Foxp3+ T Cells Regulate Gastric Inflammation and Helicobacter pylori Colonization In Vivo

GASTROENTEROLOGY 2006;131:525–537

CD25ⴙ/Foxp3ⴙ T Cells Regulate Gastric Inflammation and Helicobacter pylori Colonization In Vivo ROLAND RAD,* LENA BRENNER,* STEFAN BAUER,‡ SUSANNE SCHWENDY,‡ LAURA LAYLAND,‡ CLARISSA PRAZERES DA COSTA,‡ WOLFGANG REINDL,* ANAR DOSSUMBEKOVA,* MATHIAS FRIEDRICH,* DIETER SAUR,* HERMANN WAGNER,‡ ROLAND M. SCHMID,* and CHRISTIAN PRINZ* *Second Department of Internal Medicine and Gastroenterology, Technical University of Munich, Munich, Germany; and ‡Institute of Medical Microbiology and Immunology, Technical University of Munich, Munich, Germany

Background & Aims: Helicobacter pylori infects more than half of the world’s population. In contrast to most other pathogens, the microbe persists for the virtual life of its host. It is unclear why the immune system is unable to eliminate the infection, but recent studies suggested that CD4ⴙ/CD25ⴙ/Foxp3ⴙ regulatory T cells may be involved in this process. Methods: By using a mouse model of infection and gastric biopsies from 108 patients, we performed a detailed descriptive and functional characterization of the Helicobacter-induced CD25ⴙ/Foxp3ⴙ T-cell response. Results: In C57BL/6 mice, H pylori induced a marked gastric Foxp3ⴙ T-cell response, which increased over several months together with the severity of inflammation, until a stable homeostatic situation became established. Accordingly, in Helicobacter-infected patients, but not in uninfected individuals, large numbers of gastric Foxp3ⴙ T cells were detected immunohistochemically. To define the functional in vivo relevance of this response, CD25ⴙ cells were depleted systemically in mice by using an antiCD25 monoclonal antibody (PC61). Already 4 weeks after infection, PC61-treated mice, but not untreated animals, developed a severe gastritis with heightened cytokine expression and increased numbers of mucosal T cells, B cells, and macrophages. This was accompanied by increased titers of H pylori-specific IgG1 and IgG2c antibodies in the sera of PC61-treated mice. This increased gastric inflammatory response in CD25-depleted mice was associated with reduced bacterial loads. Conclusions: CD25ⴙ/Foxp3ⴙ T cells actively participate in the immune response to H pylori. In vivo depletion of these cells in infected mice leads to increased gastric inflammation and reduced bacterial colonization.

elicobacter pylori has colonized the human stomach since the earliest times of human evolution.1 In 10%–20% of infected individuals, the H pylori–induced chronic gastric inflammation progresses to peptic ulcer, mucosa-associated lymphoid tissue lymphoma, or gastric

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adenocarcinoma.2– 4 Infection occurs mainly in childhood, and unlike other mucosal pathogens, H pylori persists for the virtual life of its host.5,6 The reasons for this extraordinary persistence are poorly understood. Infected patients as well as different mouse strains challenged with H pylori exhibit gastric inflammation as well as a marked systemic humoral immune response.7,8 Despite the severe gastric inflammatory response, the immune system is not able to eliminate the bacterium. Immunization experiments in animal models, however, have shown that microbial eradication can be achieved by sufficient amplification of the Helicobacter-induced Th1 or Th2 immune response.9 It is currently poorly understood why this protective immunity does not arise under physiological conditions. Regulatory T (Treg) cells play a central role in the negative control of pathological as well as physiological immune responses. They are crucial for the maintenance of self-tolerance, with their dysfunction leading to severe or even fatal autoimmune diseases.10,11 Information on the role of Treg cells in the control of immune responses against infectious agents is still limited. However, recent studies implicate a function of Treg cells in the homeostasis of immune responses to several microbial infections.12 In this context, pathogen-specific Treg cells have been shown to down-regulate the Th1 response toward Bordetella pertussis and Leishmania major, leading to prevention or retardation of pathogen eradication.13,14 Several subsets of CD4⫹ T lymphocytes with regulatory properties have been described, including T regulaAbbreviations used in this paper: FACS, fluorescence-activated cell sorter; IL, interleukin; mAb, monoclonal antibody; PBS, phosphatebuffered saline; RT-PCR, reverse-transcription polymerase chain reaction; TNF, tumor necrosis factor; TGF, transforming growth factor; Treg, regulatory T cell. © 2006 by the American Gastroenterological Association Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.05.001

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tory-1 (Tr1) cells, Th3 cells, and thymus-derived CD4⫹CD25⫹ Treg cells.10 Tr1 and Th3 cells produce high amounts of interleukin (IL)-10 or transforming growth factor & beta (TGF-␤), respectively. Their suppressive activity is mainly mediated by these immunosuppressive cytokines and therefore cell-contact independent.10 The best characterized Treg cells are CD4⫹/ CD25⫹ T cells, which constitute about 5%–10% of peripheral mouse and human CD4⫹ T cells. They have been termed natural Treg cells because they arise during the normal process of maturation in the thymus and survive in the periphery as Treg cells. Their repertoire of antigen specificities is as broad as that of naive T cells, and they are capable of recognizing self- and nonselfantigens.10 They suppress proliferation and cytokine production of effector T cells in a contact-dependent manner in vitro.15 Furthermore, CD80 and CD86 seem to be essential in CD25⫹ cell-mediated suppression in vitro because their absence from effector T cells results in notably reduced susceptibility to suppression. It has been shown that CD80/86 expressed on effector T cells transmit a suppressive signal in these cells after engagement by the Treg cell-surface molecule cytotoxic T-lymphocyte–associated antigen 4 in vitro and in vivo.15 Recently, the forkhead/winged helix transcription factor Foxp3 has been identified as a marker of CD4⫹/CD25⫹ Treg cells because it plays a key role for their development and function.16 –18 CD4⫹/CD25⫹ T cells express Foxp3, whereas other leukocytes do not.16,17 Foxp3-deficient mice fail to develop CD4⫹/CD25⫹ T cells and succumb to a variety of autoimmune diseases, which can be prevented by transfer of normal CD4⫹/CD25⫹ T cells. Retroviral transduction or transgenic expression of Foxp3 in CD4⫹/CD25⫺ T cells phenotypically and functionally converts to natural Treg-like cells, which are able to suppress proliferation of effector T cells in vitro and the development of autoimmune diseases in vivo.16 –18 In addition, transduction of CD4⫹/CD25⫺ T cells with Foxp3 up-regulates the expression of Treg-cell–associated molecules, such as CD25 (IL-2 receptor alpha chain), cytotoxic T-lymphocyte–associated antigen 4, and glucocorticoid-inducible tumor necrosis factor receptor.16 Thus, Foxp3 is considered a “master control gene” for the development and function of CD4⫹/CD25⫹ T cells.10,19 Recent studies provided evidence for the development of immunosuppressive H pylori–specific Treg cells in infected hosts.20 –22 Here we describe the nature and dynamics of H pylori–induced CD25⫹/Foxp3⫹ T-cell responses in humans and mice and show the functional relevance of this response in vivo. H pylori–induced CD25⫹/Foxp3⫹ T cells play an important role in the

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negative control of antimicrobial immunity, thereby limiting the extent of H pylori–induced immunopathology. At the same time, however, this response is associated with increased bacterial colonization.

Materials and Methods Bacteria The mouse-adapted H pylori strain SS1, kindly provided by Richard Ferrero (Institute Pasteur, Paris), was used for mouse inoculation experiments.8 Bacteria were grown at 37°C under microaerophilic conditions (85% N2, 10% CO2, and 5% O2) on Wilkins Chalgren agar plates (WC agar; Oxoid, Basingstoke, England). Agar plates were supplemented with 10% horse serum (Gibco, Eggenstein, Germany), a standard Helicobacter-selective antibiotic mixture (DENT supplement; Oxoid, Basingstoke, England). For quantitative H pylori culture after recovery of bacteria from the mouse stomach, WC agar plates were further supplemented with Bacitracin (200 ␮g/mL), Nalidixic acid (10 ␮g/mL), and Polymyxin B (3 ␮g/mL) as described previously.23

Infection of Mice C57BL/6 mice were purchased from Harlan Winkelmann (Borchen, Germany). All animals were kept under specific pathogen-free conditions. Experiments were conducted according to the German animal protection law and were approved by the government of Upper Bavaria. Six-week-old male C57BL/6 mice were infected with the mouse-adapted H pylori strain SS1 by gastric intubation with a feeding needle (.1 mL of a suspension at an OD590 of 5/mL, corresponding to ⬃5 ⫻ 108 organisms). Bacteria were harvested directly from 24- to 48-hour plate cultures. Infection was repeated twice on day 3 and 5. Control mice received Brucella broth alone. Mice remained infected for 30, 60, 140, and 180 days. After anesthesia with Ketamin (provided by Dr E. Gräub AG, Bern, Switzerland) and Xylacin (Xylacinhydrochloride; provided by Bayer, Leverkusen, Germany), blood was collected by intracardial injection and archived at ⫺20°C. Subsequently, mice were killed by cervical dislocation, and gastric tissue was processed for further analyses as described later.

Quantitative H pylori Culture From Gastric Tissue An antral strip was placed in .5 mL Brucella broth, weighed, and vortexed for 10 minutes; 10, 100, and 1000 fold dilutions were prepared and plated onto Helicobacter-selective agar as described earlier. Bacterial counts were determined after 5 days of growth and normalized to the weight of corresponding stomach pieces.

Mouse Gastric Tissue Analyses After sacrificing the mice, stomachs were removed and washed with sterile water. A longitudinal segment along the greater curvature from the esophagus to the intestine was used

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for histological and immunohistochemical examinations. To allow visualization of the whole tissue strip on 1 microscopic section, the tissue segment was ligated at the esophagus and the duodenum and stretched in a horizontal position for formalin fixation and paraffin embedding. The remaining stomach was dissected along the small curvature into 2 identical pieces. One part was used for quantitative H pylori culture, as described earlier. The other part was snap frozen in liquid nitrogen, homogenized, and stored at ⫺80°C until RNA and DNA isolation.

In Vivo Depletion of CD25ⴙ T Cells The hybridoma producing anti-mouse CD25 monoclonal antibody (mAb) (clone PC61, rat IgG1) was obtained from American Type Culture Collection. Culture supernatant was collected, and PC61 was affinity purified by using protein G columns (Amersham Biosciences, Freiburg, Germany). Sixweek-old male C57BL/6 mice were treated intraperitoneally with either phosphate-buffered saline (PBS) or a single, 1-mg dose of anti-CD25 mAb (PC61). The success of depletion was analyzed after 3 and 30 days in a subset of mice by fluorescence-activated cell sorter (FACS) analysis of isolated splenocytes.

Flow Cytometry Fluorescence staining of isolated mouse splenocytes was performed as described previously.24 Antibodies were purchased from BD-Biosciences and were as follows: anti-CD4 mAb (allophycocyanin labeled), anti-CD25 mAb (phycoerythrin labeled), and anti-CD45RB mAb (PE labeled). Fluorescence was analyzed by using a FACSCalibur (Becton Dickinson, Heidelberg, Germany) flow cytometer and CELLQuest software (Becton Dickinson).

Patients and Biopsies Five gastric antral biopsies were collected from 108 German patients after receiving informed consent. Patients underwent endoscopy because of abdominal complaints. The study was approved by the local ethics committee at the Technical University of Munich. Infection with H pylori was investigated by histology and by vacAs1/s2 polymerase chain reaction (PCR). Sixty-five patients were H pylori positive (31 male; 34 female; mean age, 58.2 [39 – 65 years]), and 43 persons were not infected (20 male; 23 female; mean age, 57.1 [35– 68 years]). Patients taking nonsteroidal anti-inflammatory drugs or receiving antisecretory therapy as well as patients with ulcer disease or gastric carcinoma were excluded from the study. Two antral and corpus sections were paraffin embedded for further H&E and immunohistochemical staining. Histopathological evaluation was performed according to the Sydney classification system. The remaining antral biopsy specimens were stored in liquid nitrogen and homogenized before DNA/RNA isolation. After tissue lysation with proteinase K, DNA isolation was performed with QIAamp tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s in-

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structions. RNA was isolated by the phenol/chloroform method.

Histology and Determination of Gastric Inflammatory Scores in Mouse Gastric Tissue H&E staining was performed according to standard procedures. The extent of gastric inflammation was assessed after H&E staining based on the following criteria: grade 0, no change from normal tissue; grade 1, unifocal mild cellular infiltration in the lamina propria; grade 2, few (2–5) multifocal lesions of moderate inflammatory cell infiltrates in the lamina propria; grade 3, lesions involved a large area of the mucosa or were more frequent than grade 2; and grade 4, diffuse and severe inflammation, transmural inflammation invariably present. Sections were examined independently and blinded by 2 persons, yielding identical results in almost all cases.

Immunohistochemistry in Mouse and Human Gastric Tissue Formalin-fixed paraffin-embedded tissue sections were deparaffinized with xylene and ethanol. Pretreatment was performed as follows: microwave for 20 minutes in EDTA, pH ⫽ 8 (for human Foxp3 staining and mouse CD3 staining); steampressure boiling for 7 minutes (for H pylori staining and mouse B220 staining); and enzymatic digestion with .05% trypsine for 20 minutes at 37°C (for mouse F4/80 staining). Subsequently, sections were incubated with one of the following primary antibodies for 60 –90 minutes: 1:40 diluted mouse anti-human Foxp3 IgG1 mAb (clone 236A/E7) [Abcam, Cambridge, UK], 1: 200 diluted rat anti-mouse CD3 IgG1 mAb (clone CD3-12) [Linaris, Wertheim-Bettingen, Germany], 1:50 diluted rat anti-mouse CD45R (B220; clone RA3-6B2b) IgG2a mAb (Linaris), 1:10 diluted rat anti-mouse F4/80 (clone A3-1) IgG2b mAb [Serotec, Oxford, UK], 1:200 diluted rabbit anti–H pylori mAb [Dako, Hamburg, Germany], and control sections were incubated without primary mAb. The streptavidin-biotin method was used for further staining. Sections were therefore incubated with rat anti-mouse mAb using DAKO-LSAB-KITT (Dako) for human Foxp3 staining; rabbit anti-rat mAb using Vectastain ABC-KIT (Vector Laboratories, Burlingame, CA) for mouse CD3, B220, and F4/80 staining; and goat anti-rabbit mAb by using DAKO-LSAB-KITT (Dako). Quantification of leukocyte subsets was performed at the antrum-corpus junction. Therefore, the number of positively stained cells was counted in 6 representative visual fields at a magnification of 1000⫻ and expressed as cells/mm2.

Real-Time PCR and Reverse-Transcription PCR Quantitative expression analysis from human and mouse gastric tissue was performed by real-time quantitative TaqMan reverse-transcription (RT-PCR) as described previously.25 In brief, RNA was transcribed into complementary DNA by using MultiScribe reverse transcriptase (Applied Bio-

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Table 1. Sequences of Primers and Probes Used for Real-Time Quantitative PCR or RT-PCR Reverse primer

Fluorogenic probe

ⴱMouse ␤-act-DNA Mouse ␤-actin Mouse Foxp3 Mouse IL-12R␤2

5=-TTCAACACCCCAGCCATGTA-3= 5=-CGTGAAAAGATGACCCAGATCA-3= 5=-TTCGAGGAGCCAGAAGAGTTTC-3= 5=-ACCTGAGCTCTGCGAAATTCA-3=

Forward primer

5=-TGTGGTACGACCAGAGGCATAC-3= 5=-CACAGCCTGGATGGCTACGT-3= 5=-GGGCCTTGCCTTTCTCATC-3= 5=-CCTGGGCTGTAGGCTGCTT-3=

Mouse IFN-␥

5=-CAGCAACAGCAAGGCGAAA-3=

5=-CTGGACCTGTGGGTTGTTGAC-3=

Mouse IL-4 Mouse IL-12A

5=-CGCCATGCACGGAGATG-3= 5=-ACTAGAGAGACTTCTTCCACAACAA GAG-3= 5=-CTACAGCACCAGCTTCTTCATCA-3= 5=-CATCTTCTCAAAATTCGAGTGACAA-3= 5=-ACAAGTCGGAGGCTTAATTACACAT-3= 5=-CCAGAGCCACATGCTCCTAGA-3=

5=-CGAGCTCACTCTCTGTGGTGTT-3= 5=-GCACAGGGTCATCATCAAAGAC-3=

5=-TAGCCATCCAGGCTGTGCTGTCCC-3= 5=-TTTGAGACCTTCAACACCCCAGCCA-3= 5=-CAAGCACTGCCAAGCAGATCATCTCCT-3= 5=-TACCGACGCTCTCAAAACTCACATC CAA-3= 5=-AGGATGCATTCATGAGTATTGCCAAGTT TGA-3= 5=-TGCCAAACGTCCTCACAGCAACG-3= 5=-AGCTGCCTGCCCCCACAGAAGA-3=

Mouse Mouse Mouse Mouse

IL-12B TNF-␣ IL-6 IL-10

Mouse TGF-␤1 Mouse CD4 Mouse CD8 Mouse CD25 Human GAPDH Human FOXP3 ⴱH pylori UreB-DNA

5=-TCAAAGGCTTCATCTGCAAGTTC-3= 5=-CCAGCTGCTCCTCCACTTG-3= 5=-TTGCCATTGCACAACTCTTTTC-3= 5=-GGTCCTTTGTTTGAAAGAAAGTC TTC-3= 5=-GCTCTTGTGACAGCAAAGATAACAA-3= 5=-CGCCCCGACGTTTGG-3= 5=-AACAGGAAAGAGGAGGTGGAGTT-3= 5=-CCCTTGCAACAGGCTGGTA-3= 5=-GCTACCACAGGAGCCGAAAG-3= 5=-TGGGCTTGCCTTCCTGTCT-3= 5=-GAGTGAGACTTCCTGCCCCATA-3= 5=-TCTCCGTCATTGCAGTTGTTTC-3= 5=-GGGAAGCTTGTCATCAATGGA-3= 5=-CGCCCCACTTGATTTTGG-3= 5=-ATGCCTCCTCTTCTTCCTTGAA-3= 5=-GGGCATCCACCGTTGAGA-3= 5=-CAACAAATCCCTACAGCTTTTGC-3= 5=-CCATCAGCAGGGCCAGTT-3=

5=-CATCATCAAACCAGACCCGCCCA-3= 5=-CCTGTAGCCCACGTCGTAGCAAACCA-3= 5=-TTCTCTGGGAAATCGTGGAAATG-3= 5=-CTGCGGACTGCCTTCAGCCAGG-3= 5=-CCACGTGGAAATCAACGGGATCAGC-3= 5=-TGGGTGTTCAAAGTGACCTTCAGTCCG-3= 5=-TTTGCAAATGTCCCAGGCCGCT-3= 5=-CCACCACAGACTTCCCACAACCCAC-3= 5=-ATCCCATCACCATCTTCCAGGAGCG-3= 5=-CCCATGCCACCATCGCAGCTG-3= 5=-AGCGGTGTAACAACGATGATTGGTGGC-3=

NOTE. Asterisks indicate primer and probe sets used for quantitative DNA-PCR. The remaining primers and probes were used for expression analysis by quantitative RT-PCR. Probes were labeled with the reporter dye FAM at the 5=, and the quencher dye TAMRA at the 3= end.

systems, Weiterstadt, Germany), according to the manufacturer’s instructions. To determine absolute messenger RNA copy numbers, standard curves were generated by using plasmid dilution series containing the corresponding target sequence. Therefore, PCR products were cloned into TOPO TA vector (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Analyzing plasmid dilution series, the threshold cycle value was a linear function of the starting complementary DNA input over a wide dynamic range, with coefficients of correlation of .95 to 1. Cytokine messenger RNA copy numbers were normalized to GAPDH or ␤-actin copies for human and mouse tissues, respectively. TaqMan primers (MWG, Ebersberg, Germany) and probes (Applied Biosystems, Weiterstadt, Germany) were designed to span exon junctions or to lie in different exons to prevent amplification of genomic DNA. Primer and probe sequences are shown in Table 1. Probes were labeled with the reporter dye FAM at the 5= and the quencher dye TAMRA at the 3= end. For quantification of bacterial colonization, H pylori ureB– DNA amounts were measured quantitatively in mouse gastric tissues. After DNA isolation, ureB DNA copy numbers were determined by real-time TaqMan PCR and normalized to corresponding ␤-actin DNA copies. ␤-actin primers and probes were chosen to lay in introns and are referred to as ␤-actin–DNA primer or probe in Table 1.

Detection of Autoantibodies to Gastric Hⴙ/Kⴙ–Adenosine Triphosphatase in Mouse Sera Antiparietal cell antibodies were detected by a modified standard immunohistochemical procedure, as described

previously.26 Staining was performed on frozen or paraffinembedded mouse gastric sections. Formalin-fixed paraffin-embedded tissue sections were deparaffinized with xylene and ethanol, whereas cryostat sections were used after fixation with acetone. Sections were incubated with 1:10 and 1:100 diluted sera from PC61-treated mice and the streptavidin-biotin method was used for subsequent staining by a rat anti-mouse mAb (DAKO-LSAB-KITT). The monoclonal antibody 2B6 (MBL, Woburn, MA), specific for the ␤ subunit of the H⫹/ K⫹–adenosine triphosphatase (ATPase) served as a control.

H pylori–Specific IgG1 and IgG2c Antibodies in Mouse Sera H pylori–specific serum antibody titers were determined by enzyme-linked immunosorbent assay. Ninety-sixwell microtiter plates were coated with 50 ␮L of 50 ␮g/mL soluble H pylori extracts overnight at 4°C. Wells were then blocked with 1.0% nonfat milk in PBS and washed 3 times with PBS 0.05% Tween-20. Serial dilutions of mouse sera (1:10 to 1:1000) were then added to the wells and incubated for 2 hours at 37°C. Shown are values obtained at a serum dilutions of 1:1000. Bound-specific antibodies were detected with biotin-conjugated anti-mouse IgG2c (IgG2ab; clone 5.7; BD Pharmingen) or IgG1 (clone 85-1; BD Pharmingen) at a dilution of 1:1000. After incubation for 20 minutes with streptavidin horseradish peroxidase, the color reaction was developed by incubation at 37°C for 5 minutes with tetramethylbenzidine solution. The reaction was stopped with 10 N H2SO4, and the OD450 was determined in an ELISA reader.

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Statistical Analysis Statistical analysis was performed by using a Mann– Whitney rank sum test or a Student t test, depending on the dataset of concern. The tests applied are indicated in the figure legends. Values of P ⬍ .05 were considered to be significant.

Results Dynamics of Gastric Foxp3ⴙ T-Cell Responses in a Mouse Model of Chronic H pylori Infection To investigate if H pylori induces a Foxp3⫹ T-cell response, we chronically infected 6-week-old male C57BL/6 mice with the mouse-adapted H pylori strain SS1. Over several months of infection, mice developed a robust inflammatory response, which, however, is not capable to eliminate the bacterium; 30, 60, 140, and 180 days after infection, mice were sacrificed. Gastric expression of Foxp3 and of pro- and anti-inflammatory cytokines was analyzed quantitatively by real-time RT-PCR. As shown in Figure 1, infection of C57BL/6 mice with H pylori resulted in increased gastric expression of IL12p40, tumor necrosis factor ␣, and IL-12 receptor ␤2 chain (IL-12R␤2; specific marker of Th1 cells). Expression of IL-4 also increased with the duration of infection. However, absolute IL-4 expression remained very low. Analyzing gastric Foxp3 expression, we found that Foxp3 messenger RNA amounts were absent or very low in noninfected animals (Figure 1). One month after infection, Foxp3 expression increased significantly in H pylori– challenged mice. The extent of this response increased with the duration of infection. Six months after infection, mean gastric Foxp3 expression was almost 100-fold higher than in noninfected mice (Figure 1). In addition to Foxp3, expression of the anti-inflammatory cytokines IL-10 and TGF-␤ increased after H pylori infection (Figure 1). Thus, after H pylori acquisition, gastric pro- and anti-inflammatory responses increase over several months of infection, until a stable homeostatic situation gets established. Systemic In Vivo Depletion of CD25ⴙ T Cells in the Mouse Model of H pylori Infection Naturally occurring Foxp3⫹ T cells have been shown to constitutively express IL-2R␣ (CD25) chain and represent about 10% of peripheral blood CD4⫹ T cells. Foxp3⫹ T cells with a regulatory phenotype are mainly present in the CD4⫹/CD25high population.10 CD25 is predominantly expressed by CD4⫹ T cells but not by other lymphoid cells. Accordingly, it has been shown that anti-CD25 mAb was capable of depleting

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CD25⫹ cells in vivo without altering CD8, B, natural killer, dendritic cell, granulocyte, and macrophage populations.27 We injected 1mg anti-CD25 mAb (PC61) intraperitoneally into C57BL/6 mice. The success of depletion was investigated by flow cytometry by using splenocytes isolated at day 3 and 30 after PC61 application. Three days after injection of the antibody, a reduction of the CD25⫹ T-cell population from more than 10% to ⬃1% was observed (not shown). Infection with H pylori was performed at day 4 after PC61 injection. Figure 2(A and B) shows FACS analysis of splenocytes isolated from non-treated and PC61-treated mice at day 30 after mAb application, respectively. Whereas in the nontreated mouse ⬃11% of CD4⫹ cells were CD25⫹ (Figure 2A), fewer than 1% of CD4⫹ cells are CD25⫹ in the PC61-treated mouse (Figure 2B). Figure 2C shows the average number of splenic CD4⫹/CD25⫹ T cells from 8 treated or nontreated mice. In all mice, a highly significant reduction of the CD4⫹/CD25⫹ T-cell number could be achieved after PC61 treatment. Furthermore, the CD4⫹/CD45RBlow population was also reduced (Figure 2D), reflecting previously reported partial coexpression of CD25 and CD45RBlow on Treg cells.11 Collectively, these results show that PC61 treatment of mice leads to ⬃90% systemic loss of CD25⫹ T cells for at least 30 days. In Vivo Depletion of CD25ⴙ T Cells Leads to Increased Gastric Inflammation in Response to H pylori Infection To investigate the functional relevance of the T-cell response, PC61-treated C57BL/6 mice were infected or not with H pylori. Control mice did not receive PC61 treatment before infection. After 4 weeks, mice were sacrificed, and gastric inflammatory responses were analyzed. Previous studies have shown that in the susceptible BALB/c mouse strain, thymectomy during the critical neonatal period (2– 4 days after birth) results in the development of severe autoimmune gastritis because of the absence of CD4⫹CD25⫹ T cells.28 To exclude spontaneous development of autoimmune gastritis in PC61-treated noninfected mice, gastric inflammatory responses were evaluated in those animals. As expected, no signs of gastritis were observed in noninfected PC61treated C57BL/6 mice 4 weeks after PC61 application (n ⫽ 15). A hallmark of autoimmune gastritis is the development of antiparietal cell antibodies. The presence of those antibodies in mouse sera was investigated in all PC61-treated mice by immunohistochemistry. Whereas application of a control antiparietal cell antibody leads to specific parietal cell staining on mouse stomach tissue (Figure 3C), sera from mice used in the study had no CD25⫹

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positive reaction with mouse gastric parietal cells (representative image in Figure 3D). Analyzing gastric inflammatory responses in nontreated infected mice, a very scarce gastric infiltration with mononuclear cells could be observed 4 weeks after H pylori inoculation. Figure 3A and E show the gastric mucosa at the antrum-corpus junction of a representative mouse, with only few mucosal lymphocytes. In contrast, markedly increased gastric inflammation occured in PC61-treated mice 4 weeks after infection. Figure 3B and F show that large numbers of mononuclear cells infiltrate the gastric lamina propria and submucosa. A hyperplastic thickening of the gastric wall with extensive inflammatory cell infiltrates between the glands can be observed. Already 4 weeks after infection, a partial loss of specialized cells (parietal and chief cells) and replacement with proliferating cells or mucus secreting cells takes place. Figure 3G compares the histological scores in PC61-treated and nontreated mice. To further characterize the nature of the inflammatory response in PC61-treated animals, we investigated the presence of important leukocyte subsets in the gastric mucosa of infected mice. T cells, macrophages, and B cells were detected by immunohistochemical staining of their surface markers CD3, F4/80, and B220, respectively. As shown in Figure 3H–P, highly increased numbers of T cells, macrophages, and B cells were found in H pylori–infected PC61treated mice compared with nontreated animals. Depletion of CD25ⴙ T Cells Leads to Increased Cytokine Expression in the H pylori–Infected Stomach To investigate the influence of PC61 treatment on gastric cytokine expression, we determined mucosal cytokine profiles in H pylori–infected nontreated and PC61treated mice (Figure 4). As expected, anti-CD25 treatment resulted in markedly increased gastric expression of different proinflammatory cytokines (Figure 4). In particular, expression of the Th1-type cytokines, IL-12, tumor necrosis factor ␣, and interferon ␥ was highly increased. Three- to 50-fold higher levels of these cytokines were measured in

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 1. Dynamics of gastric pro- and anti-inflammatory gene expression during chronic H pylori infection of mice. Male C57BL/6 mice were infected with H pylori SS1 and sacrificed at different time points after challenge (30 –180 days). Noninfected control animals (Hp-) received Brucella broth. Gastric expression of cytokines, cytokine receptors, and transcription factors was analyzed by real-time RT-PCR and normalized to ␤-actin expression. Bars indicate the arithmetic mean and standard error of the mean from pooled data including 2 identical experiments with 5 mice per group. The 2-sided P value was calculated by a Student t test. Asterisks indicate significant differences compared with the H pylori–negative controls (*P ⬍ .05; **P ⬍ .01; ***P ⬍ .001).

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Figure 2. Anti-CD25 mAb PC61 depletes CD4⫹CD25⫹ T cells in vivo for at least 30 days. C57BL/6 mice were treated intraperitoneally with either PBS or a single, 1-mg dose of anti-CD25 mAb (PC61). Thirty days later, splenocytes were isolated and prepared for flow cytometry analysis by using the combination of CD4APC and either CD25-PE or CD45RB-PE antibodies. Dot plots from 1 representative untreated and 1 PC61-treated animal are shown in A and B, respectively. Values represent the percentage of cells in each quadrant. Columns in C show the arithmetic mean and standard error of the mean of absolute splenic CD4⫹/CD25⫹ T-cell numbers from groups of PC61-treated and untreated mice (n ⫽ 8). Correspondent values for CD4⫹/CD45RBlow cells are shown in D. P values were calculated by a 2-sided Student t test. Asterisks indicate significant differences (*P ⬍ .01; **P ⬍ .001). Similar results were obtained 3 days after PC61 application.

the gastric mucosa of PC61-treated mice compared with nontreated animals. In accordance, expression of the specific Th1 cell marker IL-12 receptor ␤2 chain (IL-12R␤2) was higher in CD25-depleted animals than in control mice (Figure 4). IL-4 expression did not increase significantly in PC61-treated mice. However, the increased gastritis in PC61-depleted animals was accompanied by heightened expression of the anti-inflammatory cytokines IL-10 and TGF-␤. We further assessed the expression of Foxp3 and of the surface markers CD4, CD8, and CD25 in the gastric mucosa of infected animals. Whereas CD4 and CD8 expression was significantly increased in PC61-treated mice compared with nontreated animals (P ⬍ .001), Foxp3 and CD25 expression was not increased significantly in PC61treated mice. Figure 4J–M shows the relative expression of Foxp3 and CD25 in relation to CD4 and CD8 in the 2 groups. The ratios of Foxp3/CD4, Foxp3/CD8, CD25/CD4, and CD25/CD8 expression were markedly reduced in PC61-treated mice compared with nontreated animals, suggesting an altered balance between regulatory and effector cell responses in PC61-treated mice. Collectively, these results show that upon H pylori infection, pro- and antiinflammatory cytokines are increased in PC61-treated mice compared with nontreated animals. However, the heightened expression of anti-inflammatory cytokines seems not to

be sufficient to fully compensate the altered Foxp3⫹/ CD25⫹ T-cell response in PC61-treated animals, resulting in severe gastric inflammation in these mice. H pylori–Specific Serum Levels of IgG1 and IgG2c as Surrogate Markers for Th2/Th1 Responses Th2 or Th1 responses are associated with an immediate ability to induce immunoglobulin class switching to immunoglobulin (Ig)G1 or IgG2a, respectively.29 In C57Bl/6 mice, the gene encoding IgG2a is deleted. Instead, these mice express the corresponding IgG2c (IgG2ab) isotype, with more than 80% homology in the amino acid sequence to IgG2a.30 To further investigate the consequences of PC61 treatment on the development of immunity to H pylori, we determined H pylori–specific serum IgG1 and IgG2c levels as described previously.31 Figure 5 shows that titers of both H pylori–specific IgG1 as well as IgG2c were increased in CD25-depleted mice compared with nontreated infected animals. Depletion of CD25ⴙ T Cells Leads to Reduced H pylori Colonization Densities We next investigated the consequences of the increased inflammatory response for bacterial colonization. H

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pylori colonization densities were determined 4 weeks after infection in nontreated and PC61-treated mice. Figure 6(A) shows that the number of H pylori colony-forming units/g tissue was significantly lower in PC61-treated mice than in

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control animals. To further evaluate these results, we quantified bacterial ureB-DNA in infected stomachs by real-time PCR. As shown in Figure 6B, lower levels of H pylori ureB-DNA were found in the stomachs of anti-CD25–

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Figure 4. Expression of cytokines and T-cell markers in the stomach of H pylori–infected PC61-treated and nontreated mice. (A–I) messenger RNA expression of cytokines and cytokine receptors in the gastric mucosa of PC61-treated and nontreated mice 4 weeks after H pylori infection. Messenger RNA amounts were determined by real-time TaqMan PCR and are presented as copies per 106 ␤-actin copies. (J and K) Relative ratio of ␤-actin–normalized Foxp3 expression to ␤-actin–normalized CD4 and CD8 expression, respectively. (L and M) Relative ratio of ␤-actin– normalized CD25 expression to ␤-actin-normalized CD4 and CD8 expression, respectively. Bars indicate the arithmetic mean and standard error of the mean from 1 experiment including 10 mice per group. Similar results were obtained in 2 analogous experiments. The 2-sided P value was calculated by a Student t test or a Mann–Whitney rank sum test. Asterisks indicate significant differences (*P ⫽ .05; **P ⬍ .01; ***P ⬍ .001).

treated animals compared with nontreated mice. Thus, the increased inflammatory response in PC61-treated mice is associated with a reduced bacterial burden. FOXP3ⴙ T Cells in the Chronically Infected Human Gastric Mucosa We next investigated whether FOXP3⫹ T cells are present in the human gastric mucosa. FOXP3⫹ cells were detected by immunohistochemistry by using a mouse anti-human FOXP3 mAb (clone 236A/E7).32 Figure 7A and B show the nuclear staining of the forkhead family transcription factor. In noninfected patients, FOXP3⫹ cells were absent in most cases (Figure 7C). Rarely, single FOXP3⫹ cells in the lamina propria or submucosa could be observed. In contrast, large numbers of Foxp3⫹ cells are present in the chronically infected human stomach (Figure 7A and B). Clearly, FOXP3 is not expressed by gastric epithelial cells, which is consis-

tent with the reported restricted expression of FOXP3 within lymphocytes.16,32 We next quantified FOXP3 expression in gastric biopsies from 65 H pylori–infected and 43 noninfected patients. As shown in Figure 7D, FOXP3 expression was absent or very low in noninfected persons. In contrast, up to 50-fold–increased FOXP3 levels were observed in H pylori–positive biopsies.

Discussion Several lines of evidence indicate that during the long common history of humans and H pylori there has been selective pressure on the bacterium to avoid the induction of severe inflammation. On the one hand, intense inflammation, as seen in IL-10 – knockout mice or in immunized mice, leads to dramatic reduction of colonization and even clearance of Helicobacter species from the stomach.33 On the other

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 3. Severe gastritis but no autoimmune gastritis in H pylori–infected PC61-treated mice. (A and B and E and F) H&E-stained stomach sections from representative nontreated and PC61-treated animals 4 weeks after H pylori infection. Only few scattered leukocytes can be seen in the submucosa and lamina propria (arrowhead) of a nontreated mouse 4 weeks after infection. Arrows indicate lymphocytes between the glands. Severe gastric inflammation is present in the PC61-treated mouse with dense leukocytic infiltration in the submucosa and mucosa (arrowhead). Leukocytic aggregates between the glands (arrows) are spanning the entire width of the mucosa. A concomitant hyperplasia with thickening of the gastric wall as well as gastric atrophy can be observed. (G) Histological scoring of gastritis in nontreated and PC61-treated animals. (H–P) Leukocyte subsets were stained immunohistochemically in the gastric mucosa of nontreated and PC61-treated animals 4 weeks after H pylori infection. Representative sections are shown at a magnification of 200⫻. Mucosal T cells (H–J), macrophages (K–M), and B cells (N–P) were stained with anti-F4/80, anti-CD3, and anti-B220 antibodies, respectively. The number of stained cells per mm2 are presented in J, M, and P. Each triangle represents one animal. Shown is 1 experiment including 10 mice per group. Similar results were obtained in 2 analogous experiments. The 2-sided P value was calculated by a Mann–Whitney U test. (C) Immunohistochemical staining of H⫹/K⫹-ATPase on paraffinembedded mouse stomach sections by using the monoclonal Ab 2B6. (D) Representative immunohistochemical staining of H⫹/K⫹-ATPase by using serum from a PC61-treated animal at a dilution of 1:10. None of the sera from PC61-treated mice showed reactivity with parietal cells. Photographs are taken at 100⫻ (A–D), 200⫻ magnification (E–O), or 400⫻ (cut-out in C).

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Figure 5. H pylori–specific IgG2c and IgG1 levels in PC61-treated and nontreated mice. H pylori–specific immunoglobulin titers were determined in 1:1000 diluted sera of H pylori–infected mice 4 weeks after infection. Bars indicate the arithmetic mean and standard error of the mean from 1 experiment including 10 mice per group. Similar results were obtained in 2 analogous experiments. The 2-sided P value was calculated by a Mann–Whitney U test. Asterisks indicate significant differences (**P ⫽ .005; ***P⬍ .001).

hand, severe inflammation often progresses to gastric atrophy with destruction of the glandular structure and function. This results in loss of the bacterial niche and often in the disappearance of H pylori from stomachs with atrophic gastritis.34 Thus, there seems to be a need for H pylori to circumvent induction of strong inflammatory responses. The mechanisms by which H pylori represses the host immune system are just beginning to be understood.6 Regulatory T cells actively engage in the negative control of a broad spectrum of immune responses to self, quasi-self, or nonself-antigens as in tumor immunity, organ transplantation, allergy, and microbial immunity.10 –12 Recent studies have shown that H pylori induces a CD4⫹/CD25⫹ T-cell response.20 –22 We confirm and extend those findings by providing a descriptive and functional characterization of the H pylori–induced CD25⫹/

Figure 6. Decreased bacterial colonization in PC61-treated mice. Nontreated or PC61-treated mice were sacrificed 4 weeks after H pylori infection, and bacterial colonization density was determined by quantitative culture (A) or quantification of bacterial ureB DNA (B). In A, values are expressed as colony-forming units/g stomach tissue. Each triangle represents 1 animal. Quantification of bacterial ureB DNA in B was performed by real-time PCR and normalized to ␤-actin DNA amounts. P values were calculated by a Student t test. One of 3 experiments yielding similar results is shown.

Figure 7. FOXP3⫹ T cells in the H pylori–infected human gastric mucosa. (A and B) Immunohistochemical staining of the transcription factor FOXP3. Staining is clearly localized to the nucleus (insert in B). FOXP3⫹ cells are present in the H pylori–infected human gastric mucosa (A and B) but not in the uninfected stomach (C). Glandular epithelial cells are FOXP3 negative. (E) FOXP3 expression in the gastric mucosa of H pylori– infected (n ⫽ 43) and noninfected patients (n ⫽ 65). FOXP3 expression was analyzed by real-time quantitative RT-PCR and normalized to GAPDH expression. Bars within the box plots represent median values (50th percentile). The ends of the bars indicate the 25th and 75th percentiles, the 10th and 90th percentiles are represented by error bars, and the 5th and 95th percentiles are shown by filled circles. The P value was calculated by a Mann–Whitney U test. Photographs were taken at 100⫻ (A and D), 200⫻ (B), and 1000⫻ (cut-out in B).

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Foxp3⫹ T-cell response. In a murine infection model, we analyzed the nature and dynamics of the H pylori– induced Foxp3⫹ T-cell response. By means of depletion experiments, we showed the importance of CD25⫹ T cells for the host as well as for the microbe in vivo. In mice, early upon bacterial inoculation, Foxp3⫹ T cells infiltrated the site of infection, indicating that H pylori induces the trafficking of these cells into the gastric mucosa. This response increased with the duration of infection (over several months) and with the magnitude of gastric inflammation until a stable homeostatic situation became established. In accordance with these findings, large numbers of FOXP3⫹ T cells were detected in the H pylori–infected but not in the noninfected human gastric mucosa. The physiological importance of these results is supported by the finding that already at Treg/ T-effector cell ratios of 1:10, Treg cells inhibit up to 60% of proliferation and cytokine secretion from stimulated effector cells in vitro.35 To investigate the functional relevance of C25⫹ T cells in the context of H pylori infection in vivo, we depleted CD25⫹ T cells systemically in mice before challenge. As a consequence, the proportion of CD25⫹ cells among CD4⫹ cells was reduced from ⬎10% to ⬃1% for at least 30 days. In the absence of CD25⫹ T cells, infection with H pylori resulted in a severe gastritis as soon as 4 weeks after infection. In contrast, only scarce mononuclear cell infiltrates were observed in nondepleted infected mice at this time point. Thus, CD25⫹ T cells seem to be important for the negative control of the H pylori–induced gastric pathogenesis. These results are further supported by recent findings of Raghavan et al,36 who investigated H pylori infection in T-cell– deficient nu/nu mice.36 In that report, infected T-cell– deficient mice that were reconstituted with CD25⫺ T cells had more severe gastritis than animals reconstituted with nonsorted T-cell populations (consisting of CD25⫺ and CD25⫹ T cells36). The mechanisms of suppression by CD4⫹/CD25⫹ T cells have been addressed by many investigators. CD4⫹/ CD25⫹ T cells produce rather low amounts of cytokines, and their primary suppressive effect in vitro is strictly cell-contact dependent.15 However, in vivo, soluble factors such as IL-10 seem to be important for tolerance induction by CD4⫹/CD25⫹ T cells as well.11 On the basis of in vivo observations and in light of the relative physiological scarcity of CD25⫹ T cells, it has been proposed that in vivo CD25⫹ cells would use mechanisms to amplify their suppressive action.15 Indeed, recent work has shown that human CD4⫹/CD25⫹ T cells can confer a suppressive phenotype to conventional CD4⫹ T cells in a contact-dependent manner (eg, by

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inducing the development of IL-10 –producing Tr1 cells).37,38 In support of this, we found a strong positive correlation between Foxp3 and IL-10 expression in the H pylori–infected gastric mucosa of mice and humans (data not shown). For the host, the homeostatic situation between suppressor and effector cell responses established after months of infection may be a favorable balance between microbe-induced pathology and immune-mediated pathology. However, another important consequence of the H pylori induced CD25⫹ T-cell response may be an enhanced bacterial survival. We observed decreased bacterial colonization densities in CD25-depleted mice. The accumulation of CD25⫹ T cells at the site of infection seems to limit the extent and efficacy of antimicrobial immune responses and thereby may favor pathogen survival. In animal models, establishment of chronic H pylori infection requires challenge with high bacterial loads.23 Although no information is available on the minimal infectious dose in humans, we speculate that dense colonization could contribute to successful transmission of H pylori from person to person. Recent studies in humans and mice indicate that CD4⫹/CD25⫹/Foxp3⫹ T cells consist of 2 distinct populations.19 One population is generated in the thymus.39 Such CD25⫹ T cells are naturally occurring and may be attracted to the site of infection after H pylori encounter. In vivo, such naturally occurring Tregs are capable of extensive Ag-induced proliferation. Thereby, they develop increased suppressive activity and inhibit the proliferation and cytokine production of antigenically stimulated effector T cells.40,41 A second population of Foxp3⫹ Tregs can be generated extrathymically from naive CD4⫹/CD25⫺/Foxp3⫺ cells after polyclonal or antigen-specific stimulation in vitro42– 45 and in vivo.46 – 48 In mice, these peripherally de novo– generated Foxp3⫹ cells are indistinguishable in cell-surface phenotype and functional properties from intrathymically developing natural Treg and are capable to suppress effector cell function in vitro45 and in vivo.46 – 48 Similarly, de novo extrathymically generated human CD25⫹/FOXP3⫹ T cells have a suppressive phenotype19,42– 44,49 (although the magnitude of suppression varies in different studies19,43,49). One recent study even showed that these “induced” CD25⫹/FOXP3⫹ T cells inhibit effector cell function in an antigen-specific manner.44 It could be speculated that such a second population of CD25⫹/ Foxp3⫹ T cells may be induced in the periphery (stomach) by H pylori. It should be considered that there are some differences between human and mouse Foxp3. Whereas Foxp3 expression is a necessary and sufficient condition for the

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generation of bona fide Treg in mice, ectopic FOXP3 expression in human T cells seems to induce only a modest suppressive activity.50 Thus, although FOXP3 is necessary for the Treg development in humans, it is not sufficient for the generation of fully suppressive Treg and additional signals are needed.50 Nevertheless, the requirement and crucial role of human FOXP3 for Treg development is mirrored by the fact that mutations of human FOXP3 result in IPEX, a syndrome that is the human equivalent of mouse scurfy.51 At a very young age, patients with this syndrome present with massive lymphoproliferation and a multitude of severe autoimmune diseases because of a lack of Treg.19,51 The mechanisms of CD25⫹ T-cell priming, recruitment, and survival in response to H pylori infection will be an interesting issue to address in future studies. Recent reports have shown that pathogens can trigger the production of chemokines that selectively favor Tregcell recruitment.12 Furthermore, Treg cells express several Toll-like receptors (TLRs), including TLR 4, 5, 7, and 8. It has been shown that Treg-cell function can be directly regulated by microbial products via TLR signalling.52 Another mechanism by which pathogens manipulate immune responses is to create an environment that favors Treg-cell survival and retention at the site of infection. In this context, TGF-␤, which is highly expressed in the H pylori–infected gastric mucosa, has been shown to be an important factor for the local survival and function of natural CD25⫹ Treg cells in vivo.11,12 To summarize, we found that CD25⫹/Foxp3⫹ T cells actively participate in the immune response to H pylori. CD25⫹/Foxp3⫹ T cells limit immune-mediated pathology but are associated with dense bacterial colonization. From an evolutionary point of view, bacterial survival may therefore represent a “compromise” achieved by the host with the microbe and may reflect the excellent adaptation of H pylori to humans over thousands of years of coevolution.1

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Received September 9, 2005. Accepted April 12, 2006. Address requests for reprints to: Roland Rad, MD, Klinikum Rechts der Isar der Technischen Universität München, II. Medizinische Klinik, Ismaningerstrabe 22, 81675 München, Germany. e-mail: Roland.Rad@ lrz.tum.de; fax: (49) 89-4140-7366. Roland Rad is recipient of a grant from the “Bund der Freunde der Technischen Universität München.” Supported by Else Kröner Fresenius Stiftung, Germany. We wish to thank Richard Ferrero for kindly providing us with mouse-adapted H pylori strains. We are grateful to Karin Bink and Ulrike Buchholz for excellent technical advice.