Transmaternal Helicobacter pylori exposure reduces allergic airway inflammation in offspring through regulatory T cells.

BACKGROUND: Transmaternal exposure to tobacco, microbes, nutrients, and other environmental factors shapes the fetal immune system through epigenetic processes. The gastric microbe Helicobacter pylori represents an ancestral constituent of the human microbiota that causes gastric disorders on the one hand and is inversely associated with allergies and chronic inflammatory conditions on the other. OBJECTIVE: Here we investigate the consequences of transmaternal exposure to H pylori in utero and/or during lactation for susceptibility to viral and bacterial infection, predisposition to allergic airway inflammation, and development of immune cell populations in the lungs and lymphoid organs. METHODS: We use experimental models of house dust mite- or ovalbumin-induced airway inflammation and influenza A virus or Citrobacter rodentium infection along with metagenomics analyses, multicolor flow cytometry, and bisulfite pyrosequencing, to study the effects of H pylori on allergy severity and immunologic and microbiome correlates thereof. RESULTS: Perinatal exposure to H pylori extract or its immunomodulator vacuolating cytotoxin confers robust protective effects against allergic airway inflammation not only in first- but also second-generation offspring but does not increase susceptibility to viral or bacterial infection. Immune correlates of allergy protection include skewing of regulatory over effector T cells, expansion of regulatory T-cell subsets expressing CXCR3 or retinoic acid-related orphan receptor γt, and demethylation of the forkhead box P3 (FOXP3) locus. The composition and diversity of the gastrointestinal microbiota is measurably affected by perinatal H pylori exposure. CONCLUSION: We conclude that exposure to H pylori has consequences not only for the carrier but also for subsequent generations that can be exploited for interventional purposes.

Influenza A Virus Induces Autophagosomal Targeting of Ribosomal Proteins.

Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.

Macroautophagy Proteins Control MHC Class I Levels on Dendritic Cells and Shape Anti-viral CD8(+) T Cell Responses.

The macroautophagy machinery has been implicated in MHC class II restricted antigen presentation. Here, we report that this machinery assists in the internalization of MHC class I molecules. In the absence of the autophagy factors Atg5 and Atg7, MHC class I surface levels are elevated due to decreased endocytosis and degradation. Internalization of MHC class I molecules occurs less efficiently if AAK1 cannot be recruited via Atg8/LC3B. In the absence of Atg-dependent MHC class I internalization, dendritic cells stimulate CD8(+) T cell responses more efficiently in vitro and in vivo. During viral infections, lack of Atg5 results in enhanced influenza- and LCMV-specific CD8(+) T cell responses in vivo. Elevated influenza-specific CD8(+) T cell responses are associated with better immune control of this infection. Thus, the macroautophagy machinery orchestrates T cell immunity by supporting MHC class II but compromises MHC class I restricted antigen presentation.

CD56(bright)perforin(low) noncytotoxic human NK cells are abundant in both healthy and neoplastic solid tissues and recirculate to secondary lymphoid organs via afferent lymph.

As limited information is available regarding the distribution and trafficking of NK cells among solid organs, we have analyzed a wide array of tissues derived from different human compartments. NK cells were widely distributed in most solid tissues, although their amount varied significantly depending on the tissue/organ analyzed. Interestingly, the distribution appeared to be subset specific, as some tissues were preferentially populated by CD56(bright)perforin(low) NK cells, with others by the CD56(dim)perforin(high) cytotoxic counterpart. Nevertheless, most tissues were highly enriched in CD56(bright)perforin(low) cells, and the distribution of NK subsets appeared in accordance with tissue gene expression of chemotactic factors, for which receptors are differently represented in the two subsets. Remarkably, chemokine expression pattern of tissues was modified after neoplastic transformation. As a result, although the total amount of NK cells infiltrating the tissues did not significantly change upon malignant transformation, the relative proportion of NK subsets infiltrating the tissues was different, with a trend toward a tumor-infiltrating NK population enriched in noncytotoxic cells. Besides solid tissues, CD56(bright)perforin(low) NK cells were also detected in seroma fluids, which represents an accrual of human afferent lymph, indicating that they may leave peripheral solid tissues and recirculate to secondary lymphoid organs via lymphatic vessels. Our results provide a comprehensive mapping of NK cells in human tissues, demonstrating that discrete NK subsets populate and recirculate through most human tissues and that organ-specific chemokine expression patterns might affect their distribution. In this context, chemokine switch upon neoplastic transformation might represent a novel mechanism of tumor immune escape.

Studying MHC class II transport in dendritic cells.

Professional antigen presenting cells, such as dendritic cells, are effective in activating T lymphocytes due to their unique ability to present antigens in the context of both MHC class I and II molecules. After successful loading with antigenic peptides MHC class II molecules traffic from the late endosomal loading compartment to the plasma membrane to exert their function of presenting peptides to T helper lymphocytes. Various processes play a role in this event, which are only partly understood to date. The following protocols demonstrate a strategy of how to integrate high throughput datasets to select candidates possibly involved in MHC class II transport for in depth studies. A combination of proteomics, RNA interference and biochemical experimentation can uncover novel pathways regulating transport processes in primary dendritic cells.

Exploring genome-wide datasets of MHC class II antigen presentation.

MHC class II molecules (MHCII) are critical for presenting antigens to CD4(+) T-cells. They control ignition of CD4(+) T cells and are as such involved in most auto-immune diseases. To define proteins and pathways controlling MHCII antigen presentation and expression, we performed a genome-wide flow cytometry based RNAi screen. Hits were subsequently classified by two screens that monitored the intracellular distribution and transcription of MHCII. This multi-dimensional approach allowed subclassification of hits into functional groups as a first step to defining new pathways controlling MHCII antigen presentation. The datasets from this screen are used as a template for several follow-up studies. This overview focuses on how data from genome-wide screens can be used for target-lead finding, data mining, systems biology and systematic cell biology.

Dynamics within tetraspanin pairs affect MHC class II expression.

Late endosomal multivesicular bodies (MVBs) are complicated organelles with various subdomains located at the limiting membrane and the internal vesicles (ILVs). ILVs accumulate tetraspanins such as CD63 and CD82 that might form protein assemblies, including major histocompatibility complex class II (MHC-II) and its chaperone human leukocyte antigen (HLA)-DM. Here, we studied the effect of four late endosomal tetraspanin proteins on MHC-II expression. Silencing CD9, CD63 and CD81 enhanced MHC-II expression whereas silencing CD82 did not. No effect on peptide loading was observed. Using confocal FRET technology, we measured the dynamics of CD63 and CD82 interaction with MHC-II and its chaperone HLA-DM. CD63-CD82 interactions remained unaltered in the two MVB subdomains whereas the interactions between CD63 or CD82 homologous pairs changed. CD63 stably associated with MHC-II, and CD82 with HLA-DM, on both MVB subdomains whereas the CD82-MHC-II and CD63-HLA-DM interactions changed. These data visualize for the first time the protein dynamics of tetraspanin assemblies in MVB subdomains. CD63, unlike CD82, stably interacts with MHC-II at both MVB subdomains and controls MHC-II expression.

Towards a systems understanding of MHC class I and MHC class II antigen presentation.

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.

A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation.

MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFß. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.

Routes to manipulate MHC class II antigen presentation.

MHC class II molecules (MHC-II) present antigenic fragments acquired in the endocytic route to the immune system for recognition and activation of CD4+ T cells. This ignites a series of immune responses. MHC-II strongly correlates to most autoimmune diseases. Understanding the biology of MHC-II is therefore expected to translate into novel means of autoimmunity control or immune response improvement. Although the basic cell biology of MHC-II antigen presentation is well understood, many novel aspects have been uncovered in recent years including means of antigen delivery, preparation for MHC-II loading, transport processes and vaccination strategies. We will discuss past, present and future of these insights into the biology of MHC-II.

CD40 ligation restores type 1 polarizing capacity in TLR4-activated dendritic cells that have ceased interleukin-12 expression.

Inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN)-gamma induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized not only by a dynamic immune activating but also by tolerance-inducing phenotype associated with down-modulation of cytokines previously considered to be irreversible. CD40L on activated T cells further modifies DC differentiation. Using DNA micro-arrays, we showed down-regulated mRNA levels of TLR signalling molecules, whereas CD40/CD40L signalling molecules were up-regulated at a time when LPS/IFN-gamma-activated DCs had ceased cytokine expression. Accordingly, we demonstrated that CD40/CD40L but not TLR4 or TLR3 signalling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsRNA re-established the capacity for secreting interleukin (IL)-12 in primarily LPS/IFN-gamma-activated DCs, which have exhausted their potential for cytokine secretion. The resulting TH1 polarizing DC phenotype - which lacked accompanying secretion of the crucial immune suppressive factor IL-10 - maintained the potential for activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell-mediated stimulus at an exhausted DC state, which prevents an immune tolerant DC phenotype. These findings impact on the rational design of TLR-activated DC-based cancer vaccines for the induction of anti-tumoural CTL responses.

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells.

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN-gamma for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN-gamma for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.

Semi-mature IL-12 secreting dendritic cells present exogenous antigen to trigger cytolytic immune responses.

Dendritic cells (DC) are candidates for antigen-presenting cells that present exogenous antigen on MHC class I molecules to cytotoxic T lymphocytes (CTL), a process referred to as cross-priming. We triggered interleukin (IL)-12 release from DC, which was limited to the first day after maturation induction, by a combination of lipopolysaccharide (LPS) and interferon (IFN)-gamma. To stimulate T lymphocytes, we used soluble protein derived from lysis of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) or ovalbumin loaded onto DC. Co-culture was initiated 2-6 or 48 h after maturation corresponding to "semi-mature" actively IL-12-secreting type 1 DC (sm-DC1) or a "fully mature" DC1 that had lost the ability to release IL-12 (fm-DC1), respectively. IL-12-secreting sm-DC1 but not fm-DC1 efficiently triggered cytolytic activity in autologous T lymphocytes. The combination of IL-1beta, IL-6, TNF-alpha, and prostaglandin E2 generated type 2 DC that did not secrete IL-12 (DC2) and could not prime T-cell cytolytic activity. However, supplementation of cultures using DC2 with IL-12 resulted in CTL activity while the presence of anti-IL-12 monoclonal antibodies in cultures using IL-12 secreting sm-DC1 suppressed CTL activity. Thus, actively IL-12-secreting sm-DC1 are necessary and sufficient for the antigen-specific expansion of CTL in response to exogenously provided soluble antigen.

Engraftment potential of human amnion and chorion cells derived from term placenta.

BACKGROUND: Fetal membranes are tissues of particular interest for several reasons, including their role in preventing rejection of the fetus and their early embryologic origin. which may entail progenitor potential. The immunologic reactivity and the transplantation potential of amnion and chorion cells, however, remain to be elucidated. METHODS: Amnion and chorion cells were isolated from human term placenta and characterized by immunohistochemistry, flow cytometric analysis, and expression profile of relevant genes. The immunomodulatory characteristics of these cells were studied in allogeneic and xenogeneic mixed lymphocyte reactions and their engraftment potential analyzed by transplantation into neonatal swine and rats. Posttransplant chimerism was determined by polymerase chain reaction analysis with probes specific for human DNA. RESULTS: Phenotypic and gene expression studies indicated mesenchymal stem cell-like profiles in both amnion and chorion cells that were positive for neuronal, pulmonary, adhesion, and migration markers. In addition, cells isolated both from amnion and chorion did not induce allogeneic nor xenogeneic lymphocyte proliferation responses and were able to actively suppress lymphocyte responsiveness. Transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues. CONCLUSIONS: Human amnion and chorion cells from term placenta can successfully engraft neonatal swine and rats. These results may be explained by the peculiar immunologic characteristics and mesenchymal stem cell-like phenotype of these cells. These findings suggest that amnion and chorion cells may represent an advantageous source of progenitor cells with potential applications in a variety of cell therapy and transplantation procedures.