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A receptor which binds secretory IgA (sIgA) is expressed on human T cells from patients with systemic lupus erythematosus, rheumatoid arthritis, Behcet's syndrome and IgA nephropathy and on normal T cells following phytohaemagglutinin (PHA) stimulation. The specificity of this receptor was initially probed with a panel of normal serum immunoglobulins in competitive inhibition assays with sIgA using two-colour immunofluorescence. While the receptor showed the strongest affinity for IgA1 (IC(50)10(-6) M), IgD which has a similarly glycosylated hinge region to IgA1, also bound to the receptor (IC50 10(-5) M). IgA2, which lacks the 'O'-glycosylated hinge region, did not significantly inhibit the binding at these concentrations suggesting that the IgA determinants for this receptor might be the oligosaccharides present in the hinge region of IgA1. IgA1 has up to 10 'O'-linked oligosaccharides and four N-linked oligosaccharides per molecule. In order to probe the role of the 'O'-linked hinge sugars in the binding event, a sugar library was prepared from IgA1 by a procedure designed to release 'O'-linked oligosaccharides preferentially, and to retain them in the natural closed ring formation. The sugars were released by hydrazinolysis at 65 degrees and the resulting oligosaccharide library analysed by high voltage paper electrophoresis (HVE) and P4 gel permeation chromatography. Competitive inhibition studies demonstrated that both the library and the individual 'O'-linked sugars associated with IgA1 were implicated in the binding of IgA1 to this receptor (IC50 between 1 x 10(-5) M and 6 x 10(-5) M). Within this range the individual sugars showed small differences in their affinity for the receptor in the following order: Gal beta 3GalNAc = NeuNAc2 alpha 3(6)Gal beta 3GalNAc > NeuNAc2 alpha 3(6)Gal beta 3[NeuNAc2 alpha 6]GalNAc > or = GalNAc.

Two human natural anti-carbohydrate antibodies appeared in critical evolutionary events that brought primates and hominins to brink of extinction. The first is the anti-Gal antibody, produced in Old-World monkeys (OWM), apes and humans. It binds the carbohydrate-antigen 'α-gal epitope' (Galα1-3Galβ1-4GlcNAc-R) on carbohydrate-chains (glycans) synthesized by non-primate mammals, lemurs and New-World monkeys (NWM). The second is anti-N-glycolylneuraminic-acid (anti-Neu5Gc) antibody binding Neu5Gc on glycans synthesized by OWM, apes and most non-primate mammals. Ancestral OWM and apes synthesized α-gal epitopes and were eliminated ~20-30 million-years-ago (mya). Only few accidentally mutated offspring lacking α-gal epitopes, produced anti-Gal and survived. Hominin-populations living ~3 mya synthesized Neu5Gc and were eliminated, but few mutated offspring that accidently lost their ability to synthesize Neu5Gc, produced natural anti-Neu5Gc antibody. These hominins survived and ultimately evolved into present-day humans. It is argued that these two near-extinction events were likely to be the result of epidemics caused by highly virulent and lethal enveloped viruses that killed parental-populations. These viruses presented α-gal epitopes or Neu5Gc synthesized in host-cells of the parental-populations. Mutated offspring survived the epidemics because they were protected from the lethal virus by the natural anti-Gal or anti-Neu5Gc antibodies they produced due to loss of immune-tolerance to α-gal epitopes or to Neu5Gc, respectively., (© 2022 John Wiley & Sons Ltd.)

Trained immunity (TRAIM) may be defined as a form of memory where innate immune cells such as monocytes, macrophages, dendritic and natural killer (NK) cells undergo an epigenetic reprogramming that enhances their primary defensive capabilities. Cross-pathogen protective TRAIM can be triggered in different hosts by exposure to live microbes or microbe-derived products such as heat-inactivated Mycobacterium bovis or with the glycan α-Gal to elicit protective responses against several pathogens. We review the TRAIM paradigm using two models representing distinct scales of immune sensitization: the whole bacterial cell and one of its building blocks, the polysaccharides or glycans. Observations point out to macrophage lytic capabilities and cytokine regulation as two key components in non-specific innate immune responses against infections. The study of the TRAIM response deserves attention to better characterize the evolution of host-pathogen cooperation both for identifying the aetiology of some diseases and for finding new therapeutic strategies. In this field, the zebrafish provides a convenient and complete biological system that could help to deepen in the knowledge of TRAIM-mediated mechanisms in pathogen-host interactions., (© 2022 The Authors. Immunology published by John Wiley & Sons Ltd.)

Pulmonary fibrosis involves various types of immune cells and soluble mediators, including TGF-β and IL-35, a recently identified heterodimeric cytokine that belongs to the IL-12 cytokine family. However, the effect of regulatory IL-35 may play an important role in fibrotic diseases. The aim of this paper is to explore the immunoregulatory role of IL-35 in the development of fibrosis in interstitial lung disease (ILD). To gain a better understanding of this issue, the concentrations of IL-35 and different profibrotic cytokines in fibrotic (F-ILD) and non-fibrotic (NF-ILD) patients by ELISA were compared to that of intracellular IL-35 and IL-17 on CD4+ T cells stimulated in the presence of BAL or with different ratios of recombinant IL-35 (rIL-35) and TGF-β (rTGF-β), which were evaluated by flow cytometry. We observed that BAL concentration of IL-35 was lower in F patients (p < 0.001) and was negatively correlated with concentrations of TGF-β (p < 0.001) and IL-17 (p < 0.001). In supplemented cell cultures, BAL from NF but not F patients enhanced the percentage of IL-35 + CD4+ T (p < 0.001) cells and decreased the percentage of IL-17 + CD4+ T cells (p < 0.001). The percentage of IL-35 + CD4+ T cells correlated positively with BAL concentration of IL-35 (p = 0.02), but correlated negatively with BAL concentrations of IL-17 (p = 0.007) and TGF-β (p = 0.01). After adjusting the concentrations of recombinant cytokines to establish a TGF-β: IL-35 ratio of 1:4, an enhanced percentage of IL-35 + CD4+ T cells (p < 0.001) but a decreased percentage of IL-17 + CD4+ T cells (p < 0.001) was observed. After adding recombinant IL-35 to the BAL from F patients until a 1:4 ratio of TGF-β: IL-35 was reached, a significantly increased percentage of IL-35 + CD4+ T cells (p < 0.001) and a decreased percentage of IL-17 + CD4+ T cells (p = 0.003) was found. These results suggest that IL-35 may induce an anti-fibrotic response, regulating the effect of TGF-β and the inflammatory response on CD4+ T cells. In addition, the TGF-β: IL-35 ratio in BAL has been shown to be a potential biomarker to predict the outcome of F patients with ILD., (© 2022 John Wiley & Sons Ltd.)

In the past few years, the evolution of immunotherapy has resulted in a shift in cancer treatment models. However, with immunosuppressive effects of the tumour microenvironment continues to limit advances in tumour immunotherapy. The tumour microenvironment induces metabolic reprogramming in cancer cells, which results in competition for nutrients between tumour cells and host immunocytes. Metabolic and waste products originating in tumour cells can influence the activation and effector properties of immunocytes in numerous ways and ultimately promote the survival and propagation of tumour cells. In this paper, we discuss metabolic reprogramming in tumour cells and the influence of metabolite by-products on the immune microenvironment, providing novel insights into tumour immunotherapy., (© 2021 John Wiley & Sons Ltd.)

Some human antibodies may paradoxically inhibit complement activation on bacteria and enhance pathogen survival in humans. This property was also claimed for IgG antibodies reacting with terminal galactose-α-1,3-galactose (Galα3Gal; IgG anti-αGal), a naturally occurring and abundant antibody in human plasma that targets numerous different pathogens. To reinvestigate these effects, we used IgG anti-αGal affinity isolated from a pool of normal human IgG and human hypogammaglobulinaemia serum as a complement source. Flow cytometry was performed to examine antibody binding and complement deposition on pig erythrocytes, Escherichia coli O86 and Streptococcus pneumoniae serotype 9V. Specific nanobodies were used to block the effect of single complement factors and to delineate the complement pathways involved. IgG anti-αGal was capable of activating the classical complement pathway on all the tested target cells. The degree of activation was exponentially related to the density of bound antibody on E. coli O86 and pig erythrocytes, but more linearly on S. pneumoniae 9V. The alternative pathway of complement amplified complement deposition. Deposited C3 fragments covered the activating IgG anti-αGal, obstructing its detection and highlighting this as a likely general caveat in studies of antibody density and complement deposition. The inherent capacity for complement activation by the purified carbohydrate reactive IgG anti-αGal was similar to that of normal human IgG. We propose that the previously reported complement inhibition by IgG anti-αGal relates to suboptimal assay configurations, in contrast to the complement activating property of the antibodies demonstrated in this paper., (© 2020 John Wiley & Sons Ltd.)

There have been substantial strides forward in our understanding of the contribution of regulatory T (Treg) cells to cancer immunosuppression. In this issue, we present a series of papers highlighting emerging themes on this topic relevant not only to our understanding of the fundamental biology of tumour immunosuppression but also to the design of new immunotherapeutic approaches. The substantially shared biology of CD4 + conventional T (Tconv) and Treg cells necessitates a detailed understanding of the potentially opposing functional consequences that immunotherapies will have on Treg and Tconv cells, a prominent example being the potential for Treg-mediated hyperprogressive disease following anti-PD-1 therapy. Such understanding will aid patient stratification and the rational design of combination therapies. It is also becoming clear, however, that Treg cells within tumours exhibit distinct biological features to both Tconv cells and Treg cells in other tissues. These distinct features provide the opportunity for development of targeted immunotherapies with greater efficacy and reduced potential for inducing systemic toxicity., (© 2019 John Wiley & Sons Ltd.)

MHC class I-related gene protein (MR1) is a non-polymorphic MHC class IB antigen-presenting molecule that is the restricting molecule for mucosal-associated invariant T (MAIT) cells, a prominent population of innate-like antibacterial T cells. The MAIT cell-MR1 axis represents a new paradigm in antigen presentation, with the MR1 ligand derived from vitamin B compounds or their metabolic precursors. Many bacteria and some fungi produce the activating ligand for MR1. In evolution, MR1 is highly conserved in most, but not all, mammals. In humans and rodents it is expressed in a broad range of cell types, both haematopoietic and non-haematopoietic, although cell surface expression has been difficult to detect. Although MR1 trafficking shares features with both the MHC class I and MHC class II pathways, it is distinct. Several strands of evidence suggest that the intracellular location where MR1 is loaded differs for soluble ligand and for ligand derived from intact bacteria. The regulation of MR1 surface expression may also vary between different cell types. This paper will review what is currently known about the expression and trafficking of MR1 and propose a model for the loading and trafficking of MR1., (© 2017 John Wiley & Sons Ltd.)

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti-schistosome sera. A pair of cross-reactive peanut molecules at ~30 000-33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti-S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α-1 and κ-5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α-1 or κ-5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross-reactivities. The results are consistent with the antigenic cross-reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross-reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on 'blocking antibodies' could provide an insight for the inverse relationship observed between schistosome infection and allergies., (© 2017 John Wiley & Sons Ltd.)

The nuclear factor-κB (NF-κB) family of transcription factors play an essential role for the regulation of inflammatory responses, immune function and malignant transformation. Aberrant activity of this signalling pathway may lead to inflammation, autoimmune diseases and oncogenesis. Over the last two decades great progress has been made in the understanding of NF-κB activation and how the response is counteracted for maintaining tissue homeostasis. Therapeutic targeting of this pathway has largely remained ineffective due to the widespread role of this vital pathway and the lack of specificity of the therapies currently available. Besides regulatory proteins and microRNAs, long non-coding RNA (lncRNA) is emerging as another critical layer of the intricate modulatory architecture for the control of the NF-κB signalling circuit. In this paper we focus on recent progress concerning lncRNA-mediated modulation of the NF-κB pathway, and evaluate the potential therapeutic uses and challenges of using lncRNAs that regulate NF-κB activity., (© 2016 John Wiley & Sons Ltd.)

B cells have an important pathogenic role in the development of type 1 diabetes in the non-obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACI high -expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation-inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up-regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down-regulate TACI. Availability of the TACI ligand B-cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4-hydroxy-3-nitrophenylacetyl hapten-conjugated hen egg lysozyme, NOD mice produced higher titres of low-affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse., (© 2016 John Wiley & Sons Ltd.)

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency., (© 2012 Blackwell Publishing Ltd.)

Natural killer (NK) cells provide an immediate first line of defence against viral infections. Memory responses, maintained by CD4(+) T cells, require exposure to viral antigen and provide long-term protection against future infections. It is known that NK cells can promote the development of the adaptive response through cytokine production and cross-talk with antigen-presenting cells. In this paper however, we summarize a series of recent publications, in mouse models and for the first time in man, with the unifying message that rapid viral antigen control by the innate immune system limits antigen exposure to CD4(+) cells thereby abrogating the development of a memory response. We discuss the significant implication of these studies on viral treatment strategies and immunization models., (© 2012 Blackwell Publishing Ltd.)

As the amount of biomedical information available in the literature continues to increase, databases that aggregate this information continue to grow in importance and scope. The population of databases can occur either through fully automated text mining approaches or through manual curation by human subject experts. We here report our experiences in populating the National Institute of Allergy and Infectious Diseases sponsored Immune Epitope Database and Analysis Resource (IEDB, http://iedb.org), which was created in 2003, and as of 2012 captures the epitope information from approximately 99% of all papers published to date that describe immune epitopes (with the exception of cancer and HIV data). This was achieved using a hybrid model based on automated document categorization and extensive human expert involvement. This task required automated scanning of over 22 million PubMed abstracts followed by classification and curation of over 13 000 references, including over 7000 infectious disease-related manuscripts, over 1000 allergy-related manuscripts, roughly 4000 related to autoimmunity, and 1000 transplant/alloantigen-related manuscripts. The IEDB curation involves an unprecedented level of detail, capturing for each paper the actual experiments performed for each different epitope structure. Key to enabling this process was the extensive use of ontologies to ensure rigorous and consistent data representation as well as interoperability with other bioinformatics resources, including the Protein Data Bank, Chemical Entities of Biological Interest, and the NIAID Bioinformatics Resource Centers. A growing fraction of the IEDB data derives from direct submissions by research groups engaged in epitope discovery, and is being facilitated by the implementation of novel data submission tools. The present explosion of information contained in biological databases demands effective query and display capabilities to optimize the user experience. Accordingly, the development of original ways to query the database, on the basis of ontologically driven hierarchical trees, and display of epitope data in aggregate in a biologically intuitive yet rigorous fashion is now at the forefront of the IEDB efforts. We also highlight advances made in the realm of epitope analysis and predictive tools available in the IEDB., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

As a commentary on a recently published paper in Immunology, this article summaries the principle of norovirus P particle as a promising vaccine against noroviruses. It emphasizes the importance of P particle formation in the immune enhancement of the vaccine and methods for production/verification of high quality P particles which may be easily neglected by researchers., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

The bacterial superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes are potent stimulators of polyclonal T-cell proliferation. They are the causes of toxic shock syndrome but also induce CD25(+)  FOXP3(+) regulatory cells in the CD4 compartment. Several studies have recently described different forms of antigen-induced regulatory CD8(+) T cells in the context of inflammatory diseases and chronic viral infections. In this paper we show that bacterial superantigens are potent inducers of human regulatory CD8(+) T cells. We used four prototypic superantigens of S. aureus (toxic shock syndrome toxin-1 and staphylococcal enterotoxin A) and Str. pyogenes (streptococcal pyrogenic exotoxins A and K/L). At concentrations below 1 ng/ml each toxin triggers concentration-dependent T-cell receptor Vβ-specific expression of CD25 and FOXP3 on CD8(+) T cells. This effect is independent of CD4(+) T-cell help but requires antigen-presenting cells for maximum effect. The cells also express the activation/regulatory markers cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumour necrosis factor receptor-related protein and skin homing adhesins CD103 and cutaneous lymphocyte-associated antigen. Superantigen-induced CD25(+)  FOXP3(+)  CD8(+) T cells were as potent as freshly prepared naturally occurring CD4(+) regulatory T cells in suppressing proliferation of CD4(+)  CD25(-) T cells in response to anti-CD3 stimulation. Although superantigen-induced CD8(+)  CD25(+) FOXP3(+) express interleukin-10 and interferon-γ their suppressive function is cell contact dependent. Our findings indicate that regulatory CD8(+) T cells may be a feature of acute bacterial infections contributing to immune evasion by the microbe and disease pathogenesis. The presence and magnitude of regulatory CD8(+) T-cell responses may represent a novel biomarker in such infections. Superantigen-induced regulatory CD8(+) T cells also have therapeutic potential., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

This paper describes an experimental model of neuroinflammation based on the production of interleukin-6 (IL-6) by neural glial cells infected with Theiler's murine encephalomyelitis virus (TMEV). Production of IL-6 mRNA in mock-infected and TMEV-infected SJL/J murine astrocytes was examined using the Affymetrix murine genome U74v2 DNA microarray. The IL-6 mRNA from infected cells showed an eightfold increase in hybridization to a sequence encoding IL-6 located on chromosome number 5. Quantitative real-time reverse transcription PCR (qPCR) was used to study the regulation of IL-6 expression. The presence of IL-6 in the supernatants of TMEV-infected astrocyte cultures was quantified by ELISA and found to be weaker than in cultures of infected macrophages. The IL-6 was induced by whole TMEV virions, but not by Ad.βGal adenovirus, purified TMEV capsid proteins, or UV-inactivated virus. Two recombinant inflammatory cytokines, IL-1α and tumour necrosis factor-α were also found to be potent inducers of IL-6. The secreted IL-6 was biologically active because it fully supported B9 hybridoma proliferation in a [(3) H]thymidine incorporation bioassay. The cerebrospinal fluid of infected mice contained IL-6 during the acute encephalitis phase, peaking at days 2-4 post-infection. Finally, this in vitro neuroinflammation model was fully inhibited, as demonstrated by ELISA and qPCR, by five selective oestrogen receptor modulators., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

Myeloid-derived suppressor cells (MDSC) are important to the tumour microenvironment as they actively suppress the immune system and promote tumour progression and metastasis. These cells block T-cell activation in the tumour microenvironment, preventing anti-tumour immune activity. The ability of a treatment to alter the suppressive function of these cells and promote an immune response is essential to enhancing overall therapeutic efficacy. Interleukin-12 (IL-12) has the potential not only to promote anti-tumour immune responses but also to block the activity of cells capable of immune suppression. This paper identifies a novel role for IL-12 as a modulator of MDSC activity, with implications for IL-12 as a therapeutic agent. Treatment with IL-12 was found to alter the suppressive function of MDSC by fundamentally altering the cells. Interleukin-12-treated MDSC exhibited up-regulation of surface markers indicative of mature cells as well as decreases in nitric oxide synthase and interferon-γ mRNA both in vitro and in vivo. Treatment with IL-12 was also found to have significant therapeutic benefit by decreasing the percentage of MDSC in the tumour microenvironment and increasing the percentage of active CD8(+) T cells. Treatment with IL-12 resulted in an increase in overall survival accompanied by a reduction in metastasis. The findings in this paper identify IL-12 as a modulator of immune suppression with significant potential as a therapeutic agent for metastatic breast cancer., (© 2011 The Authors. Immunology © No claim to original US government works.)

Removal of apoptotic cells from inflammatory sites by macrophages is an important step in the resolution of inflammation. However, the effect of inflammatory modulators on phagocytic clearance of apoptotic cells remains to be clarified. In this paper, we demonstrate that lipopolysaccharide (LPS), a potent inflammatory agent, inhibits the phagocytosis of apoptotic neutrophils by mouse peritoneal macrophages. This inhibition can be attributed to both LPS-mediated induction of tumour necrosis factor (TNF-α) and suppression of growth arrest-specific gene 6 (Gas6) in macrophages. We found that LPS-induced TNF-α production inhibited phagocytic ability of macrophages in an autocrine manner. In contrast, Gas6 expression in macrophages was blocked by LPS, which also contributes to the inhibition of macrophage phagocytosis by LPS. Our data suggest that phagocytic clearance of apoptotic neutrophils by macrophages can be regulated by local pro- and anti-inflammatory factors in two opposite states.

Peripheral blood CD4(+) CD45RO(+) T cells activated in vitro are able to induce expression of tumour necrosis factor-α (TNF-α) in monocytes via a contact-dependent mechanism. Activation is achieved either with interleukin-2 (IL-2)/IL-6/TNF-α over an 8-day period or cross-linking CD3 using anti-CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen-activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti-CD3 activated T cells, p38 MAPK is a negative regulator for anti-CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte-derived TNF-α) or production of T-cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine-induced effector function and promotes cell proliferation and cytokine production.

The ultimate outcome of T cell receptor recognition is determined by the context in which the antigen is encountered. In this fashion both antigen-presenting cells and T cells must integrate multiple environmental cues in the form of pathogen-associated molecular patterns, cytokines and accessory molecule signals. The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays a central role in integrating environmental signals critical to regulating metabolism and cell survival. In this paper we review the data demonstrating that mTOR integrates signals from the immune microenvironment and therefore facilitates the generation of the adaptive immune response. Specifically, we review the role of mTOR in promoting dendritic cell activation and maturation, in regulating full T cell activation versus anergy, and influencing the induction of regulatory T cells.

Mannose-binding lectin (MBL) exists in the serum as a complex with MBL-associated serine protease (MASP). A recent paper described how MASP-free recombinant rat MBL stimulates the phagocytosis of Escherichia coli and Staphylococcus aureus by rat Kupffer cells through an increase in the level of a phagocytosis receptor. We have examined the effect of human MBL on the phagocytic action of human macrophages. Purified recombinant human MBL stimulated the phagocytosis of E. coli by THP-1 macrophages, leaving that of latex beads, apoptotic human cells, zymosan particles or S. aureus unchanged. This stimulatory effect was observed when either phagocytes or targets were preincubated with MBL. Furthermore, MBL bound to THP-1 macrophages as well as to E. coli, but not to S. aureus, through lipid A. These results indicated that human MBL in the absence of MASP stimulates macrophage phagocytosis of E. coli by bridging targets and phagocytes.

In the early 1970s a spate of papers by research groups around the world provided evidence for a negative regulatory role of thymus-derived lymphocytes (T cells). In 1971, Gershon and Kondo published a seminal paper in Immunology entitled 'Infectious Immunological Tolerance' indicating that such negative regulation could be a dominant effect that prevented otherwise 'helpful' T cells from mediating their function. Over the next decade, suppressor T cells, as these negative regulatory cells became known, were intensively investigated and a complex set of interacting cells and soluble factors were described as mediators in this process of immune regulation. In the early 1980s, however, biochemical and molecular experiments raised questions about the interpretation of the earlier studies, and within a few years, the term 'suppressor T cell' had all but disappeared from prominence and research on this phenomenon was held in poor esteem. While this was happening, new studies appeared suggesting that a subset of T cells played a critical role in preventing autoimmunity. These T cells, eventually dubbed 'regulatory T cells', have become a major focus of modern cellular immunological investigation, with a predominance that perhaps eclipses even that seen in the earlier period of suppressor T cell ascendancy. This brief review summarizes the rise and fall of 'suppressorology' and the possibility that Tregs are a modern rediscovery of suppressor T cells made convincing by more robust models for their study and better reagents for their identification and analysis.

Pemphigus vulgaris (PV) is considered to be an autoimmune disease affecting skin and mucous membranes. Traditionally, PV autoantibodies are thought to recognize antigens located in the intercellular substance (ICS) of keratinocytes; antigens represented mainly by the desmosomal cadherin desmoglein 3 (Dsg3). Accordingly, titres of anti-ICS and anti-Dsg3 immunoglobulin G (IgG) are considered to be major laboratory criteria when making a diagnosis of PV. In this paper, we demonstrated for the first time that PV IgG bind antigen(s) expressed on the surface of peripheral blood mononuclear cells (PBMC), as revealed by immunofluorescence studies. This novel autoantigen is immunoprecipitated by PV IgG as a 130 000 molecular weight protein. However, Western blot analysis of the immunocomplexes failed to show reactivity with anti-Dsg3 monoclonal and polyclonal antibodies. Taken together, our data provide strong evidence that PV autoimmunity targets a 130 000 antigen other than Dsg3 on PBMC. This shifting from epidermis to blood cells may open new perspectives for a better understanding of pemphigus autoimmunity and more rational approaches to its treatment.

CCL25/TECK and CCL28/MEC are CC chemokines primarily expressed in thymic dendritic cells and mucosal epithelial cells. The cognate receptors of CCL25 and CCL28, CCR9 and CCR10, respectively, are mainly expressed on T and B lymphocytes. In human, mouse and pig, CCL25 and CCL28 play a key role in the segregation and the compartmentalization of the mucosal immune system through recruitment of immune cells to specific locations. However, little is known about their role in the ontogeny of the mucosal immune system during fetal development. In the present paper, we report the cloning and the sequencing of ovine CCL25, CCL28, CCR9 and CCR10 and the subsequent assessment of their mRNA expression by q-polymerase chain reaction in several tissues, including thymus, gut-associated lymphoid tissue and mammary gland, from young and adult sheep and in the fetal lamb during the development of the immune system. CCL25 mRNA was highly expressed in thymus and gut while CCL28 mRNA was more expressed in large intestine, trachea, tonsils and mammary gland, especially at the end of gestation. These results are consistent with observations in other species suggesting similar roles for these chemokines in sheep. In fetuses, mRNA of CCL25, CCL28 and their receptors are expressed early in the thymus and mucosal tissues, including the small intestine and the nasal mucosa. Furthermore, their expression increased towards the end of gestation. Consequently, we hypothesize that CCL25 and CCL28 play an important role in the lymphocyte colonization of fetal tissues, enabling the development of a functional immune system.

It has been known for 15 years that the chicken epidermis contains ATPase+ and major histocompatibility complex class II-positive (MHCII+) dendritic cells. These cells were designated as Langerhans cells but neither their detailed phenotype nor their function was further investigated. In the present paper we demonstrate a complete overlapping of ATPase, CD45 and vimentin staining in all dendritic cells of the chicken epidermis. The CD45+ ATPase+ vimentin+ dendritic cells could be divided into three subpopulations: an MHCII+ CD3- KUL01+ and 68.1+ (monocyte-macrophage subpopulation markers) subpopulation, an MHCII- CD3- KUL01- and 68.1- subpopulation and an MHCII- CD3+ KUL01- and 68.1- subpopulation. The first population could be designated as chicken Langerhans cells. The last population represents CD4- CD8- T-cell receptor-alphabeta- and -gammadelta- natural killer cells with cytoplasmic CD3 positivity. The epidermal dendritic cells have a low proliferation rate as assessed by bromodeoxyuridine incorporation. Both in vivo and in vitro experiments showed that dendritic cells could be mobilized from the epidermis. Hapten treatment of epidermis resulted in the decrease of the frequency of epidermal dendritic cells and hapten-loaded dendritic cells appeared in the dermis or in in vitro culture of isolated epidermis. Hapten-positive cells were also found in the so-called dermal lymphoid nodules. We suggest that these dermal nodules are responsible for some regional immunological functions similar to the mammalian lymph nodes.

Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca(2+)-dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a band of approximately 53,000 MW in the reduced state and approximately 138,000 MW in the unreduced state. Porcine SP-D was sensitive to collagenase digestion and N-deglycosylation, which reduced the molecular mass to approximately 24,000 MW and approximately 48,000 MW respectively, in the reduced state. N-deglycosylation of the collagen-resistant fragment, reduced the molecular mass to approximately 21,000 MW showing the presence of an N-glycosylation site located in the CRD. Porcine SP-D bound to solid-phase mannan in a dose and Ca(2+)-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP-D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkuhn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

The human mast cell line (HMC)-1 cell line is growth-factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC-1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC-1, named HMC-1(560 ) and HMC-1(560,816 ), with different phenotypes and designated by the locations of specific mutations in the c-kit proto-oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing trade mark method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c-kit gene causing an amino acid substitution of Gly-560 for Val. In contrast, only HMC-1(560,816) cells have the c-kitV816 mutation found in mast cell neoplasms causing an Asp-->Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3'-kinase (PI 3-kinase) in both variants of HMC-1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC-1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.

CD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti-CD26 monoclonal antibody (mAb) 1F7 inhibits human T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines and human T-cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti-CD26 mAb 1F7 appears to be induced through activation of extracellular signal-regulated kinase (ERK) pathway. These data thus suggest that anti-CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft-vs.-host disease.

Macrophages can process and present exogenous antigens on major histocompatibility complex (MHC) class I molecules through an alternative mechanism involving the internalization of antigens and the secretion of peptides loading MHC class I molecules at the cell surface. In this paper, we found that interferon-gamma (IFN-gamma) -activated macrophages infected with Salmonella typhimurum secreted peptides able to load empty MHC Kb molecules on co-cultured TAP-2-deficient RMA-S cells, added as targets for peptide loading. The increase in class I Kb on the RMA-S cells, resulting from the macrophage-derived peptides, exhibited a comparable stability as the direct addition of an exogenous Kb-binding peptide (OVA257-264) to the RMA-S cells. In both cases, the Kb complexes were stable for at least 3 hr after separating the RMA-S cells from the macrophages. The endosomal inhibitors, leupeptin and ammonium chloride, did not inhibit the release of peptides and the increase in Kb staining on the RMA-S cells in the co-culture systems. Brefeldin A also had no effect. P815 cells previously co-cultured with Salmonella-infected macrophages became targets for cytotoxic T lymphocytes isolated from Salmonella-infected BALB/c mice. Taken together, our data suggest that IFN-gamma-activated macrophages process exogenous antigens in an intracellular compartment where serine proteases generate peptides released to the external environment for loading empty MHC class I molecules at the cell surface. This TAP-independent mechanism for the MHC class I presentation may be involved in priming cytotoxic T lymphocytes against intracellular pathogens in vivo.

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF-alpha in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF-alpha due to serum could be inhibited by the phosphatidylinositol (PI) 3-K inhibitors, wortmannin and LY294002, suggesting that PI 3-K is involved in the translational control of TNF-alpha by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF-alpha secretion and this was up-regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3-Kalpha increased the production of TNF-alpha in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen-activated protein kinase (MAPK), potentially downstream of PI 3-kinase, PD98059 and SB203580. Differently from with PI 3-K inhibitors, the accumulation of TNF-alpha mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3-K is an important step in controlling TNF-alpha protein synthesis in response to growth factors.

Secondary lymphoid tissue consists of two major populations of cells: lymphoid cells and stromal cells. It is generally accepted that these two cell populations influence each other however, factors mediating these processes are poorly understood. In this paper we characterize one of the possible means of communication between stroma and lymphocytes namely through hepatocyte growth factor/c-met receptor interactions. Hepatocyte growth factor (HGF) is a pleiotropic factor that is mainly produced by mesenchymal cells and acts on cells of epithelial origin which express the HGF receptor c-met. Here we demonstrate that biologically active HGF is constitutively produced by fibroblast-like stromal cells from human lymphoid tissues. HGF secretion from stromal cells was increased by direct contact with activated T cells. This increase was abrogated when activated T cells were separated physically from stromal cells. Using neutralizing antibody or cytokine inhibitors we provide evidence that enhancement of HGF production was due to additive effects of T-cell membrane-associated interleukin-1 (IL-1) and CD40 ligand. Finally, we also show that B lymphocytes activated with CD40L/anti-mu or phorbol 12-myristate 13-acetate (PMA) express c-met receptor. Co-culture of activated B cells with stromal cells from spleen leads to enhanced production of immunoglobulins. This can be partially inhibited by introduction of anti-HGF neutralizing antibodies to the culture system. Substitution of stromal cells with recombinant HGF did not produce enhancement of immunoglobulin secretion. On the other hand stimulation of c-met receptor with HGF leads to enhanced integrin-mediated adhesion of activated B cells to vascular cell adhesion molecule (VCAM-1) and fibronectin. On the basis of the above experiments we conclude that HGF production by fibroblast-like stromal cells can be modulated by activated T cells, thus providing signals for the regulation of adhesion of c-met expressing B cells to extracellular matrix proteins. In this way HGF may indirectly influence immunoglobulin secretion by B cells.

An immunodominant epitope of human immunodeficiency virus-1 (HIV-1) gp160 recognized by Dd class I major histocompatibility complex (MHC) molecule-restricted, CD8+ cytotoxic T lymphocytes (CTL) was originally identified as a peptide composed of 15 amino acids (P18IIIB: RIQRGPGRAFVTIGK). However, further study has indicated that a 10-mer peptide, I-10 (RGPGRAFVTI), within P18IIIB is the minimal-sized epitope and the trimming step(s) of two carboxyl terminal amino acids (GK) is essential to produce I-10 from P18IIIB. In the processing, angiotensin-1-converting enzyme (ACE), found in sera, plays a central role in generating I-10. Target cells could be sensitized with I-10 under conditions where ACE activity in the sera was abrogated. In contrast, in the case of P18IIIB, requiring further processing to delete the C-terminus of two amino acids in order to act, sensitization of target cells was completely abrogated under the conditions. Pretreatment of target cells with brefeldin A (BFA), preventing the presentation of endogenous antigens from the class I MHC molecule pathway, did not inhibit the presentation of P18IIIB. Moreover, glutaraldehyde-fixed cells, which can not process native protein, though they could present the exogenously added peptides, were also sensitized by P18IIIB. These results clearly demonstrate that the fine processing to produce I-10 occurred in the extracellular milieu. Furthermore, our result suggests that the longer P18IIIB can bind to the class I molecules on the cell surface, and then be trimmed by ACE while it is bound. The mechanisms behind the extracellular processing outlined in this paper will offer important information for designing peptide-based vaccines to elicit MHC molecule-restricted effectors.

We previously demonstrated that the majority (>/= 90%) of porcine ileal Peyer's patch (IPP) follicular cells are immature B cells destined to die by apoptosis, when incubated at 37 degrees. In this paper we approached the mechanisms responsible for positive selection of porcine IPP follicular immature B-cell selection, by screening for various cell types, cytokines and polyclonal and monoclonal antibodies for promoting the survival of IPP B cells. Of these reagents, only CD3 cross-linked purified T cells from mesenteric lymph nodes were able to rescue IPP follicular B cells from apoptosis, although polyclonal anti-IPP lymphocyte antibodies delayed apoptosis. This survival effect could be reproduced simply by incubating IPP follicular B cells with soluble and cell membrane-expressed CD154, an observation consistent with the demonstrated presence of CD40 and CD154 on porcine IPP follicular B cells and activated T cells, respectively. The IPP follicular B cells rescued in this manner expressed a more mature surface marker phenotype. Immunohistology and fluorescence-activated cell sorter analysis demonstrated that subpopulations of IPP follicular T cells (less than 0.5%) express CD154. Thus, perhaps unexpectedly, CD154 on T cells may play a role in the positive selection of immature B cells in the porcine IPP. The origin and control of the activated T cells identified within the porcine IPP remains to be investigated.

As is well known in the case of Langerhans cells, dendritic cells (DCs) play a crucial role in the initiation of immunity to simple chemicals such as noted in the contact hypersensitivity. Because DCs are scattered in non-lymphoid organs as immature cells, they must be activated to initiate primary antigen-specific immune reactions. Therefore, we hypothesized that some simple chemicals must affect the function of DCs. In this paper, we first demonstrated that human monocyte-derived DCs responded to such simple chemicals as 2, 4-dinitrochlorobenzene (DNCB), 2,4,6-trinitrochlorobenzene (TNCB), 2, 4-dinitrofluorobenzene (DNFB), NiCl2, MnCl2, CoCl2, SnCl2, and CdSO4 by augmenting their expression of CD86 or human leucocyte antigen-DR (HLA-DR), down-regulating c-Fms expression or increasing their production of tumour necrosis factor-alpha (TNF-alpha). In addition, the DCs stimulated with the chemicals demonstrated increased allogeneic T-cell stimulatory function. Next, we found that, among these chemicals, only NiCl2 and CoCl2 induced apoptosis in them. Finally, we examined the effects of these chemicals on CD86 expression by three different macrophage subsets and DCs induced from the cultures of human peripheral blood monocytes in the presence of macrophage colony-stimulating factor (M-CSF), M-CSF + interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and GM-CSF + IL-4, respectively. Among them, only DCs dramatically augmented their expression of CD86. These observations have revealed unique characteristics of DCs, which convert chemical stimuli to augmentation of their antigen presenting function, although their responses to different chemicals were not necessarily uniform in the phenotypic changes, cytokine production or in the induction of apoptosis.

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In this paper we show that the apparent monovalency of circulating IgG4 is caused by asymmetry of plasma IgG4. A large fraction of plasma IgG4 molecules have two different antigen-binding sites, resulting in bispecificity. Sera from patients with IgG4 antibodies to both house dust mite and grass pollen induced cross-linking of Sepharose-bound grass pollen antigen to radiolabelled house dust mite allergen Der p I. This bispecific binding activity was not observed in sera with IgG4 antibodies to either grass pollen or house dust mite exclusively. Depletion of IgG4 antibodies resulted in disappearance of the bispecific activity. By size exclusion chromatography we excluded the possibility that bispecific activity was caused by aggregation of IgG4 antibodies. These results indicate that circulating (polyclonal) IgG4 antibodies have two different antigen-binding sites and therefore are functionally monovalent antibodies.