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Volume 115, Issue 4, Pages 891-897 (October 1998) Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection Pierre F. Saldinger, Nadine Porta, Pascal Launois, Jacques A. Louis, Gary A. Waanders, Hanifa Bouzourène, Pierre Michetti, André L. Blum, Irène E. Corthésy–Theulaz Gastroenterology Volume 115, Issue 4, Pages 891-897 (October 1998) DOI: 10.1016/S0016-5085(98)70261-6 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 In vitro stimulation with rUreB; optimization of culture conditions for splenic CD4+ cells. Cells were obtained from infected mice that were immunized twice at weekly intervals. Optimal antigen concentration for CD4+ cell proliferation was determined after a 5-day incubation using medium only and 1, 10, and 100 μg/mL rUreB. (A) Keyhole limpet hemocyanin (*) was used as control antigen at 10 μg/mL. (B) Secretion of IFN-γ (▩) and IL-4 (■) was measured in culture supernatant after 3 and 5 days of incubation with 10 μg rUreB by ELISA and bioassay, respectively, as described in Materials and Methods. <, Cytokine level below detection. Gastroenterology 1998 115, 891-897DOI: (10.1016/S0016-5085(98)70261-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Specific (A) proliferation and (B and C) cytokine secretion of splenic CD4+ cells after mucosal immunization. Groups of 3 mice were killed at 3, 4, 5, and 6 weeks after infection and groups of 8 mice after 8 weeks. The number of immunizations that each group of mice received is indicated. Filled bars correspond to animals immunized with rUreB and CT; empty bars are control animals receiving CT. (A) [3H]Thymidine incorporation is expressed as mean ± SD from triplicate cultures. Secretion values for (B) IL-4 and (C) IFN-γ for CD4+ T cells isolated from animals immunized with rUreB and CT are expressed as mean ± SD from duplicate measurements. The secretion values of IL-4 for CD4+ T cells isolated from animals immunized with CT only are not indicated because they were all below the detection threshold of 4 pg/mL. <, Nondetectable cytokines. Gastroenterology 1998 115, 891-897DOI: (10.1016/S0016-5085(98)70261-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Anti-rUreB antibodies in the serum of infected/immunized (■) and infected (○) mice. Total (A) IgG, (B) IgG1, and (C) IgG2a concentrations were separately determined by isotype-specific ELISA. Each value represents the mean of sera from 3 mice ± SD assessed at a serum dilution of 1:200 except 3 weeks after the last immunization, when sera from 8 mice were analyzed. The titers of anti-UreB IgG1 and IgG2a antibodies in the serum of the infected/immunized mice at death were 4.6 ± 0.3 and 2.4 ± 0.1, respectively. Gastroenterology 1998 115, 891-897DOI: (10.1016/S0016-5085(98)70261-6) Copyright © 1998 American Gastroenterological Association Terms and Conditions