Tumor-infiltrating mucosal-associated invariant T (MAIT) cells retain expression of cytotoxic effector molecules.

Mucosal-associated invariant T (MAIT) cells all express a semi-invariable T cell receptor recognizing microbial metabolites presented on the MHC class I-like molecule MR1. Upon activation, they rapidly secrete cytokines and increase their cytotoxic potential. We showed recently that MAIT cells with Th1 phenotype accumulate in human colon adenocarcinomas. Here, we investigated the cytotoxic potential of tumor-infiltrating MAIT cells in colon adenocarcinomas, and to what extent it may be affected by the tumor microenvironment. Activation of MAIT cells from tumors induced increased Granzyme B, and to a lesser extent, perforin expression. Degranulation was assessed by surface expression of CD107a, and was also seen in response to cognate antigen recognition. The cytotoxic potential of tumor-associated MAIT cells was very similar to that of MAIT cells from unaffected colon. MAIT cells were also identified by immunofluorescence in direct contact with tumor cells in sections from colon cancer specimens. To summarize, tumor-associated MAIT cells from colon tumors have strong cytotoxic potential and are not compromised in this regard compared to MAIT cells from the unaffected colon. We conclude that MAIT cells may contribute significantly to the protective immune response to tumors, both by secretion of Th1-associated cytokines and by direct killing of tumor cells.

Human Mucosa-Associated Invariant T Cells Accumulate in Colon Adenocarcinomas but Produce Reduced Amounts of IFN-γ.

Mucosa-associated invariant T (MAIT) cells are innate-like T cells with a conserved TCR α-chain recognizing bacterial metabolites presented on the invariant MHC-related 1 molecule. MAIT cells are present in intestinal tissues and liver, and they rapidly secrete IFN-γ and IL-17 in response to bacterial insult. In colon cancer, IL-17-driven inflammation promotes tumor progression, whereas IFN-γ production is essential for antitumor immunity. Thus, tumor-associated MAIT cells may affect antitumor immune responses by their secreted cytokines. However, the knowledge of MAIT cell presence and function in tumors is virtually absent. In this study, we determined the frequency, phenotype, and functional capacity of MAIT cells in colon adenocarcinomas and unaffected colon lamina propria. Flow cytometric analyses showed significant accumulation of MAIT cells in tumor tissue, irrespective of tumor stage or localization. Colonic MAIT cells displayed an activated memory phenotype and expression of chemokine receptors CCR6 and CCR9. Most MAIT cells in unaffected colon tissues produced IFN-γ, whereas only few produced IL-17. Colonic MAIT cells also produced TNF-α, IL-2, and granzyme B. In the tumors, significantly lower frequencies of IFN-γ-producing MAIT cells were seen, whereas there were no differences in the other cytokines analyzed, and in vitro studies showed that secreted factors from tumor tissue reduced IFN-γ production from MAIT cells. In conclusion, MAIT cells infiltrate colon tumors but their ability to produce IFN-γ is substantially reduced. We suggest that MAIT cells have the capacity to promote local immune responses to tumors, but factors in the tumor microenvironment act to reduce MAIT cell IFN-γ production.

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

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.

DC-derived IL-18 drives Treg differentiation, murine Helicobacter pylori-specific immune tolerance, and asthma protection.

Persistent colonization with the gastric bacterial pathogen Helicobacter pylori causes gastritis and predisposes infected individuals to gastric cancer. Conversely, it is also linked to protection from allergic, chronic inflammatory, and autoimmune diseases. We demonstrate here that H. pylori inhibits LPS-induced maturation of DCs and reprograms DCs toward a tolerance-promoting phenotype. Our results showed that DCs exposed to H. pylori in vitro or in vivo failed to induce T cell effector functions. Instead, they efficiently induced expression of the forkhead transcription factor FoxP3, the master regulator of Tregs, in naive T cells. Depletion of DCs in mice infected with H. pylori during the neonatal period was sufficient to break H. pylori-specific tolerance. DC depletion resulted in improved control of the infection but also aggravated T cell-driven immunopathology. Consistent with the mouse data, DCs infiltrating the gastric mucosa of human H. pylori carriers exhibited a semimature DC-SIGN(+)HLA-DR(hi)CD80(lo)CD86(lo) phenotype. Mechanistically, the tolerogenic activity of H. pylori-experienced DCs was shown to require IL-18 in vitro and in vivo; DC-derived IL-18 acted directly on T cells to drive their conversion to Tregs. CD4(+)CD25(+) Tregs from infected wild-type mice but not Il18(-/-) or Il18r1(-/-) mice prevented airway inflammation and hyperresponsiveness in an experimental model of asthma. Taken together, our results indicate that tolerogenic reprogramming of DCs ensures the persistence of H. pylori and protects against allergic asthma in a process that requires IL-18.

Mucosal vaccination increases local chemokine production attracting immune cells to the stomach mucosa of Helicobacter pylori infected mice.

BACKGROUND: Vaccination is an attractive approach for the prevention of Helicobacter pylori infection and disease. In a mouse model, infection induces an accumulation of dendritic cells, macrophages, granulocytes, and B- and T cells to the stomach mucosa, which is further heightened when the infection is preceded by a mucosal immunization. We have studied the chemokines and chemokine receptors guiding infection- and vaccination-induced immune cells to the stomach and their relation to protection against H. pylori infection in mice. MATERIALS AND METHODS: C57BL/6 mice were immunized sublingually with H. pylori lysate antigens and cholera toxin adjuvant or left unimmunized, and then challenged with live H. pylori bacteria. Stomach tissue was taken at 3, 7, 14 and 21 days after challenge and bacterial colonization, chemokine and chemokine receptor gene expression, and influx of cells into the stomach mucosa were evaluated. RESULTS: RT-PCR array screening revealed differential expression of a broad range of chemokine and chemokine receptor genes between immunized and unimmunized mice. A significant upregulation of chemokines known to attract, among other cells, eosinophils (CCL8), T cells (CXCL10, CXCL11) and neutrophils (CXCL2, CXCL5) and of their cognate receptors CCR3, CXCR3 and CXCR2, preceded or coincided with vaccine-induced protection, which was first evident 7 days after infection and was then sustained at the later time-points. Consistent with the increase in chemokines and chemokine receptors flow cytometric analysis indicated a sequential accumulation of CD4(+) T cells, eosinophils, neutrophils and CD103(+) dendritic cells in the gastric lamina propria of immunized mice. CONCLUSIONS: This study provides insights into vaccination-induced chemokines that guide the influx of protective immune cells into the stomach of H. pylori infected mice.

Enhanced M1 macrophage polarization in human helicobacter pylori-associated atrophic gastritis and in vaccinated mice.

BACKGROUND: Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined. METHODOLOGY/PRINCIPAL FINDINGS: By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion. CONCLUSIONS/SIGNIFICANCE: These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.

Helicobacter pylori and its effect on innate and adaptive immunity: new insights and vaccination strategies.

Infection with the gastric bacterium Helicobacter pylori invariably leads to active chronic gastritis, and is strongly correlated to peptic ulcer disease, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. The infection leads to local accumulation of inflammatory cells and strong activation of B- and T-cell immunity. Still, the immune response can not eliminate the bacteria, and unless antibiotic treatment is used, the infection is usually lifelong. During the last few years, several immunomodulatory properties of H. pylori have been described, which probably contribute to the inability of the immune system to eradicate the bacterium. Another factor promoting bacterial persistence is probably the induction of a substantial regulatory T-cell response by the infection. Several different immunization schedules have resulted in protective immunity in animal models, while in humans no reliable vaccine is available as yet. In this article, we describe the innate and adaptive immune responses to H. pylori, and the attempts to create an effective vaccine.

Salmonella induces death of CD8alpha(+) dendritic cells but not CD11c(int)CD11b(+) inflammatory cells in vivo via MyD88 and TNFR1.

Dendritic cells (DCs), whose lifespan influences their ability to stimulate the immune system, are potent APCs that are critical for initiating immunity. Here, we show that oral infection with Salmonella enterica serovar Typhimurium induces death of DCs in the gut-draining lymph nodes. Although CD8alpha(+) DCs were sensitive to Salmonella-induced death, CD8alpha(-) DCs and in particular recruited CD11c(int)CD11b(+) inflammatory cells, were resistant. Infecting mice deficient for MyD88 revealed that Salmonella-induced death of CD8alpha(+) DCs was dependent on this adaptor for TLR signaling. In addition, CD8alpha(+) DCs in infected, TNFR1-deficient mice were resistant to Salmonella-induced death. These data, combined with the strict MyD88-dependent production of TNF in Salmonella-infected mice, suggest that MyD88-dependent TNF mediates DC death. As recruited CD11c(int)CD11b(+) cells were resistant to Salmonella-induced death, they could compensate for the infection-induced loss of DCs if they function as APCs. However, in contrast to DCs, CD11c(int)CD11b(+) cells could not present the model antigen OVA expressed in Salmonella to OVA-specific CD4 T cells. These results show that Salmonella induces DC death after oral infection via MyD88 and TNFR1, which could have a negative impact on the initiation of antibacterial immunity.

Early cellular responses to Salmonella infection: dendritic cells, monocytes, and more.

SUMMARY: Dendritic cells (DCs), monocytes, macrophages, and neutrophils are myeloid-derived phagocytes critical to controlling bacterial infections, and these cells have complementary functions to ensure host survival. Recent data have shed light on the dynamics and function of myeloid cells at the early stage of infection. In particular, murine infection models with Salmonella enterica serovar Typhimurium have been useful for understanding the host response required to develop immunity to systemic salmonellosis. This review summarizes the early cellular responses in the intestinal lymphoid tissues to Salmonella and discusses Peyer's patch-dependent and -independent penetration of bacteria through the intestinal epithelium. Once Salmonella accesses host tissue, phagocytes respond by recruitment, redistribution, and activation in intestinal tissues. Recruited monocytes are specialized in controlling bacterial replication by producing anti-microbial molecules but are poor antigen-presenting cells. In contrast, DCs undergo maturation by direct (bacteria-mediated) and indirect (cytokine-mediated) pathways in vivo to optimize their antigen presentation capacity, and directly matured DCs have unique mechanisms to ensure T-cell stimulation. Toll-like receptor signaling is critical to DC maturation and myeloid cell recruitment during Salmonella infection, and the role of myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways as well as proinflammatory cytokines and type 1 interferons in these processes are discussed.

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection.

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-alphabeta produced during infection. In infected MyD88(-/-)IFN-alphabetaR(-/-) mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-alphabeta, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella-containing DCs from MyD88(-/-) or MyD88(-/-)IFN-alphabetaR(-/-) mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.

TNF-alpha-dependent and -independent maturation of dendritic cells and recruited CD11c(int)CD11b+ Cells during oral Salmonella infection.

Maturation of dendritic cells (DC) is crucial for their ability to induce adaptive immunity. Although several mediators of DC maturation have been found, their contributions to DC maturation during infection are poorly understood. In this study we show that murine conventional (CD11c(high)) DC up-regulate costimulatory molecules in a subset-specific manner after oral Salmonella infection. Although both CD8alpha+ and CD8alpha- subsets increase CD86 expression, CD40 was preferentially up-regulated on CD8alpha+ DC, and CD80 was preferentially increased on CD8alpha- DC. In addition, high levels of CD80 and CD86 were found on CD11c(int)CD11b+ cells that accumulated in infected organs. Costimulatory molecules were simultaneously induced on CD11c(high) and CD11c(int)CD11b+ cells in Peyer's patches, mesenteric lymph nodes and spleen 5 days after infection despite different kinetics of peak bacterial burden in these organs. Up-regulation of costimulatory molecules occurred on all DC within the respective subset. Moreover, <1% of CD11c-expressing cells associated with Salmonella expressing enhanced GFP in vivo. Thus, DC maturation did not depend on bacterial uptake. Rather, infection-induced up-regulation of CD80, CD86, and CD40 on CD11c-expressing cells of mesenteric lymph nodes was dependent on TNFR type I (TNFRI) signaling. Although indirect up-regulation of costimulatory molecules on DC and CD11c(int)CD11b+ cells was TNFRI dependent, cells directly associated with Salmonella were able to mature independently of TNFRI signaling. Thus, Salmonella-induced TNF-alpha is an important mediator of indirect DC maturation during infection, whereas a TNF-alpha-independent maturation pathway contributes to direct maturation of bacteria-associated DC.

In vivo compartmentalization of functionally distinct, rapidly responsive antigen-specific T-cell populations in DNA-immunized or Salmonella enterica serovar Typhimurium-infected mice.

The location and functional properties of antigen-specific memory T-cell populations in lymphoid and nonlymphoid compartments following DNA immunization or infection with Salmonella were investigated. Epitope-specific CD8+ -T-cell expansion and retention during the memory phase were analyzed for DNA-immunized mice by use of a 5-h peptide restimulation assay. These data revealed that epitope-specific gamma interferon (IFN-gamma)-positive CD8+ T cells occur at higher frequencies in the spleen, liver, and blood than in draining or peripheral lymph nodes during the expansion phase. Moreover, this distribution is maintained into long-term memory. The location and function of both CD4+ and CD8+ Salmonella-specific memory T cells in mice who were given a single dose of Salmonella enterica serovar Typhimurium was also quantitated by an ex vivo restimulation with bacterial lysate to detect the total Salmonella-specific memory pool. Mice immunized up to 6 months previously with S. enterica serovar Typhimurium had bacterium-specific CD4+ T cells that were capable of producing IFN-gamma or tumor necrosis factor alpha (TNF-alpha) at each site analyzed. Similar findings were observed for CD8+ T cells that were capable of producing IFN-gamma, while a much lower frequency and more restricted distribution were associated with TNF-alpha-producing CD8+ T cells. This study is the first to assess the frequencies, locations, and functions of both CD4+ and CD8+ memory T-cell populations in the same Salmonella-infected individuals and demonstrates the organ-specific functional compartmentalization of memory T cells after Salmonella infection.

Immunity to Salmonella from a dendritic point of view.

Dendritic cells (DC) are the key link between innate and adaptive immunity. Features of DC, including their presence at sites of antigen entry, their ability to migrate from peripheral sites to secondary lymphoid organs, and their superior capacity to stimulate naïve T cells places them in this pivotal role in the immune system. DC also produce cytokines, particularly IL-12, upon antigen encounter and can thus influence the ensuing adaptive immune response. As DC are phagocytic antigen-presenting cells located at sites exposed to bacterial invaders, studies have been performed to gain insight into the role of DC in combating bacterial infections. Indeed, studies with Salmonella have shown that DC can internalize and process this bacterium for peptide presentation on MHC-II as well as MHC-I. DC can also act as bystander antigen--presenting cells by presenting Salmonella antigens after internalizing neighbouring cells that have undergone Salmonella-induced apoptotic death. DC also produce IL-12 and TNF-alpha upon Salmonella encounter. Moreover, studies in a murine infection model have shown that splenic DC increase surface expression of co-stimulatory molecules during infection, and DC contain intracellular bacteria. In addition, quantitative changes occur in splenic DC numbers in the early stages of oral Salmonella infection, and this is accompanied by redistribution of the defined DC subsets in the spleen of infected mice. DC from Salmonella-infected mice also produce cytokines and can stimulate bacteria-specific T cells upon ex vivo co-culture. In addition, DC may play a role in the traversal of bacteria from the intestinal lumen. Studying the function of DC during Salmonella infection provides insight into the capacity of this sophisticated antigen-presenting cell to initiate and modulate the immune response to bacteria.

Dendritic cells as inducers of antimicrobial immunity in vivo.

Models of infection have provided important insight into the function of dendritic cells (DC) in vivo. Several microbial products induce DC maturation via Toll-like receptors, a process that is crucial for the ability of DC to initiate adaptive immune responses. Splenic DC have also been shown to produce IL-12 during infection in vivo. This DC-derived IL-12 might be important to skew T cell responses towards Th1. Microbial infections also induce changes in the DC populations of lymphoid organs, often in a subset-specific manner, manifested as an accumulation and redistribution of DC. Furthermore, data are emerging pointing at an absolute requirement of DC in priming of naïve T cells in vivo.