Volume 128, Issue 5, Pages 1327-1339 (May 2005) A Secreted Low--Molecular-Weight Protein From Helicobacter pylori Induces Cell-Cycle Arrest of T Cells  Markus Gerhard, Christian Schmees, Petra Voland, Nicole Endres, Markus Sander, Wolfgang Reindl, Roland Rad, Madlene Oelsner, Thomas Decker, Martin Mempel, Ludger Hengst, Christian Prinz  Gastroenterology  Volume 128, Issue 5, Pages 1327-1339 (May 2005) DOI: 10.1053/j.gastro.2005.03.018 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 H pylori inhibits proliferation of stimulated human PBL. (A) PBL were isolated from healthy, H pylori–negative volunteers and proliferation was determined by incorporation of [3H]-thymidine. PBL were stimulated with PHA (5 μg/mL) and IL-2 (20 U/mL) and simultaneously incubated with H pylori strain 26695 (▩) for 48 hours at MOI 1-10. ■, Unstimulated PBL controls. The inhibitory effect increased in a dose-dependent manner, and at bacterial/cell ratios between 5 and 10 the proliferation rate was suppressed to basal levels. E coli had no inhibitory effect on PBL at the same concentrations (□). One of 10 independent experiments using different samples is shown. In further experiments, a MOI of 5 was used if not indicated differently. (B) Several H pylori strain types (26695, G27, TX30a, P12) (3, 4, 5, 6) and defined knock-out mutants (7, 8, 9, 10) were tested for their inhibitory effect. All strains showed a similar potency to inhibit mitogen-induced T-cell proliferation. Analysis of the inhibitory activity of H pylori bacteria after physical disruption of the bacterial cells (12, 13) or separation by transwell culture insets (11). Water extracts were prepared as described and heat treated (16, 17) or digested with Proteinase K (15) (n = 4 independent experiments). (C) Elution profile (absorption at 280 nm) of HPWEs separated by gel filtration chromatography (top), and corresponding inhibitory activity of the eluted fractions (bottom). Elution of proteins used as size markers are indicated (arrows). The fraction containing VacA activity is represented by an extended arrow (19), the grey bar indicates the fractions exhibiting inhibitory activity in lymphocyte proliferation assay (lower graph). (D) Dose-response curve of HPWE from wild-type and VacA knock-out strains. Data represent n = 3 independent experiments. The median inhibitory concentration was 2.5 μg/mL (±.5) HPWE. (E) VacA mutant genotype and phenotype were confirmed by Western blotting (upper graph) and polymerase chain reaction (lower graph). Gastroenterology 2005 128, 1327-1339DOI: (10.1053/j.gastro.2005.03.018) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Inhibition of lymphocyte proliferation by HPWE. (A) CD3+/CD28+ T cells were separated by positive or negative selection from PBL and stimulated with anti-CD3/CD28 T-cell expander. Co-incubation with HPWE (concentrations corresponding to MOI 5) from strain 26695 (identical results were obtained with other strains used in Figure 1B, not shown) completely inhibited proliferation (determined after 72 hours of incubation). The mean of 3 independent experiments is shown. From the CD3+ pool, CD4+, and CD8+-positive T cells then were purified further by negative selection, stimulated with anti-CD3–/CD28–coated beads, and incubated with HPWE for 72 hours. CD 19+ cells were isolated by positive selection from the CD3-negative pool. Proliferation of all cell types was inhibited to a similar extent. Data are the mean of 3 independent experiments. (B) T-cell proliferation and inhibition by HPWE in the presence of cytokines or blocking antibodies. T cells were purified as described for A and stimulated with anti-CD3–/CD28–coated beads before incubation with HPWE. Antibodies to transforming growth factor β, IL-1β, or tumor necrosis factor-α were added during stimulation experiments, as well as IL-2 (20 U/mL) and L-NMMA (10−4 mol/L) to overcome the inhibitory effect. Data represent 4 independent experiments. Gastroenterology 2005 128, 1327-1339DOI: (10.1053/j.gastro.2005.03.018) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effect of HPWE on antigen-specific activation of T cells. LT12 CD8+ T cells were cocultured for 60 hours with MART-1 peptide–loaded dendritic cells and exposed to HPWE simultaneously. (A) IFN-γ production (■) was measured 72 hours after addition of HPWE (10 μg/mL); proliferation (▩) of the same samples was assessed by [3H]-thymidine incorporation (n = 3). (B) (Left) FACS histograms of CD69 expression on LT12 T cells. Mean fluorescence (mf) was determined 24 hours after incubation with water extract from mock-inoculated culture broth as basal control (upper graph), MART-1 peptide (middle), or HPWE (lower graph). (Right) Expression of CD25 on primary CD3+ lymphocytes stimulated with CD3/CD28 beads (upper graph) and co-incubated with HPWE (10 μg/mL) from wild-type 60190 (middle graph) or VacA knock-out mutant (lower graph). The mean fluorescence was similar in all samples and thus not indicated. At lower concentrations, HPWE induced up-regulation of CD25 (not shown). (C) IL-2 production from CD3+ lymphocytes treated as indicated was determined by enzyme-linked immunosorbent assay after 48 hours. NS, not significant. (D) Nuclear translocation of green fluorescent protein–tagged NFAT was assessed by laser scanning confocal microscopy under indicated conditions. (E) NFAT reporter assays using B3Z cell line stably transfected with NFAT-βGal reporter plasmid. Cells were stimulated with dendritic cells loaded with 2 μg/mL SIINFEKL in PBS for 24 hours with or without HPWE. β-galactosidase activity was measured after the addition of chlorophenol red–β-galactoside as substrate, and extinction was read at 570 nm. Experiments were performed in triplicate, and data are expressed as the mean. Two independent experiments were performed. *P values <.05 as determined by Student t test were considered significant compared with the stimulation control. NFAT is activated at 10 μg/mL, and only is inhibited using 250 μg/mL HPWE of the wild-type strain but not the VacA knock-out mutant. Gastroenterology 2005 128, 1327-1339DOI: (10.1053/j.gastro.2005.03.018) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Determination of apoptotic percentage and cell-cycle analysis of PHA-stimulated PBL after incubation with HPWE. (Left histograms) PHA-stimulated cells. (Right histograms) PHA-stimulated cells incubated with HPWE. (A) Propidium iodide staining of PBL after 72 hours incubation; the percentages of apoptotic cells (sub-G1, left) and cells in S/G2/M-phases (right bar) are indicated (1 of 3 representative experiments). (B) TUNEL staining of PBL was performed after 72 hours; positive cells were calculated from triplicates. (C) Statistical evaluation of 6 independent experiments with PBL prepared from different patients and analyzed by propidium iodide staining according to A. The upper graph shows the mean percentages in S/G2/M-phase, the lower graph shows the percentage of apoptotic cells (sub-G1) after stimulation with PHA and the addition of HPWE. Results were identical using life H pylori, sonicated H pylori, or HPWE (not shown). (D) FACS diagrams of annexin-labeled human CD4 T cells after stimulation with CD3/CD28 and different concentrations of HPWE from strain G27. Percentage of gated annexin-positive cells is indicated. Other wild-type strains gave comparable results (not shown). (E) Comparison of proliferation and apoptosis as determined in D by using different concentrations of HPWE and wild-type or VacA mutant strains from 3 independent experiments. Gastroenterology 2005 128, 1327-1339DOI: (10.1053/j.gastro.2005.03.018) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Western blot analysis of cell-cycle regulatory proteins in PBL stimulated with PHA and HPWE. (A) Lymphocytes (108) were stimulated with PHA for 24, 48, and 72 hours and simultaneously incubated with HPWE or RPMI 1640 medium as control. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed with 30 μg total protein per lane. Phosphorylated Rb (as a shift to the hyperphosphorylated form of lower mobility) was detected 48 hours after stimulation and was sustained after 72 hours. This phosphorylation was abolished completely by HPWE. PHA induces up-regulation of cyclins D3, E, and A; concomitant incubation with HPWE reduces or prevents this up-regulation. p27 down-regulation was observed 48 hours after stimulation with PHA and was inhibited by HPWE, whereas cdk 2 and cdk 4 levels were not altered. (B) p27 protein expression was investigated at 24 hours with indicated concentrations of HPWE. Actin was used as loading control. Gastroenterology 2005 128, 1327-1339DOI: (10.1053/j.gastro.2005.03.018) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 MEFs from wild-type (p27+/+) and p27 knock-out mice (p27−/− MEF) were stimulated with HPWE. (A) Wild-type and p27−/− MEFs were incubated with HPWE from wild-type and VacA knock-out strains at indicated concentrations. Proliferation was assessed by [3H]-thymidine incorporation. Data are presented as percent inhibition, with HPWE at 10 μg/mL, which completely inhibited proliferation, serving as reference (100% inhibition, not shown). Two independent experiments were performed, and samples were calculated as a mean of 3 independent wells. *P values <.05 as determined by Student t test were considered significant. (B) Absence of p27 protein in p27 knock-out animals was verified by immunoblotting. Gastroenterology 2005 128, 1327-1339DOI: (10.1053/j.gastro.2005.03.018) Copyright © 2005 American Gastroenterological Association Terms and Conditions