien Tsai, MD, Hirokazu Kawasaki, PhD, Chien-Ho Chen, PhD, Beverly Plunkett, MS, Reiko T. Lee, PhD, Yuan C. Lee, PhD, Shau-Ku Huang, PhD Journal of Allergy and Clinical Immunology Volume 119, Issue 6, Pages 1522-1528 (June 2007) DOI: 10.1016/j.jaci.2007.01.034 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The relative levels of serum-specific antibodies in sensitized, C3H/hej, and TLR4 wild-type C3H/HeOuj mice. A, Serum titration analysis of specific IgE and IgG1 levels. B and C, The relative levels of serum IgE and IgG2a antibodies in sensitized C3H/hej (Fig 1, B) and TLR4 wild-type (Fig 1, C) mice. N = 9 to 12 mice per group. ∗P < .05. NS, Normal saline. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Levels (mean ± SE) of the cytokines IL-4, IL-13, and IFN-γ in spleen cells from sensitized C3H/hej mice with saline (NS) or antigen (BSA or Lex-BSA) stimulated with NS, BSA, or Lex-BSA. N = 9 mice per group. ∗P < .05. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Analysis of antibody and cytokine levels in C3H/hej mice receiving DC transfers. A, Relative levels (mean ± SE) of serum-specific antibodies in mice receiving BM-DC transfers. B, Levels (mean ± SE) of the cytokines IL-4, IL-13, and IFN-γ in spleen cells of adoptively transferred mice. N = 9 to 12 mice per group. ∗P < .05. NS, Normal saline. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Analysis of antigen (BSA)–induced antibody and cytokine levels in BALB/c mice. A, Relative levels (mean ± SE) of serum BSA-specific antibodies in sensitized BALB/c mice. B, Levels (mean ± SE) of the TH2 cytokines IL-4 and IL-13 in spleen cells. N = 9 to 12 mice per group. C, Analysis of serum IL-12p70 levels in mice sensitized with saline (–), BSA, or Lex-BSA. N = 4 mice per group. ∗P < .05. NS, Normal saline. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Measurement of antigen (OVA)–induced antibody and cytokine levels in BALB/c mice. A, Relative levels (mean ± SE) of serum OVA-specific antibodies in sensitized BALB/c mice. B, Levels (mean ± SE) of IL-4, IL-13, and IFN-γ in spleen cells. N = 6 to 9 mice per group. ∗P < .05. NS, Normal saline. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Analysis of cytokines in BM-DCs. A and B, Levels (mean ± SE) of IL-12p70 (Fig 1, A) and IL-10 (Fig 1, B) in BALB/c BM-DCs are shown. DCs were incubated with medium alone or LPS in the presence or absence of varying concentrations of glycoforms as indicated, with their respective structures and linked positions for fucose shown. ∗P < .05 versus LPS alone. C, Levels (mean ± SE) of LPS-induced IL-12p70 and IL-10 production in BM-DCs. ∗P < .05 versus LPS alone. D, Mannan blocks the inhibitory effect of Lex-BSA on LPS-induced gene expression for IL-12p40 in BM-DCs. E, Relative level (mean ± SE) of nuclear NF-κB (nNF-κB) in BM-DCs. ∗P < .05. N = 3 experiments. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Levels of antibodies and cytokines in IL-12−/− mice. A, Relative levels (mean ± SE) of serum BSA-specific antibodies. B, Levels (mean ± SE) of the TH2 cytokines IL-4 and IL-13 in spleen cells from BSA- or Lex-BSA–sensitized mice stimulated with BSA. N = 9 to 12 mice per group. ∗P < .05. Journal of Allergy and Clinical Immunology 2007 119, 1522-1528DOI: (10.1016/j.jaci.2007.01.034) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions