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The Journal of Immunology, 2006, 176: 5306-5313.
Copyright © 2006 by The American Association of Immunologists

Induction of CTLA-4-Mediated Anergy Contributes to Persistent Colonization in the Murine Model of Gastric Helicobacter pylori Infection1

Kathleen M. Anderson*, Steven J. Czinn{dagger}, Raymond W. Redline* and Thomas G. Blanchard2,{dagger}

* Department of Pathology and {dagger} Department of Pediatrics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Helicobacter pylori infection induces gastric inflammation but the host fails to generate protective immunity. Therefore, we evaluated the immunologic mechanisms that contribute to the failure of the T cells to promote active immunity to H. pylori in the mouse model of H. pylori infection. Spleen cells from infected C57BL/6 mice underwent significantly less proliferation and cytokine production than cells from immune mice upon in vitro stimulation with H. pylori lysate. Similar results were observed when stimulating with Ag-pulsed macrophages demonstrating that hyporesponsiveness was not due to a direct effect of H. pylori virulence factors on the T cells. Ag-specific hyporesponsiveness could be reversed by the addition of high-dose IL-2 but not by removal of CD4+CD25+ T cells, indicating that hyporesponsiveness was due to anergy and not due to active suppression. Cells from infected mice lacked significant suppressor activity as shown by the failure to reduce the recall response of cells from immune mice in coculture at physiologic ratios. Direct blockade of CTLA-4 using anti-CTLA-4 Fabs or indirect blockade using CTLA-4 Ig plus anti-CD28 Ab resulted in significantly increased T cell activation in vitro. The importance of CTLA-4 in establishing anergy was confirmed in an in vivo model of H. pylori infection in which mice that received anti-CTLA-4 Fabs responded to H. pylori challenge with significantly greater inflammation and significantly reduced bacterial load. These results suggest that CTLA-4 engagement induces and maintains functional inactivation of H. pylori-specific T cells during H. pylori infection resulting in a reduced immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Gram-negative bacterium Helicobacter pylori is a pathogen of the gastric mucosa. Infection induces inflammation and an active immune response, but the host fails to generate protective immunity. Without treatment therefore, H. pylori establishes persistent colonization that results in chronic histologic gastritis (1). The study of H. pylori pathogenesis has largely focused on its causative role in the development of symptomatic gastritis, peptic ulcer disease (2), and gastric cancer (3). The importance of elucidating the pathogenic mechanisms of H. pylori is underscored by its prevalence in over 50% of the population worldwide. However, over 80% of infected individuals remain asymptomatic for life (1). Because most people infected with H. pylori remain healthy despite persistent bacterial colonization, the possibility exists that the bacteria are capable of limiting the host immune response, thereby decreasing immunopathology and facilitating continued bacterial colonization.

Several lines of evidence are in accordance with a limited immune response to H. pylori in most patients. Recall assays in which cytokine production and cell proliferation of PBMCs and gastric lamina propria mononuclear cells from infected subjects and noninfected control subjects was measured and demonstrated that cells from infected subjects failed to respond more strongly than cells from control subjects (4, 5, 6, 7). In most cases, cells from infected subjects actually had weaker responses than controls. More recently, Lundgren et al. (8) were able to increase the H. pylori-specific in vitro recall response of PBMCs isolated from H. pylori-infected subjects by removing CD25+ regulatory T cells. Increased activity could not be demonstrated using PBMCs from uninfected controls indicating that T cells from infected subjects had recognized Ag in vivo but were being prevented from responding to subsequent exposure.

A murine model of H. pylori infection has been developed to study pathogenesis and immunity (9, 10). Infection of mice with H. pylori lasts for the life of the animal and results in histologic gastritis similar to that induced in humans (10). Protective immunity can be achieved in this model by several different immunization protocols (11). Challenge to immunized mice results in the rapid induction of severe gastric inflammation termed "postimmunization gastritis" that is significantly more severe than the gastritis induced by chronic infection (12, 13, 14). The generation of postimmunization gastritis results in a significant decrease in bacterial load in as little as 2 wk after challenge (15). CD4+ T cells have been shown to be a necessary component of both chronic gastritis (16, 17) as well as protective postimmunization gastritis (18, 19). However, similar to the observations we described for humans, H. pylori-specific T cells isolated from experimentally infected mice have weak recall responses to antigenic stimulation in vitro (20).

The goal of the present study was to identify the mechanism responsible for the reduced immune response during H. pylori infection in mice. We now show that hyporesponsiveness to H. pylori infection may be due to the induction of anergy in CD4+ T cells recruited to the gastric mucosa. We also show that this functional inactivation is, at least in part, due to expression of CTLA-4 on the T cell surface, which may prevent the necessary costimulation from occurring when APCs engage the TCR. Blocking of CTLA-4 during H. pylori challenge results in an increased immune response and an ability to eradicate H. pylori from the gastric mucosa in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

IL-2 Duoset ELISA kit and purified CTLA-4 Ig were purchased from R&D Systems. IFN-{gamma} quantification ELISA and recombinant human IL-2 were purchased from BD Biosciences. RPMI 1640 and FBS were purchased from Invitrogen Life Technologies. MACS CD4+ purification reagents and columns were purchased from Miltenyi Biotec. Anti-CD28 Ab was purchased from eBioscience, and anti-CD25 and anti-CD69 Abs were purchased from BD Biosciences. Control hamster IgG was purchased from Jackson ImmunoResearch Laboratories. Anti-CTLA-4 mAb was purified by protein G chromatography from supernatants of the clone UC10-4F10-11 hybridoma obtained from American Type Culture Collection. Fabs of anti-CTLA-4 mAb were generated by papain digestion obtained from Pierce and purified by passage over a protein A column (Pierce). Complement reagents and gradient (Low-Tox M rabbit complement and Lymphocyte-M) were purchased from Cedarlane Laboratories. H. pylori lysates were prepared by probe sonication of H. pylori Sidney Strain organisms in PBS. Sonicate was sterile filtered using 0.2-µm acrodisc filters from Pall.

Bacteria

H. pylori Sidney strain (9) were grown on Columbia agar (Difco) containing 7% horse blood (Cleveland Scientific) under microaerobic conditions (5% O2, 10% CO2) at 37°C for 96 h. Media was supplemented with trimethoprim (20 µg/ml), vancomycin (6 µg/ml), amphotericin B (2.5 µg/ml), and cefsulodin (16 µg/ml) (Sigma-Aldrich). Before inoculation of mice, bacteria were transferred to 10-ml Brucella broth (Difco) supplemented with 10% FBS (Invitrogen Life Technologies) and amphotericin B (2.5 µg/ml). Liquid cultures were established in T25 flasks and maintained at 37°C with 5% CO2.

Mice

Six- to 10-wk-old C57BL/6 female mice were purchased from The Jackson Laboratory and were housed under specific pathogen-free conditions in microisolator units at Case Western Reserve University. All studies involving the use of mice were reviewed and approved by the Case Western Reserve University Institutional Animal Care and Use Committee. The Case Western Reserve University animal facility is fully accredited by the American Association for the Accreditation of Laboratory Animal Care International.

Infection and immunization

All infections and challenges were performed with H. pylori Sidney strain using flexible tubing on the end of an 18-gauge needle. Each mouse received a gavage of 500 µl of an actively growing bacterial culture of at least 0.3 nm OD450 on 2 consecutive days. Mice were sacrificed no sooner than 4 wk postinfection. Immunization was accomplished by intranasal administration of 100 µg of H. pylori lysate plus 5 µg of cholera toxin adjuvant on day 0, 7, 14, and 28 as previously described (20). Mice were sacrificed no earlier than 1 wk after the final immunization.

In vitro recall assay

Spleens were removed from mice infected with H. pylori or immunized with H. pylori lysate and cholera toxin. RBCs were removed by lysis and the bulk splenocytes were then plated in triplicate in 96-well plates at 1 x 106 cells/well in 200 µl of RPMI 1640/10% FBS medium (complete RPMI). For suppression assays, bulk cells from infected mice were plated alone or at the indicated ratios with bulk cells from immunized mice and stimulated with H. pylori lysate. Groups of wells were treated with 100 µg/ml anti-CTLA-4 Fabs, 10 µg/ml CTLA-4 Ig, or high-dose (>1000 U/ml) IL-2 for 1 h before stimulation with 10 µg/ml H. pylori lysate. Cells were incubated at 37°C, and supernatants were removed at 12 or 36 h to determine the level of IL-2 or IFN-{gamma} secretion, respectively, by ELISA.

Proliferation assay

Bulk splenocytes prepared as described above were suspended in complete RPMI medium and distributed in 96-well flat-bottom tissue culture plates at 2 x 105 cells/well. H. pylori lysate was added to a final concentration of 1 µg/ml, and stimulation proceeded for the period of time indicated in each experiment. Each well was pulsed with 0.5 µCi [3H]thymidine 18 h before harvest. Cells were harvested using a TomTec Harvester and Wallac Betaplate scintillation counter to determine the degree of [3H]thymidine incorporation.

T cell activation assay

Bulk splenocytes were prepared as described. For purification of CD4+ cells, bulk splenocytes were incubated at 4°C with CD4+ MACS Ab and positively selected according to manufacturer’s instructions using a MACS column on a magnetic support. The CD4+ cells were eluted from the column and plated at 2 x 105 cells/well in 96-well plates containing i.p. macrophages previously pulsed for 6 h with H. pylori lysate. Macrophages were washed multiple times to eliminate exogenous Ag before the addition of CD4+ cells.

Flow cytometry

Splenocytes isolated from immunized or infected mice were plated as described and stimulated with H. pylori lysate. A total of 2 x 105 cells was then incubated with Fc{gamma}RIII for 15 min followed by incubation with FITC-CD69 Ab according to manufacturer’s instructions. Analysis was performed using a Coulter Epics XL-MCL.

Complement-mediated cytotoxicity

To prepare CD25– cells, spleen cell suspensions were prepared from mice infected with H. pylori. RBCs were removed by lysis and incubated with 0.65 µg of anti-mouse CD25 Ab in 2% FBS/PBS per 1 x 107 cells for 30 min at 4°C and washed in PBS. The cells were resuspended in a solution of PBS and Low-Tox M rabbit complement (20/1 v/v) and incubated at 37°C for 45 min. Dead cells were removed and lymphocytes were concentrated using a Lymphocyte-M gradient followed by several washing steps in PBS. The deletion of CD25+ cells was confirmed by analyzing PE-conjugated anti-CD25 Ab labeled cells by flow cytometry. A subpopulation of cells was also stained with FITC-conjugated anti-rat Ab to confirm that all CD25+ cells were lysed, and that the unlabeled anti-CD25 Ab used for complement-mediated killing was not blocking the binding of PE-conjugated anti-CD25 Ab. CD25– cells were then plated in triplicate in 96-well plates at 5 x 105 cells/well in 200 µl of complete RPMI medium. The cells were stimulated as described for assessment of cytokine production in response to H. pylori Ags.

In vivo study

Anti-CTLA-4 Fab or control hamster Fab in PBS (200 µg) were injected into the i.p. cavity of mice starting at day 0 and then every other day until 4 days before sacrifice. On days 1 and 3, mice were infected with H. pylori by gastric gavage as we described. Mice were sacrificed at 14 or 28 days postinfection. At harvest, gastric tissue was collected for analysis. Blood was collected from the superior vena cava and the serum was stored at –20°C until analyzed. Gastric biopsy strips were removed from the entire length of the greater curvature of the stomach. One biopsy was placed in a preweighed tube of 200-µl Columbia broth, weighed again, homogenized and used to make serial 10-fold dilutions. The 10-µl aliquots of each dilution were plated as described for CFU determination. A second strip was pinned flat, fixed in 10% buffered formalin, and processed for H&E staining at the Willard Alan Bernaum Cystic Fibrosis Research Center core facility (Case Western Reserve University School of Medicine). The degree of inflammation was determined as previously described (19, 21). Briefly, the area of the tissue section displaying the most severe inflammation was scored on a scale from 1 to 5 and the mean ± SD was calculated for each separate group. Bulk splenocytes were prepared as described and plated at 5 x 106 cells/well in 500 µl of complete RPMI medium in 48-well tissue culture dishes. The cells were stimulated as described for assessment of cytokine production in response to H. pylori Ags.

Statistics

Differences between experimental groups in each experiment was evaluated by Student’s t test. Differences were considered statistically significant for values of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
T cells from infected mice have a limited recall response to in vitro stimulation with H. pylori Ag

To compare the activation status of T cells from chronically infected mice with mice that have been protectively immunized, we generated single cell suspensions from the spleens of these mice and stimulated the cells with H. pylori lysate Ag in vitro. Cells from infected mice produced significantly less IL-2 (Fig. 1A; p < 0.0001) and IFN-{gamma} (Fig. 1B; p < 0.005) than cells from immune mice when stimulated for 12 and 36 h, respectively. The proliferative response of cells from infected mice was also significantly lower than cells from immune mice following stimulation with H. pylori Ag as measured at 70 and 94 h (Fig. 1C; p < 0.005). Additionally, expression of CD69, a T cell activation surface marker, was increased after in vitro stimulation of cells from immunized mice, whereas cells isolated from infected mice displayed only moderately increased levels as determined by flow cytometry (data not shown).


Figure 1
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  		FIGURE 1. Spleen cells from H. pylori-infected mice have reduced recall responses relative to cells from immunized mice. Splenocytes isolated from H. pylori-immunized or H. pylori-infected mice were stimulated in vitro with either 1 or 10 µg/ml H. pylori lysate Ags. Cytokine production was determined at 12 h poststimulation for IL-2 (A) and 36 h poststimulation for IFN-{gamma} (B). Cells from infected mice failed to produce IL-2 (p < 0.0001) and produced significantly less IFN-{gamma} (p < 0.005) than cells from immunized mice. C, The kinetics of proliferation was compared for splenocytes stimulated with 1 µg/ml H. pylori lysate as determined by incorporation of [3H]thymidine. Proliferation of cells isolated from H. pylori-infected mice was significantly reduced at both 70 and 94 h poststimulation compared with cells from immunized mice (p < 0.005). The data are one representative of five independent experiments with three mice per group per experiment.

 
Hyporesponsiveness of T cells from infected mice is not due to direct interactions with H. pylori proteins

The vacuolating cytotoxin A (VacA) of H. pylori has been shown to interfere with the cell cycle of the T cell and arrest its activation (22, 23, 24, 25). Therefore, to determine whether the limited recall response of the T cells from infected mice was due to direct interactions with H. pylori proteins, we stimulated CD4+ T cells with i.p. macrophages that had been pulsed with H. pylori lysate and washed repeatedly to eliminate free Ags. Fig. 2 shows that when stimulation of CD4+ T cells is performed in the context of Ag presentation, T cells from infected mice display the same limited IL-2 production as observed when adding lysate Ag directly to the bulk cells as performed in Fig. 1A, and this value was significantly lower than the amount of IL-2 produced by stimulated cells from immune mice (p < 0.002).


Figure 2
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  		FIGURE 2. Decreased T cell activation of cells from infected mice does not require direct contact with H. pylori Ags. The i.p. macrophages were pulsed with H. pylori lysate Ag for 6 h and then washed to remove exogenous lysate. The pulsed macrophages were then used to stimulate CD4+ splenocytes from either H. pylori-immunized or H. pylori-infected mice. IL-2 production was measured at 12 h poststimulation as a marker of T cell activation. Pulsed macrophages stimulated significant levels of IL-2 production from the cells of immune mice but failed to stimulate cells from infected mice (p < 0.002). Cells were pooled from three mice in each group.

 
Cells from infected mice fail to suppress the activation of cells from immunized mice

Several studies have described the presence of CD4+CD25+ regulatory cells in infected humans and mice that can suppress the H. pylori-specific immune response (8, 26, 27). These studies suggest the decrease in T cell activation during H. pylori infection may be due to active suppression. We cocultured cells from immunized mice with various ratios of cells from infected mice to determine whether suppressive regulatory cells might be present in infected mice that are capable of limiting the response of cells from immune mice. Activation was monitored by a change in IFN-{gamma} production following stimulation with H. pylori lysate Ag. As illustrated in Fig. 3, cells isolated from infected mice were unable to significantly suppress the response of cells isolated from immunized mice unless the number of cells from infected mice was increased to 10 times that of the cells from immunized mice. Analysis of a bulk cell population from infected mice by flow cytometry demonstrated that 11% of the cells were CD25+ (regulatory) cells (data not shown) confirming a physiologic ratio of 10:1 nonregulatory to regulatory cells. This value is consistent with what others have observed in peripheral tissues (28, 29). Furthermore, a similar ratio has been described in the gastric mucosa during H. pylori infection as 12% of CD3+ cells are CD25+, confirming a 10:1 ratio (30). Therefore, the suppression observed in Fig. 3 required 10 times the physiologic ratio.


Figure 3
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  		FIGURE 3. T cells from H. pylori-infected mice have weak regulatory activity. Splenocytes from H. pylori-immunized mice were cocultured with varying ratios of cells from H. pylori-infected mice to determine whether the hyporesponsive cells were capable of suppressing the activity of the cells from immunized mice. IFN-{gamma} production was measured as a marker of T cell activation 36 h poststimulation. A 10-fold increase in the number of cells from infected mice was required to achieve reduced activation in cells from immunized mice. The data are one representative of two independent experiments.

 
H. pylori-specific hyporesponsiveness is reversed by the addition of high-dose IL-2

The induction of anergy during H. pylori infection could explain the hyporesponsiveness observed in the T cells from infected mice. Anergy can be reversed by the addition of high-dose IL-2 and Ag in vitro. We administered high-dose (>1000 U/ml) recombinant human IL-2 to cells from infected mice stimulated with H. pylori lysate Ag and measured the production of IFN-{gamma} as an indicator of cell activation. As shown in Fig. 4, the addition of exogenous recombinant human IL-2 restored the ability of H. pylori-specific cells from infected mice to respond to stimulation. Treatment of the cells with IL-2 resulted in a significant increase in IFN-{gamma} production compared with cells treated with lysate in the absence of exogenous cytokine (p < 0.001). Unlike human systems of in vitro analysis in response to H. pylori Ag, the use of the mouse model allows for a direct comparison to a protective immune response. Reversal of anergy resulted in a recall response that resembled that of cells from immunized mice, although the difference between these two groups was still significant (p < 0.01).


Figure 4
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  		FIGURE 4. Unresponsive H. pylori-specific splenocytes from H. pylori-infected mice are activated by the addition of exogenous high-dose IL-2. Splenocytes isolated from H. pylori-infected mice were stimulated with H. pylori lysate Ag in the presence or absence of high-dose (>1000 U/ml) recombinant human IL-2. IFN-{gamma} was measured as a marker of T cell activation. The addition of IL-2 induced significantly more IFN-{gamma} production compared with cells stimulated with Ag alone (p < 0.001). The data represent one of three independent experiments with three mice per group per experiment.

 
H. pylori-specific hyporesponse is maintained in the absence of CD25+ cells

To more specifically address the possibility that H. pylori-specific T cells are actively suppressed by the presence of regulatory T cells, we deleted the CD25+ cells from spleen cells of H. pylori-infected mice. CD25+ cells were removed by complement depletion as described in Materials and Methods, and the remaining CD25– cells were stimulated in vitro with H. pylori lysate in the absence of CD25+ regulatory T cells. As demonstrated in Fig. 5, CD25– cells isolated from H. pylori-infected mice remain hyporesponsive despite the absence of suppressive CD25+ cells as measured by the production of IFN-{gamma} and IL-2. Conversely, CD25+ cell depletion of cells from immunized mice did not abrogate the ability of the CD25– cells to respond to stimulation with H. pylori Ag, suggesting that removal of CD25+ cells does not remove all activated cells (data not shown).


Figure 5
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  		FIGURE 5. H. pylori-specific CD25– cells remain hyporesponsive in the absence of CD25+ cells. Splenocytes isolated from H. pylori-infected mice were depleted of CD25+ cells. The remaining CD25– population was stimulated in vitro with 10 µg/ml H. pylori lysate and tested for IFN-{gamma} (A) and IL-2 (B) production at 36 and 12 h, respectively. The data are one representative of five independent experiments.

 
CTLA-4 expressed by T cells from H. pylori-infected mice prevents the costimulation necessary for activation

Anergy can be induced by engagement of the TCR in the absence of a suitable costimulatory event. To determine the role of costimulation in both hyporesponsiveness and protective immunity we performed a series of in vitro blocking experiments. Cells from immunized mice were treated with CTLA-4 Ig to bind the costimulatory ligands CD80/CD86 present on APCs. As seen in Fig. 6A, blocking of CD80/CD86-mediated costimulation significantly reduced the recall response of cells from immunized mice (p < 0.0001). The reduced levels of activation observed in cells from immunized mice in the absence of costimulation suggest that lack of appropriate costimulation may be a contributing factor in the weak recall response of infected mice. To determine whether the hyporesponsiveness observed during H. pylori infection was due to the absence of costimulation at the time of T cell stimulation, we investigated the role of CTLA-4, a coreceptor implicated in down-regulation of T cells. The CD80/CD86 APC costimulatory ligands were blocked with CTLA-4 Ig, similar to the experiment performed above in Fig. 6A. However, we then added exogenous anti-CD28 to provide a positive costimulatory signal to the T cells to initiate a recall response. The response of cells from infected mice was significantly increased in the absence of CTLA-4 engagement by CD80/CD86 (Fig. 6B; p < 0.001). We also directly blocked CTLA-4 in the cells from infected mice using anti-CTLA-4 Fabs derived from the UC10-4F10 hybridoma cell line. As shown in Fig. 6C, stimulation with H. pylori Ag in the absence of CTLA-4 results in increased IFN-{gamma} production relative to stimulation with H. pylori alone (p < 0.001). Therefore, using indirect (Fig. 6B) and direct (Fig. 6C) methods to block CTLA-4 engagement in vitro, T cell activation of cells isolated from H. pylori-infected mice can be significantly increased.


Figure 6
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  		FIGURE 6. CTLA-4-dependent inhibiti on contributes to the decreased activity of H. pylori-specific T cells from infected mice. A, The addition CTLA-4 Ig to block CD80/CD86 molecules on APCs significantly reduced the activation of splenocytes from H. pylori-immunized mice as measured by IL-2 production (p < 0.0001). Data are one representative of two experiments with three mice per group. B, Direct cross-linking of the CD28 coreceptor with anti-CD28 Ab significantly increases IFN-{gamma} production of splenocytes from H. pylori-infected mice when the CTLA-4-B7 interaction is simultaneously blocked by CTLA-4 Ig (p < 0.001). Data represent one of four experiments with three mice per group. C, Direct blocking of CTLA-4 engagement with anti-CTLA-4 Fabs significantly increases T cell activation in the splenocytes isolated from H. pylori-infected mice as measured by IFN-{gamma} production (p < 0.001). The data are one representative of two experiments with three mice per group. D, Depletion of CD25+ cells prevents the increase in T cell activation observed after direct blocking of CTLA-4 engagement with anti-CTLA-4 Fab. Data represent one of two experiments with two mice per group.

 
CTLA-4 is constitutively expressed on CD25+ regulatory T cells, whereas expression on CD25– cells is induced only after activation. Therefore, to more precisely determine the cell type expressing CTLA-4 in our model, we removed CD25+ cells using complement depletion as described. As demonstrated in Fig. 6D, stimulation of CD25– cells from H. pylori-infected mice in the absence of CTLA-4 results in no significant change in T cell activation. The observation that CTLA-4 blockade results in increased activation of a bulk cell population (Fig. 6, B and C) with no observed change in CD25– purified cells (Fig. 6D) suggests that CTLA-4 expression on CD25+ cells contributes to the decreased T cell activation during H. pylori infection and reinforces the notion that there is a population of CD25– T cells that remain hyporesponsive even in the absence of regulatory T cells.

Blocking of CTLA-4 during H. pylori infection results in significantly increased gastritis

We next performed in vivo analysis to determine whether CTLA-4 expression helps limit the T cell response during H. pylori infection. CTLA-4 was blocked in H. pylori-infected mice by regular injections of anti-CTLA-4 Fabs as described in Materials and Methods. Mice were harvested 4 wk postchallenge, and the degree of gastric inflammation was compared with mice treated with control hamster Fab, and to non-Ab-treated mice that had been immunized before challenge. As demonstrated in previous experiments, immunized mice responded to challenge with more severe inflammation that was significantly greater than inflammation found in naive controls (Fig. 7; p < 0.02). Control Fab-treated mice developed a mild gastritis that was not significantly greater than naive animals. Mice treated with anti-CTLA-4-specific Fabs developed mucosal inflammation that was equivalent to the postimmunization gastritis observed in immune mice (inflammation score 2.1 ± 1.1 and 2.1 ± 1.3, respectively) and was also significantly greater than found in naive mice (p < 0.01). Therefore, in the absence of CTLA-4 engagement, mice respond to H. pylori infection with an inflammatory response that resembles the increased inflammation achieved in immunized mice.


Figure 7
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  		FIGURE 7. Blocking of CTLA-4 binding in vivo results in increased gastritis in response to H. pylori infection. Immunized and naive mice were challenged with H. pylori. Two groups of nonimmune infected mice were injected with either anti-CTLA-4 blocking Fabs or control Fab for the duration of infection. Mice were sacrificed at 4 wk, and the degree of gastric inflammation of each group was compared with naive control mice.

 
In vivo neutralization of CTLA-4 results in decreased colonization in H. pylori-challenged mice

The reduction in bacterial load in H. pylori-immunized mice has been shown to follow the induction of postimmunization gastritis. We quantified the number of H. pylori CFU in the gastric mucosa of anti-CTLA-4 Fab-treated mice 4 wk postchallenge to determine whether the increased gastritis achieved in these mice resulted in any degree of protection. As shown in Fig. 8, control hamster Fab-treated mice were colonized with levels of H. pylori equivalent to nontreated infected mice. Immunized mice had significantly reduced numbers of H. pylori compared with nontreated infected mice (p = 0.008). Analysis of anti-CTLA-4 specific Fab-treated mice also revealed a significantly reduced bacterial load compared with nontreated (p = 0.012) and control Ab treated mice (p = 0.01). The level of infection in this group was not significantly different from that of immunized mice. These data demonstrate that the persistence of H. pylori in the gastric mucosa is dependent, at least in part, on the engagement of CTLA-4.


Figure 8
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  		FIGURE 8. Blocking of CTLA-4 binding in vivo results in decreased bacterial load at the gastric mucosa. Immunized and naive mice were challenged with H. pylori. Two groups of nonimmune infected mice were injected with either anti-CTLA-4 blocking Fabs or control Fab for the duration of infection. Mice were sacrificed at 4 wk, and the number of H. pylori per gram of stomach tissue was determined. Anti-CTLA-4 Fab treatment results in a significant decrease in bacterial colonization relative to infected mice (p = 0.012) and control Fab-treated mice (p = 0.01). Data shown are from eight mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite the recruitment of activated T cells to the gastric mucosa of humans and mice infected with H. pylori, the host fails to eradicate the bacteria from the gastric lumen. This is in stark contrast to the ability of the adaptive immune response activated by immunization to reduce or eliminate H. pylori (11). Functional differences between nonprotective T cells activated during chronic H. pylori infection and protective T cells activated by vaccination have only recently begun to be elucidated (8, 27, 31). In the present investigation, we provide evidence that H. pylori-specific T cells from infected mice are distinct from H. pylori-specific T cells from immunized mice in their ability to respond to subsequent exposure to H. pylori Ags. We noted a marked hyporesponsiveness in the T cells from infected mice and that these cells were relatively ineffective at suppressing the recall response of H. pylori-specific T cells from immunized mice. The anergic state of these cells could be reversed by the addition of Ag plus IL-2. Nonresponsiveness could also be abrogated by blocking CTLA-4 on the surface of the T cells, or by direct Ab cross-linking of CD28 on the T cells while blocking B7 expressed on the APCs. The importance of CTLA-4 in mediating hyporesponsiveness was confirmed in our in vivo model of H. pylori infection in which mice that received anti-CTLA-4 Fabs gained the ability to significantly reduce the bacterial load.

Pursuit of the mechanisms that might explain the inability of H. pylori-specific CD4+ T cells to protect the host has become a topic of increased investigation. The H. pylori vacuolating cytotoxin A (VacA) protein has now been shown to directly interact with T cells and interfere with cell cycle progression thus preventing proliferation and IL-2 production (22, 23, 25). A second soluble factor has also been identified with similar effects on T cell proliferation (24). Additionally, TLRs have recently been described on T cells, indicating that bacterial Ags may act directly on T cells to influence activation (32, 33). However, we observed a decrease in T cell activation using cells isolated from infected mice even in the absence of T cell contact with H. pylori lysate. When soluble Ag was removed from our system, Ag-pulsed macrophages were sufficient to achieve hyporesponsiveness.

Another possible mechanism to explain the nonprotective nature of the chronic inflammation that accompanies H. pylori infection is the suppression of the T cell response by regulatory CD4+CD25+ cells. CD25 is the surface receptor for the T cell stimulatory cytokine IL-2 (34). CD4+CD25+ cells are generally considered to be down-regulatory and have been shown to play an important role in limiting immunity in a number of models including intestinal inflammation, pulmonary inflammation, and autoimmunity (34, 35, 36). Recently, CD4+CD25+ cells have also been implicated in suppressing the cellular immune response against H. pylori. In humans, removal of CD25+ cells from a bulk population of PBMCs results in an increased recall response to H. pylori as measured by proliferation (8). CD4+CD25+ cells have also been described with increased frequency in gastric biopsy of H. pylori-infected subjects compared with uninfected controls (26, 30). In the mouse model, transfer of CD25+-depleted lymph node cells into nude mice recipients before challenge with H. pylori resulted in increased gastritis and a reduced bacterial load compared with mice reconstituted with bulk lymph node cells (27). These data suggest that suppression of the active immune response by CD4+CD25+ cells may contribute to the hyporesponsiveness observed during H. pylori infection.

Most models of regulatory T cell activity propose that regulation is accomplished with limited numbers of regulatory cells (34, 37, 38). The suppression demonstrated by in vitro analysis of human and murine lymphocytes from H. pylori-infected donors however was achieved by using equal numbers of CD25+ and CD25– cells (8, 39). We also observed suppressive activity as demonstrated by the ability of cells from infected mice to limit the in vitro recall response of cells from immunized mice. However, it was necessary to increase the ratio of cells from infected mice to cells of immune mice by 10-fold to obtain equal numbers of CD25+ cells from infected mice to CD25– cells from immune mice thereby achieving significant levels of suppression. Therefore, although regulatory T cells may play a role in limiting the inflammation at the gastric mucosa in response to H. pylori infection, it is possible that other mechanisms participate in H. pylori-specific hyporesponsiveness.

Our data support a role for anergy in H. pylori immunopathogenesis. Administration of high-dose IL-2 during H. pylori stimulation of cells isolated from H. pylori-infected mice resulted in T cell activation levels similar to that of cells from immunized mice suggesting that T cells responding during H. pylori infection are anergic. We note that the study described above by Lundgren et al. (8) in which the CD25– PBMC population from infected patients proliferated in response to H. pylori Ags failed to produce IFN-{gamma}. Those results are also consistent with CD25+ cell-independent hyporesponsiveness. A population of anergic T cells in the gastric mucosa would also help explain the success of therapeutic immunization in H. pylori-infected subjects and animals (40, 41, 42). If lack of protection is predominantly due to the presence of suppressive T cells responding to initial infection, it could be argued that these cells would also suppress T cells responding after subsequent immunization. Therefore, we hypothesize that cells responding to infection become functionally inactive, thereby permitting cells responding to subsequent therapeutic immunization to clear the infection upon migrating to the gastric mucosa.

Our data suggest that the host response to H. pylori infection is restricted in a CTLA-4-mediated manner. Blocking the CTLA-4 receptor allows the T cell to promote heightened inflammation at the gastric mucosa resulting in a reduced bacterial load. Our data are consistent with models of induced immunity against other bacteria where the use of the CTLA-4 blocking Ab UC10-4F10 results in increased immunity or inflammation in the host (43, 44, 45). Watanabe et al. (31), however, observed reduced gastritis in H. pylori-infected mice when CTLA-4 was blocked in vivo compared with untreated H. pylori-infected mice. Additionally, the bacterial load in mice treated with anti-CTLA-4 Ab was comparable to untreated mice. The most likely explanation for the discrepancy between results of the present study and those of Watanabe et al. (31) is the protocol for administering neutralizing Abs. Whereas we continued to apply Fab for the duration of H. pylori infection, their study stopped administering Ab after 7 days even though the experiment was conducted for a total of 6 wk. Other laboratories that have administered CTLA-4-neutralizing Ab sacrifice the animals shortly after cessation of Ab treatment. Additionally, it has been demonstrated by others that the CTLA-4-neutralizing Ab UC10-4F10 is depleted from the circulation within 10 days after cessation of treatment (46).

The recent resurgence in the study of T cell suppressor activity has helped explain a wide variety of models for immunologic quiescence as well as immunopathogenesis. However, the characteristics of the cells isolated from mice infected with H. pylori are consistent with other models in which anergic cells have been characterized (47, 48). Anergic cells remain hyporesponsive even in the continued presence of both Ag and costimulation. Although IL-2 production is inhibited in anergic cells, the cells retain the ability to respond to IL-2. However, both Ag presentation and IL-2 are required to reverse anergy. Anergy has also been shown to be dependent upon CTLA-4 expression. We presently demonstrate the importance of CTLA-4 expression on CD25+ cells through in vitro experiments designed to circumvent the inhibitory effects of CTLA-4 binding, and through in vivo experiments in which CTLA-4 is neutralized. Others have demonstrated that T cells lacking CTLA-4 were resistant to tolerance induction (46, 49).

The distinct immune responses induced by chronic H. pylori infection of the gastric mucosa and by H. pylori immunization are indicative of the different roles the respective tissues play in responding to bacterial stimuli. Whereas immunization targets bacterial Ags to secondary lymph nodes where active immunity is favored, colonization of the gastrointestinal mucosa leads to the activation of T cells within the lamina propria where T cell activity against commensal organisms can be detrimental to the host. It is possible that the host response to H. pylori is similar to the response in other noninvasive bacterial colonizers of the mucosa and consequently uses immune mechanisms that favor hyporesponsiveness. In the periphery, CTLA-4-mediated down-regulation helps limit an active immune response to prevent continued activity subsequent to the establishment of immunity. The role of CTLA-4 in the mucosa may be quite distinct. The constant exposure of lymphocytes to food Ags and commensal bacteria demands a mechanism that precludes undesirable immune responses before they develop. Therefore, it remains possible that the mucosal environment of the stomach during H. pylori infection favors CTLA-4 expression thereby preventing the development of an active immune response.

Direct examination of CD4+ T cells isolated from the gastric mucosa in mice is technically difficult and does not provide sufficient numbers of purified cells required to perform in vitro assays or adoptive transfer experiments. These difficulties are compounded when assessing immunized mice that lack gastric mucosal lymphocytes unless challenged with live H. pylori. Therefore, to investigate the H. pylori-specific immune response in detail, we have relied upon the investigation of splenocytes from H. pylori infected or immunized mice. Several laboratories have demonstrated similarities in vitro when comparing lymphocytes isolated from the gastric mucosa with those isolated from PBMCs or spleens in several models of Helicobacter infection (4, 6, 50). Because H. pylori infections are restricted to the gastric mucosa, it is believed that H. pylori-specific cells present in the spleen were initially activated in the stomach and subsequently migrated to the periphery. Thus, unlike the intestines where distinct populations of cells preferentially home to the gut, the immune response in the stomach may consist of peripheral lymphocytes that respond to inflammatory signals and then recirculate to lymph nodes and spleens. The recruitment of peripheral lymphocytes to the stomach is also supported by the observation that adoptively transferred CD4+ splenocytes are sufficient to generate gastritis when immunodeficient recipients are challenged with H. pylori (16, 19, 51). A subpopulation of these cells might then migrate out of the stomach to populate the spleen and lymph nodes following activation. Cells previously activated in the intestinal mucosa have been identified in the periphery verifying the ability to recirculate after activation (52, 53). As stated earlier, the real distinction between H. pylori-specific lymphocytes responding to immunization and those responding to infection may not necessarily be the source of the lymphocytes, but rather the microenvironment where activation occurs.

Our study demonstrates for the first time that protection from H. pylori infection may be achieved in the absence of functional CTLA-4. These findings suggest that CTLA-4 may be preferentially overexpressed on T cells that are activated within the gastric lamina propria during H. pylori infection resulting in functional inactivation of T cells and a consequent inability to promote a protective inflammatory response. As CTLA-4 expression is tightly controlled to limit autoimmunity yet permit necessary T cell activation, the mechanism responsible for CTLA-4 expression during an active immune response to a mucosal bacterial infection must be explored in more detail.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by National Institutes of Health Grants AI055710 (to T.G.B.) and DK046461 (to S.J.C.) and by the Flow Cytometry Core Facility of the Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland Grant P30 CA43703. Back

2 Address correspondence and reprint requests to Dr. Thomas G. Blanchard, Department of Pediatrics, Room 737, Rainbow Babies & Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail address: tgb4{at}po.cwru.edu Back

Received for publication October 3, 2005. Accepted for publication January 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Suerbaum, S., P. Michetti. 2002. Helicobacter pylori infection. N. Engl. J. Med. 347: 1175-1186. [Free Full Text]
  2. Marshall, B., J. Warren. 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1: 1311-1315. [Medline]
  3. Parsonnet, J., G. D. Friedman, D. P. Vandersteen, Y. Chang, J. H. Vogelman, N. Orentreich, R. K. Sibley. 1991. Helicobacter pylori infection and the risk of gastric carcinoma. N. Engl. J. Med. 325: 1127-1131. [Abstract]
  4. Karttunen, R., G. Andersson, K. Poikonen, T. U. Kosunen, T. Karttunen, K. Juutinen, S. Niemelä. 1990. Helicobacter pylori induces lymphocyte activation in peripheral blood cultures. Clin. Exp. Immunol. 82: 485-488. [Medline]
  5. Sharma, S. A., G. G. Miller, G. I. Pérez-Pérez, R. S. Gupta, M. J. Blaser. 1994. Humoral and cellular immune recognition of Helicobacter pylori proteins are not concordant. Clin. Exp. Immunol. 97: 126-132. [Medline]
  6. Fan, X. J., A. Chua, C. N. Shahi, J. McDevitt, P. W. Keeling, D. Kelleher. 1994. Gastric T lymphocyte responses to Helicobacter pylori in patients with H. pylori colonisation. Gut 35: 1379-1384. [Abstract/Free Full Text]
  7. Karttunen, R., T. Karttunen, H. P. Ekre, T. T. MacDonald. 1995. Interferon {gamma} and interleukin 4 secreting cells in the gastric antrum in Helicobacter pylori positive and negative gastritis. Gut 36: 341-345. [Abstract/Free Full Text]
  8. Lundgren, A., E. Suri-Payer, K. Enarsson, A. M. Svennerholm, B. S. Lundin. 2003. Helicobacter pylori-specific CD4+CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals. Infect. Immun. 71: 1755-1762. [Abstract/Free Full Text]
  9. Lee, A., J. O’Rourke, M. C. De Ungria, B. Robertson, G. Daskalopoulos, M. F. Dixon. 1997. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112: 1386-1397. [Medline]
  10. Marchetti, M., B. Arico, D. Burroni, N. Figura, R. Rappuoli, P. Ghiara. 1995. Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 267: 1655-1658. [Abstract/Free Full Text]
  11. Blanchard, T. G., J. C. Eisenberg, Y. Matsumoto. 2004. Clearance of Helicobacter pylori infection through immunization: the site of T cell activation contributes to vaccine efficacy. Vaccine 22: 888-897. [Medline]
  12. Goto, T., A. Nishizono, T. Fujioka, J. Ikewaki, K. Mifune, M. Nasu. 1999. Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice. Infect. Immun. 67: 2531-2539. [Abstract/Free Full Text]
  13. Garhart, C. A., F. P. Heinzel, S. J. Czinn, J. G. Nedrud. 2003. Vaccine-induced reduction of Helicobacter pylori colonization in mice is interleukin-12 dependent but {gamma} interferon and inducible nitric oxide synthase independent. Infect. Immun. 71: 910-921. [Abstract/Free Full Text]
  14. Michetti, P., I. Corthesy-Theulaz, C. Davin, R. Haas, A.-C. Vaney, M. Heitz, J. Bille, J. P. Kraehenbuhl, E. Saraga, A. L. Blum. 1994. Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease. Gastroenterology 107: 1002-1011. [Medline]
  15. Garhart, C. A., R. W. Redline, J. G. Nedrud, S. J. Czinn. 2002. Clearance of Helicobacter pylori infection and resolution of postimmunization gastritis in a kinetic study of prophylactically immunized mice. Infect. Immun. 70: 3529-3538. [Abstract/Free Full Text]
  16. Roth, K. A., S. B. Kapadia, S. M. Martin, R. G. Lorenz. 1999. Cellular immune responses are essential for the development of Helicobacter felis-associated gastric pathology. J. Immunol. 163: 1490-1497. [Abstract/Free Full Text]
  17. Eaton, K. A., M. Mefford, T. Thevenot. 2001. The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice. J. Immunol. 166: 7456-7461. [Abstract/Free Full Text]
  18. Ermak, T. H., P. J. Giannasca, R. Nichols, G. A. Myers, J. Nedrud, R. Weltzin, C. K. Lee, H. Kleanthous, T. P. Monath. 1998. Immunization of mice with urease vaccine affords protection against Helicobacter pylori infection in the absence of antibodies and is mediated by MHC class II-restricted responses. J. Exp. Med. 188: 2277-2288. [Abstract/Free Full Text]
  19. Gottwein, J. M., T. G. Blanchard, O. S. Targoni, J. C. Eisenberg, B. M. Zagorski, R. W. Redline, J. G. Nedrud, M. Tary-Lehmann, P. V. Lehmann, S. J. Czinn. 2001. Protective anti-Helicobacter immunity is induced with aluminum hydroxide or complete Freund’s adjuvant by systemic immunization. J. Infect. Dis. 184: 308-314. [Medline]
  20. Rahn, W., R. W. Redline, T. G. Blanchard. 2004. Molecular analysis of Helicobacter pylori-associated gastric inflammation in naive versus previously immunized mice. Vaccine 23: 807-818. [Medline]
  21. Blanchard, T. G., S. J. Czinn, R. W. Redline, N. Sigmund, G. Harriman, J. G. Nedrud. 1999. Antibody-independent protective mucosal immunity to gastric helicobacter infection in mice. Cell. Immunol. 191: 74-80. [Medline]
  22. Gebert, B., W. Fischer, R. Haas. 2004. The Helicobacter pylori vacuolating cytotoxin: from cellular vacuolation to immunosuppressive activities. Rev. Physiol. Biochem. Pharmacol. 152: 205-220. [Medline]
  23. Gebert, B., W. Fischer, E. Weiss, R. Hoffmann, R. Haas. 2003. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 301: 1099-1102. [Abstract/Free Full Text]
  24. Gerhard, M., C. Schmees, P. Voland, N. Endres, M. Sander, W. Reindl, R. Rad, M. Oelsner, T. Decker, M. Mempel, et al 2005. A secreted low-molecular-weight protein from Helicobacter pylori induces cell-cycle arrest of T cells. Gastroenterology 128: 1327-1339. [Medline]
  25. Boncristiano, M., S. R. Paccani, S. Barone, C. Ulivieri, L. Patrussi, D. Ilver, A. Amedei, M. M. D’Elios, J. L. Telford, C. T. Baldari. 2003. The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms. J. Exp. Med. 198: 1887-1897. [Abstract/Free Full Text]
  26. Lundgren, A., E. Strömberg, Å. Sjöling, C. Lindholm, K. Enarsson, A. Edebo, E. Johnsson, E. Suri-Payer, P. Larsson, A. Rudin, et al 2005. Mucosal FOXP3-expressing CD4+CD25high regulatory T cells in Helicobacter pylori-infected patients. Infect. Immun. 73: 523-531. [Abstract/Free Full Text]
  27. Raghavan, S., M. Fredriksson, A.-M. Svennerholm, J. Holmgren, E. Suri-Payer. 2003. Absence of CD4+CD25+ regulatory T cells is associated with a loss of regulation leading to increased pathology in Helicobacter pylori-infected mice. Clin. Exp. Immunol. 132: 393-400. [Medline]
  28. Maloy, K. J., F. Powrie. 2001. Regulatory T cells in the control of immune pathology. Nat. Immunol. 2: 816-822. [Medline]
  29. Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda, T. Takahashi. 2001. Immunologic tolerance maintained by CD25+CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182: 18-32. [Medline]
  30. Strömberg, E., A. Lundgren, A. Edebo, S. Lundin, A. M. Svennerholm, C. Lindholm. 2003. Increased frequency of activated T-cells in the Helicobacter pylori-infected antrum and duodenum. FEMS Immunol. Med. Microbiol. 36: 159-168. [Medline]
  31. Watanabe, K., K. Murakami, R. Sato, T. Okimoto, K. Maeda, M. Nasu, A. Nishizono, T. Fujioka. 2004. CTLA-4 blockade inhibits induction of Helicobacter pylori-associated gastritis in mice. Clin. Exp. Immunol. 135: 29-34. [Medline]
  32. Matsuguchi, T., K. Takagi, T. Musikacharoen, Y. Yoshikai. 2000. Gene expressions of lipopolysaccharide receptors, Toll-like receptors 2 and 4, are differently regulated in mouse T lymphocytes. Blood 95: 1378-1385. [Abstract/Free Full Text]
  33. Caramalho, I., T. Lopes-Carvalho, D. Ostler, S. Zelenay, M. Haury, J. Demengeot. 2003. Regulatory T cells selectively express Toll-like receptors and are activated by lipopolysaccharide. J. Exp. Med. 197: 403-411. [Abstract/Free Full Text]
  34. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
  35. Read, S., V. Malmström, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192: 295-302. [Abstract/Free Full Text]
  36. Hori, S., T. L. Carvalho, J. Demengeot. 2002. CD25+CD4+ regulatory T cells suppress CD4+ T cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice. Eur. J. Immunol. 32: 1282-1291. [Medline]
  37. O’Garra, A., P. Vieira. 2004. Regulatory T cells and mechanisms of immune system control. Nat. Med. 10: 801-805. [Medline]
  38. Belkaid, Y., B. T. Rouse. 2005. Natural regulatory T cells in infectious disease. Nat. Immunol. 6: 353-360. [Medline]
  39. Raghavan, S., E. Suri-Payer, J. Holmgren. 2004. Antigen-specific in vitro suppression of murine Helicobacter pylori-reactive immunopathological T cells by CD4+CD25+ regulatory T cells. Scand. J. Immunol. 60: 82-88. [Medline]
  40. Doidge, C., I. Crust, A. Lee, F. Buck, S. Hazell, U. Manne. 1994. Therapeutic immunisation against Helicobacter infection. Lancet 343: 914-915. [Medline]
  41. Cuenca, R., T. G. Blanchard, S. J. Czinn, J. G. Nedrud, T. P. Monath, C. K. Lee, R. W. Redline. 1996. Therapeutic immunization against Helicobacter mustelae in naturally infected ferrets. Gastroenterology 110: 1770-1775. [Medline]
  42. Michetti, P., C. Kreiss, K. L. Kotloff, N. Porta, J. L. Blanco, D. Bachmann, M. Herranz, P. F. Saldinger, I. Corthesy-Theulaz, G. Losonsky, et al 1999. Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected adults. Gastroenterology 116: 804-812. [Medline]
  43. McCoy, K., M. Camberis, G. L. Gros. 1997. Protective immunity to nematode infection is induced by CTLA-4 blockade. J. Exp. Med. 186: 183-187. [Abstract/Free Full Text]
  44. Thompson, C. B., J. P. Allison. 1997. The emerging role of CTLA-4 as an immune attenuator. Immunity 7: 445-450. [Medline]
  45. Leach, D. R., M. F. Krummel, J. P. Allison. 1996. Enhancement of antitumor immunity by CTLA-4 blockade. Science 271: 1734-1736. [Abstract]
  46. Samoilova, E. B., J. L. Horton, H. Zhang, S. J. Khoury, H. L. Weiner, Y. Chen. 1998. CTLA-4 is required for the induction of high dose oral tolerance. Int. Immunol. 10: 491-498. [Abstract/Free Full Text]
  47. Schwartz, R. H.. 2003. T cell anergy. Annu. Rev. Immunol. 21: 305-334. [Medline]
  48. Appleman, L. J., V. A. Boussiotis. 2003. T cell anergy and costimulation. Immunol. Rev. 192: 161-180. [Medline]
  49. Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6: 411-417. [Medline]
  50. Mohammadi, M., S. Czinn, R. Redline, J. Nedrud. 1996. Helicobacter-specific cell-mediated immune responses display a predominant Th1 phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice. J. Immunol. 156: 4729-4738. [Abstract]
  51. Eaton, K. A., S. R. Ringler, S. J. Danon. 1999. Murine splenocytes induce severe gastritis and delayed-type hypersensitivity and suppress bacterial colonization in Helicobacter pylori-infected SCID mice. Infect. Immun. 67: 4594-4602. [Abstract/Free Full Text]
  52. Eksteen, B., A. E. Miles, A. J. Grant, D. H. Adams. 2004. Lymphocyte homing in the pathogenesis of extra-intestinal manifestations of inflammatory bowel disease. Clin. Med. 4: 173-180. [Medline]
  53. Pallone, F., S. Fais, O. Squarcia, L. Biancone, P. Pozzilli, M. Boirivant. 1987. Activation of peripheral blood and intestinal lamina propria lymphocytes in Crohn’s disease: in vivo state of activation and in vitro response to stimulation as defined by the expression of early activation antigens. Gut 28: 745-753. [Abstract/Free Full Text]



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