Volume 141, Issue 1, Pages 186-196.e1 (July 2011) Dendritic Cells Prevent Rather Than Promote Immunity Conferred by a Helicobacter Vaccine Using a Mycobacterial Adjuvant  Iris Hitzler, Mathias Oertli, Burkhard Becher, Else Marie Agger, Anne Müller  Gastroenterology  Volume 141, Issue 1, Pages 186-196.e1 (July 2011) DOI: 10.1053/j.gastro.2011.04.009 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 CAF01 adjuvant elicits protective immunity against H pylori. (A) Timetable of CAF01 and CT immunizations. (B) H pylori colonization levels as assessed by colony count assay (colony-forming units) for mice vaccinated orally with CT or with CAF01 adjuvant via different routes (ip, intraperitoneal; sc, subcutaneous; in, intranasal; po, oral) as well as for infected controls (inf). Horizontal lines represent median values. Combined data from 2 to 4 independent studies are shown. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Protection against H pylori (HP) requires Th1-biased immunity. (A–C) Relative levels of H pylori–specific serum IgG (A), mucosal IgA (B), and mucosal IgG (C) in immunized, infected, and uninfected mice, measured by enzyme-linked immunosorbent assay and represented as optical density values. (D) Serum and (E) mucosal H pylori–specific IgG1 and IgG2c levels of the same mice (left panels), as well as IgG2c/IgG1 ratios (right panels) for mice with antibody levels above baseline. (F) Gastric IFN-γ and IL-17 transcript levels as determined by real-time reverse transcription polymerase chain reaction, normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Groups of 4–10 animals were examined in panels A–F; data are shown as mean ± SEM. *Not detectable. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 T cells but not B cells are required for CAF01-induced protection. (A–B) Colonization levels of (A) JHT−/− and (B) MHC class II−/− mice that were vaccinated with either CT (left panels) or CAF01 SC (right panels) or remained unvaccinated (inf) in comparison to wild-type mice. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 CAF01 vaccine-induced protection is accompanied by gastric infiltration of various immune cell types. (A) Colonization levels of the mice analyzed with respect to gastric infiltration in panels B–F. (B–F) Gastric immune cell infiltration in % of total stomach cells (left panels) and representative flow cytometry plots (right panels) of CT-immunized, CAF01-immunized, and infected-only (inf) mice. Leukocytes (CD45+, B), CD4+ T cells (C), activated memory CD4+ T cells (CD44+CD62L−; D), mast cells (c-Kit+; E), and neutrophils (Ly6-G+; F) are shown. In panel D, the plots show the CD44+CD62L− fraction of all gastric CD4+ cells. Panels A–F show data from 2 combined experiments. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Mixed Th1/Th17 responses against H pylori are elicited by a CAF01-adjuvanted vaccine and both Th subsets are required for protection. (A) IFN-γ– and IL-17–producing CD4+ T cells in % of all stomach cells in CAF01-immunized, infected, and uninfected wild-type mice as determined by intracellular cytokine staining (pooled data from 2 independent experiments; upper panels). Representative flow cytometry plots are also shown (lower panels). (B) IFN-γ– and IL-17–positive fractions of total CD4+ T cells in MLN of CAF01-immunized, infected, and uninfected mice. (C) Colonization levels of wild-type, IL-23p19−/−, IL-12p35−/−, and IL-12/23p40−/− mice immunized SC with CAF01, relative to infected-only controls. (D, E) Gastric infiltration in percent of total stomach cells of leukocytes (CD45+), activated memory CD4+ T cells (CD44+CD62L−), mast cells (c-Kit+), and neutrophils (Ly6-G+), (D) as well as IFN-γ– and IL-17–producing CD4+ T cells (E) of the mice shown in panel C. (F) Real-time reverse transcription polymerase chain reaction results, normalized to glyceraldehyde-3-phosphate dehydrogenase, for IFN-γ and IL-17 expression in immunized wild-type, IL-23p19−/−, IL-12p35−/−, and IL-12/23p40−/− mice relative to infected-only (inf) wild-type controls. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Treg depletion during challenge infection improves vaccine-induced protection; DCs from H pylori–infected mice induce Treg ex vivo. (A) Colonization levels of FoxP3-EGFP-DTR tg and wild-type mice vaccinated SC with CAF01, relative to infected controls; all mice received DT. (B–D) Gastric infiltration in percent of total stomach cells of leukocytes (CD45+), activated memory CD4+ T cells (CD44+CD62L−), mast cells (c-Kit+), and neutrophils (Ly6-G+; B) as well as IFN-γ– and IL-17–producing CD4+ T cells (C–D) for the mice shown in panel A. Proportions of FoxP3+ (E), and IL-10+ (F) CD4+ T cells converted from naïve CD4+CD25− T cells upon coculture with MLN-derived DCs from immunized, infected, or uninfected mice or cultured without DCs. Pooled data from 3 independent experiments are shown in panel E. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Vaccination outcomes improve upon DC depletion. (A) Colonization levels of CT-immunized, CAF01-immunized, and infected-only CD11c-DTR tg (tg) compared to wild-type (wt) mice, treated (+DT) or not with DT every 2 days during challenge infection. (B) Gastric infiltration of leukocytes (CD45+), memory CD4+ T cells (CD44+CD62L−), mast cells (c-Kit+), and neutrophils (Ly6-G+) in immunized CD11c-DTR tg and wild-type mice, treated (+DT) or not with DT. (C) Giemsa-stained histological sections of representative CT-immunized and CT-immunized/DC-depleted mice, illustrating immune cell infiltration into the gastric mucosa. (D–E) Real-time reverse transcription polymerase chain reaction results, normalized to glyceraldehyde-3-phosphate dehydrogenase, for (D) IFN-γ and (E) IL-17 expressions in CD11c-DTR tg and wild-type immunized and/or infected mice, treated (+DT) or not with DT. Pooled data from 3 independent experiments are shown. Gastroenterology 2011 141, 186-196.e1DOI: (10.1053/j.gastro.2011.04.009) Copyright © 2011 AGA Institute Terms and Conditions