B. Samuel Lundin

Helicobacter pylori-Specific CD4+ CD25high Regulatory T Cells Suppress Memory T-Cell Responses to H. pylori in Infected Individuals

ABSTRACT Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25high T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25high regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25high T cells suppressed H. pylori-induced responses in cocultures with CD25low/− cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.

Mucosal FOXP3-expressing CD4+ CD25high regulatory T cells in Helicobacter pylori-infected patients

ABSTRACT Helicobacter pylori chronically colonizes the stomach and duodenum and causes peptic ulcers or gastric adenocarcinoma in 10 to 20% of infected individuals. We hypothesize that the inability of patients to clear H. pylori infections is a consequence of active suppression of the immune response. Here we show that H. pylori-infected individuals have increased frequencies of CD4+ CD25high T cells in both the stomach and duodenal mucosa compared to uninfected controls. These cells have the phenotype of regulatory T cells, as they express FOXP3, a key gene for the development and function of regulatory T cells, as well as high levels of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) protein. In contrast, mucosal CD4+ CD25low and CD4+ CD25− cells express little FOXP3 mRNA and low levels of the CTLA-4 protein. Mucosal CD4+ CD25high T cells are present in individuals with asymptomatic H. pylori infections as well as in duodenal ulcer patients. The frequencies of CD4+ CD25high cells are also increased in the stomachs of H. pylori-infected patients with gastric adenocarcinoma, particularly in cancer-affected tissues. These findings suggest that regulatory T cells may suppress mucosal immune responses and thereby contribute to the persistence of H. pylori infections.

Identification of protein expression signatures associated with Helicobacter pylori infection and gastric adenocarcinoma using recombinant antibody microarrays.

Antibody microarray based technology is a powerful emerging tool in proteomics, target discovery, and differential analysis. Here, we report the first study where recombinant antibody fragments have been used to construct large scale antibody microarrays, composed of 127 different antibodies against mostly immunoregulatory antigens. The arrays were based on single framework recombinant antibody fragments (SinFabs) designed for high on-chip stability and functionality and were used for the analysis of malignant and normal stomach tissue samples from Helicobacter pylori-positive and -negative patients. Our results demonstrate that distinct tumor- as well as infection-associated protein expression signatures could be identified from these complex tissue proteomes, as well as biomarkers such as IL-9, IL-11, and MCP-4, previously not found in these diseases. In a longer perspective, this study may improve the understanding of H. pylori-induced stomach cancer and lead to development of improved diagnostics.

Oral immunization with a Salmonella enterica serovar typhi vaccine induces specific circulating mucosa-homing CD4(+) and CD8(+) T cells in humans.

ABSTRACT The kinetics and homing characteristics of T-cell responses in humans after mucosal immunizations have not been well characterized. Therefore, we have investigated the magnitude and duration of such responses as well as the homing receptor expression of antigen-specific peripheral blood T cells by using an oral model vaccine, i.e., the live, attenuated Salmonella enterica serovar Typhi vaccine (Ty21a). Eight volunteers were each given three doses of the vaccine 2 days apart, and blood samples, from which CD4+ and CD8+ T cells were selected by the use of magnetic beads, were collected before vaccination and at regular intervals thereafter. To purify the potentially antigen-specific gut-homing T cells, CD45RA− integrin β7+ cells were further sorted by flow cytometry. The sorted cells were then stimulated in vitro with the serovar Typhi vaccine strain, and the proliferation of cells and the cytokine production were measured. Following vaccination, there was a large increase in both the proliferation of and the gamma interferon (IFN-γ) production by blood T cells stimulated with the vaccine strain. The responses were seen among both CD4+ and CD8+ T cells, although the CD8+ cells produced the largest amounts of IFN-γ. Peak responses were seen 7 to 14 days after the onset of vaccination. Furthermore, most of the IFN-γ produced by both CD4+ and CD8+ cells emanated from cells with the potential to home to mucosal tissues, as the integrin β7-expressing memory T cells produced around 10-fold more IFN-γ than the remaining populations. In conclusion, we demonstrate that oral vaccination with a live oral bacterial vaccine induces antigen-specific CD4+ and CD8+ memory T cells, almost all of which express the gut-homing integrin β7.

Helicobacter pylori-Specific CD4+ T Cells Home to and Accumulate in the Human Helicobacter pylori-Infected Gastric Mucosa

ABSTRACT Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4+ but similar numbers of CD8+ lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4+ T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4+ T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4+ T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4+ T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4+ T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.

Natural Killer Cells and Helicobacter pylori Infection: Bacterial Antigens and Interleukin-12 Act Synergistically To Induce Gamma Interferon Production

ABSTRACT Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-γ in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-γ), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-γ by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-γ by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor β2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-γ when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection.

CD4+CD25high regulatory T cells reduce T cell transendothelial migration in cancer patients

Cell‐mediated immunity is thought to be the main mechanism of anti‐tumour responses of the host, but it is not known if cancer disease affects T cell recruitment from blood to tissues. Therefore, we compared Heliobacter pylori‐induced T cell transendothelial migration (TEM) in H. pylori‐infected gastric carcinoma patients, colon and lung carcinoma patients and healthy volunteers. H. pylori induced significant T cell migration from all groups. However, there was a dramatic reduction of T cell TEM in gastric carcinoma patients (80%) compared to healthy individuals. A similarly reduced transmigration was also seen in colon and lung carcinoma patients. We found significantly increased frequencies of Treg cells in the blood of gastric carcinoma patients compared to healthy individuals, and depletion of Treg cells from the blood of these patients prior to TEM restored T cell migration. The effect of Treg cells was largely dependent on cell‐cell contact, but not on IL‐10 or TGF‐β. In addition, the presence of Treg cells led to reduced T cell attachment to endothelium and decreased production of T cell‐recruiting chemokines during TEM. In conclusion, Treg cell‐mediated reduction of T cell TEM may reduce T cell recruitment in patients with epithelial malignancies, thereby hampering anti‐tumour responses.

DC-LAMP+ dendritic cells are recruited to gastric lymphoid follicles in Helicobacter pylori-infected individuals.

ABSTRACT Infection with Helicobacter pylori is associated with development of ulcer disease and gastrointestinal adenocarcinoma. The infection leads to a large infiltration of immune cells and the formation of organized lymphoid follicles in the human gastric mucosa. Still, the immune system fails to eradicate the bacteria, and the substantial regulatory T cell (Treg) response elicited is probably a major factor permitting bacterial persistence. Dendritic cells (DCs) are professional antigen-presenting cells that can activate naive T cells, and maturation of DCs is crucial for the initiation of primary immune responses. The aim of this study was to investigate the presence and localization of mature human DCs in H. pylori-infected gastric mucosa. Gastric antral biopsy specimens were collected from patients with H. pylori-associated gastritis and healthy volunteers, and antrum tissue was collected from patients undergoing gastric resection. Immunohistochemistry and flow cytometry showed that DCs expressing the maturation marker dendritic cell lysosome-associated membrane glycoprotein (DC-LAMP; CD208) are enriched in the H. pylori-infected gastric mucosa and that these DCs are specifically localized within or close to lymphoid follicles. Gastric DC-LAMP-positive (DC-LAMP+) DCs express CD11c and high levels of HLA-DR but little CD80, CD83, and CD86. Furthermore, immunofluorescence analyses demonstrated that DC-LAMP+ DCs are in the same location as FoxP3-positive putative Tregs in the follicles. In conclusion, we show that DC-LAMP+ DCs with low costimulatory capacity accumulate in the lymphoid follicles in human H. pylori-infected gastric tissue, and our results suggest that Treg-DC interactions may promote chronic infection by rendering gastric DCs tolerogenic.