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Local differentiation of eosinophil precursors occurs in the human thymus. Thymic eosinophils are often positioned in the corticomedullary junction between the CD4

Background Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). Purpose To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. Methods Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. Results At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. Conclusions This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. Clinical Implications This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices.

Eosinophils differentiate and mature in the thymus, outside of the bone marrow, in healthy individuals. Locally developed thymic eosinophils may contribute to the maturation and selection of human thymocytes.

Background The prevalence of self-experienced adverse reactions to foods seems to have an increasing trend in both adults and children. However, it is unclear if the prevalence of food hypersensitivity in the Swedish adult population is still rising, what symptoms are caused by different foods and which are the most common foods to which adults are more frequently IgE-sensitised. Methods In a cross-sectional study based on questionnaire responses, interviews and clinical examinations as part of the West Sweden Asthma Study, 1042 subjects from the general population, 17–78 years of age, living in Västra Götaland, Sweden, were included. The subjects reported symptoms for 56 specified foods and blood samples were taken to examine the IgE-sensitisation pattern for 9 common foods. Results Approximately 32% of adults reported food hypersensitivity, affecting mostly women and subjects less than 61 years old. The foods most often reported to cause adverse reactions were hazelnut (8.9%), apple (8.4%), milk (7.4%) and kiwi (7.3%). Less than one percent (0.9%) reported symptoms from ingestion of meat. Symptoms mostly affected the gastrointestinal tract (15%) and the skin (2.7%). Sixteen per cent were IgE-sensitised to common foods, most often to hazelnut (13.3%), peanut (4.9%) and almond (3.0%), while 5.9% reported symptoms and were IgE-sensitised to the same food, mainly to hazelnut (5.3%). Conclusions The prevalence of self-reported food hypersensitivity in West Sweden indicates a rising trend. The correspondence between self-reported symptoms and IgE-sensitisation to foods is generally poor, except for hazelnut and almond which exhibit moderate or fair correlation.

Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers’ children exhibit higher BAFF levels in cord blood than non-farmers’ children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall’s corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.

Extensive knowledge has been gained the last years concerning mechanisms underlying the selection of single positive thymocytes in the thymic medulla. Less is known regarding other important processes in the thymic medulla such as the regulation of late stage thymocyte maturation. We have previously reported that exosomes are abundant in the thymus with a phenotype that indicates an epithelial cell origin and immunoregulatory properties. In this study we use an in vitro system to investigate the effects of thymic exosomes on the maturation of single positive thymocytes as well as effects on nTreg formation. We show that thymic exosomes promote the maturation of single positive CD4+CD25− cells into mature thymocytes with S1P1+Qa2+ and CCR7+Qa2+ phenotypes. Furthermore, we show that thymic exosomes reduce the formation of CD4+CD25+FoxP3+ thymocytes and that these exosome effects are independent of dendritic cell co-stimulation but require intact exosomal RNA content and surface proteins. An efficient direct uptake of exosomes by both thymocytes and thymic DC’s is also demonstrated. In conclusion, this study demonstrates that exosomes may represent a new route of communication within the thymus.

Thyroid-associated ophthalmopathy (TAO) has a predilection for inferior rectus muscle that has never been explained. We conducted immunohistochemical staining for the soluble cleaved form of collagen XIII alpha 1 (COL13A1) and found constitutively low expression of COL13A1 in normal human inferior rectus muscles and moderate expression of COL13A1 in normal human medial rectus muscles. COL13A1 is known to be essential to development and maintenance of neuromuscular junctions and there is some evidence to suggest it may help support normal immune function. The combination of constitutively low expression of COL13A1, high physiological and metabolic demands, and consequentially relatively high exposure to stressors via the blood stream may help explain the particular vulnerability of inferior rectus to TAO compared to other extraocular muscles.

Thyroid-associated ophthalmopathy (TAO) causes irreversible increase in extraocular fat volume that contributes to the risk of exophthalmos and compressive optic neuropathy. Collagen XIII is implicated in uncontrolled cell growth in some tumours, but we are not aware of any studies of collagen XIII in TAO-affected solid tissue to date. We conducted immunohistochemical staining for collagen XIII alpha 1 (COL13A1), present in both the transmembrane and cleaved forms of collagen XIII, in consecutive prospectively collected human extraocular tissue specimens from patients with TAO and controls. We identified overexpression of collagen XIII in active TAO-affected fat. We discuss how species and cell-type specific responses of collagen XIII to stressors may help explain the different phenotypes of TAO.

Leukoplakias (LPLs) are lesions in the oral mucosa that may develop into oral squamous cell carcinoma (OSCC). The objective of this study was to assess presence and distribution of dendritic Langerhans cells (LCs) and T cells in patients with LPLs with or without cell dysplasia and in oral squamous cell carcinoma (OSCC). Biopsy specimens from patients with leukoplakias (LPLs) with or without dysplasia and oral squamous cell carcinoma (OSCC) were immunostained with antibodies against CD1a, Langerin, CD3, CD4, CD8 and Ki67, followed by quantitative analysis. Analyses of epithelium and connective tissue revealed a significantly higher number of CD1a + LCs in LPLs with dysplasia compared with LPLs without dysplasia. Presence of Langerin + LCs in epithelium did not differ significantly between LPLs either with or without dysplasia and OSCC. T cells were found in significantly increased numbers in LPLs with dysplasia and OSCC. The number of CD4+ cells did not differ significantly between LPLs with and without dysplasia, but a significant increase was detected when comparing LPLs with dysplasia with OSCC. CD8+ cells were significantly more abundant in OSCC and LPLs with dysplasia compared with LPLs without dysplasia. Proliferating cells (Ki67+) were significantly more abundant in OSCC compared to LPLs with dysplasia. Confocal laser scanning microscopy revealed colocalization of LCs and T cells in LPLs with dysplasia and in OSCC. LCs and T cells are more numerous in tissue compartments with dysplastic epithelial cells and dramatically increase in OSCC. This indicates an ongoing immune response against cells with dysplasia.

Galectin-3, a beta-galactoside binding, endogenous lectin, takes part in various inflammatory events and is produced in substantial amounts at inflammatory foci. We investigated whether extracellular galectin-3 could participate in the phagocytic clearance of apoptotic neutrophils by macrophages, a process of crucial importance for termination of acute inflammation. Using human leukocytes, we show that exogenously added galectin-3 increased the uptake of apoptotic neutrophils by monocyte-derived macrophages (MDM). Both the proportion of MDM that engulfed apoptotic prey and the number of apoptotic neutrophils that each MDM engulfed were enhanced in the presence of galectin-3. The effect was lactose-inhibitable and required galectin-3 affinity for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins, since a mutant lacking this activity was without effect. The enhanced uptake relied on the presence of galectin-3 during the cellular interaction and was paralleled by lectin binding to apoptotic cells as well as MDM in a lactose-dependent manner. These findings suggest that galectin-3 functions as a bridging molecule between phagocyte and apoptotic prey, acting as an opsonin. The process of clearance, whereby apoptotic neutrophils are removed by macrophages, is crucial for the resolution of acute inflammation and our data imply that the increased levels of galectin-3 often found at inflammatory sites could potently affect this process.

Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

Thymocytes go through several steps of maturation and selection in the thymus in order to form a functional pool of effector T-cells and regulatory T-cells in the periphery. Close interactions between thymocytes, thymic epithelial cells, and dendritic cells are of vital importance for the maturation, selection, and lineage decision of the thymocytes. One important question that is still unanswered is how a relatively small epithelial cell population can present a vast array of self-antigens to the manifold larger population of developing thymocytes in this selection process. Here, we review and discuss the literature concerning antigen transfer from epithelial cells with a focus on exosomes. Exosomes are nano-sized vesicles released from a cell into the extracellular space. These vesicles can carry proteins, microRNAs, and mRNAs between cells and are thus able to participate in intercellular communication. Exosomes have been shown to be produced by thymic epithelial cells and to carry tissue-restricted antigens and MHC molecules, which may enable them to participate in the thymocyte selection process.

Whereas a neonate is born is sterile, immediate exposure to its mother's mucosal surfaces allows it to acquire microflora, which plays an important role in defense against potential pathogens. This initial stimulus helps the immature immune system of the newborn to develop the capacity to respond with specific immunologic tolerance while avoiding the development of allergic and autoimmune disease. Breast-feeding provides nutritional and developmental, along with anti-infectious, advantages to the infant. The significant protection conferred by breast-feeding against varied infections such as acute and prolonged diarrhea, neonatal septicemia, respiratory tract infections, acute and recurrent otitis media, and urinary tract infections is observed worldwide. Human breast milk contains numerous components, including antibodies, cytokines, hormones, enzymes, and major proteins with multiple activities (microbicidal, tumoricidal, anti-inflammatory, autoimmune, etc.). Breast-feeding can strikingly reduce infant mortality, as well as the fertility of the breast-feeding mother. In this manner, breast-feeding provides significant benefits for lactating mothers and their offspring, in addition to society as a whole. © 2015 Elsevier Inc. All rights reserved.

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto-antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4+CD25+ T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ-free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4+CD25+ T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4+CD25+ T cells from conv animals. Intracellular staining for Foxp3 and CTLA-4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4+CD25+ T cells in GF MLN expressed Foxp3 and CTLA-4, while the expression of these markers was similar amongst the CD4+CD25+ T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population.

Regulatory T cells are induced by oral administration of an antigen, but the physiological requirements and localization of the inductive sites are largely unknown. Using an adoptive transfer system of cells transgenic for ovalbumin T-cell receptor (OVA TCR tg), we found that antigen-specific CD4+ T cells were activated in the liver-draining celiac lymph node (CLN) shortly after ovalbumin feeding, and that a significantly higher proportion of the T cells in the CLN developed into the putative regulatory phenotype [co-expressing CD25 with the glucocortico-induced tumour necrosis factor (TNF) receptor family related gene (GITR), cytotoxic T-lymphocyte antigen (CTLA)-4 and CD103] than in Peyer's patches, the mesenteric and peripheral lymph nodes and the spleen. In addition, a particularly high level of expression of CD103 on the OVA-specific T cells in the CLN may favour homing to the epithelium of the intestine. While equally suppressive, OVA tg T cells isolated from the CLN of OVA-fed DO11.10 mice were less dependent on transforming growth factor (TGF)-beta for suppression than cells isolated from the peripheral and mesenteric lymph nodes, which indicates the involvement of an additional suppressive mechanism. The expression of FoxP3 was not up-regulated in any of the lymph node compartments studied. Our phenotypic and functional findings suggest that the induction of regulatory T cells in the CLN may be relevant in the control of the immune response to dietary antigens.

Fed protein undergoes processing and coupling to major histocompatibility complex (MHC) II molecules during passage through the intestinal epithelium, generating a tolerogenic form of the antigen in serum. Transfer of this factor to naïve animals induces tolerance in the recipient. In this study, we investigate what impact colonization with Gram-positive (Lactobacillus plantarum) or Gram-negative (Escherichia coli) bacteria has on tolerogenic processing in the gut. Germ-free (GF), monocolonized or conventional mice were fed ovalbumin (OVA), and their serum was collected and transferred to naïve conventional recipients that were tested for delayed-type hypersensitivity against OVA after parenteral immunization. A transferable tolerogenic factor was produced by conventional mice, but not by mice that were germ free or monocolonized with either E. coli or L. plantarum. Conventional, but neither GF nor monocolonized mice showed upregulation of MHCII expression in the epithelium of small intestine. The results suggest that a complex intestinal microflora is needed to support oral tolerance development.

The effect of fermentation by Lactobacilli and of proteolytic hydrolysis of whole milk on the IgE binding ability of β-lactoglobulin was studied using an ELISA inhibition assay. Sera from nine adult milk allergic patients were tested. The individual sera showed a similar inhibition pattern in the changes during fermentation and proteolysis. The degradation of β-lactoglobulin was studied with liquid chromatography. In general, fermentation with Lactobacilli gave little effect on IgE binding, even though chromatography data showed a gradual degradation of β-lactoglobulin. Proteolysis with trypsin, however, gave extensive degradation of β-lactoglobulin and strongly decreased IgE binding. In addition, we measured the inhibition pattern of β-lactoglobulin in various selected commercially available fermented milk products. These showed an IgE binding capacity similar to that of nonfermented high pasteurized milk. Keywords: Milk; allergenicity; IgE antibody; IgE antibody inhibition; fermentation; lactic acid bac...

SUMMARYPrevalence of allergy is increasing in many countries and might be related to changed environmental factors, such as dietary fatty acids (FA). The present study investigates whether dietary ratio of n-6 to n-3 FA influences the induction of immunological tolerance to ovalbumin (OA) in neonatal rats. During late gestation and throughout lactation Sprague-Dawley rats were fed a diet containing 7% linseed oil (n-3 diet), sunflower oil (n-6 diet) or soybean oil (n-6/n-3 diet). At 10–16 days of age the rat offspring were subsequently exposed, or not, to OA via the milk. The offspring were weaned onto the same diets as the mothers and immunized with OA and the bystander antigen human serum albumin (HSA). In the offspring on the n-3 diet exposure to OA via the milk resulted in lower delayed type hypersensitivity reaction (DTH) and antibody responses against both OA and HSA, compared to those in the offspring not exposed to OA, indicating the induction of oral tolerance. In the offspring on the n-6 diet, the exposure to OA led to depressed specific immune responses against only OA, not HSA. In the offspring on the n-6/n-3 diet oral exposure to OA did not influence immune responses against OA, or HSA. The results indicate that the dietary ratio of n-6/n-3 FA is important for the induction of neonatal oral tolerance. Thus nonoptimal feeding may have effects on the development of immunological tolerance to dietary antigen ingested by the mother. The ratio of n-6/n-3 FA in the diet may be considered in the context of increased prevalence of allergy.

The present study examines whether dietary essential fatty acid (EFA) intake influences the induction of oral tolerance to ovalbumin (OA) in neonatal and adult rats. During late gestation and throughout lactation Sprague-Dawley rats were fed a diet supplemented (S) with EFA (7% soybean oil), or a diet deficient (D) in EFA (7% hydrogenated lard). The rat offspring were subsequently exposed to OA either via the milk at 10-16 days (neonatal rats), or as adults via the drinking water at 7-9 wk of age. Oral administration of OA to the adult rats lead to suppression of the delayed-type hypersensitivity (DTH) reactivity and IgG antibody response against OA, which was not influenced by their diets. In the offspring of the dams fed the D diet antigen exposure via the milk resulted in suppression of the serum antibody levels and DTH reaction against OA indicating induction of oral tolerance. Higher transforming growth factor beta (TGF-beta) mRNA levels in the draining lymph nodes suggested this to be mediated by regulatory T cells. In contrast, OA exposure of the dams fed the S diet did not result in a suppressed OA response of their offspring. Thus, the quality of FA ingested by the mother may have effects on the development of immunological tolerance to dietary antigens in the offspring. Our results might have importance for the understanding of the increase in allergy related to the Western type of diet.

Background: The pathophysiologic interactions of inflammatory reactions between the mucosa of the respiratory tract and that of the gastrointestinal tract in individuals with allergy are poorly studied, despite the fact that allergic symptoms in the airways and the gastrointestinal tract might arise in the same patient. Objective: The objective of this study was to examine the inflammatory response histologically by enumerating eosinophils, IgE+ cells, and T cells in duodenal biopsy specimens in adult patients with IgE-mediated birch pollen allergy during the birch pollen season and off-season. Methods: Nine patients with birch pollen allergy verified by skin prick test and serum IgE antibodies were investigated toward the end of the birch pollen season and again 6 months later (off-season). Duodenal biopsy specimens were obtained and studied by immunostaining for the eosinophil major basic protein (MBP), IgE, and CD3+ T cells. Results: Oral allergy syndrome to birch-associated foods was present in all patients as indicated by questionnaire. Duodenal increases of MBP+ eosinophils and IgE-bearing cells were found in the patients during the birch pollen season as compared with off-season. No seasonal differences in the T-cell numbers in these patients were seen. Off-season, there was no significant difference between the patients and the control subjects regarding the intestinal frequencies of MBP+ eosinophils, mucosal IgE+ cells, and T cells. Conclusion: Birch pollen exposure triggered a local inflammation with an increase in duodenal eosinophils and IgE-carrying mast cells in patients with allergy. Our study gives evidence for the interplay between immunologically active cells in the airways and the gut. (J Allergy Clin Immunol 2003;112:45-51.)

Summary Child mortality in diarrhoeal disease is increased significantly by vitamin A deficiency in poor countries. The pathological mechanisms are not known in detail. However, in this paper we report that vitamin A-deficient Wistar rats had much reduced IgA+ plasma cells in the ileal lamina propria (eightfold reduction from 470 cells/mm2, P = 0·009), as well as a prominent reduction of CD4+ cells in the parafollicular regions of ileal Peyer's patches (reduction from 7200 to 105 cells/mm2, P = 0·009). IL-2Ralpha-chain (CD25) positive lymphocytes in the ileal Peyer's patches were also reduced significantly in vitamin A deficiency (from 1400 to 300 cells/mm2, P = 0·009). The density of CD8 cells tended to be increased relative to the control animals (from 5100 to 6000 cells/mm2, not statistically significant). In conclusion, the marked decrease of lamina propria IgA+ plasma cells may be one cause of the high diarrhoeal mortality in vitamin A deficiency. This, in turn, appears to be related to reduced numbers of activated or regulatory CD4+ T cells in Peyer's patches.

Objectives: The aim of this investigation was to characterise and compare the inflammatory infiltrates in patients with orofacial granulomatosis solely (OFG-S) and OFG with coexisting Crohn’s disease (OFG+CD). Study Design: Biopsy specimens with granulomas were obtained from patients with OFG-S (n=11) and OFG+CD (n=11) and immunostained with antibodies against CD1a, CD3, CD4, CD8, CD11c, CD20, CD68 and mast cell tryptase, followed by quantitative analysis. Results: Analyses of the connective tissue revealed a significantly higher number of CD3-expressing T cells and CD11c-expressing dendritic cells in the connective tissue of patients with OFG-S compared to patients with OFG+CD. Mast cells displayed a high level of activation, although no significant difference was detected when comparing the two groups. Conclusions: The results show a different composition of the inflammatory infiltrate in patients with OFG-S compared to patients with OFG+CD. The present observations support that partly divergent immune mechanisms are involved in these two different subcategories of OFG. Key words:Granulomas, autoimmunity, T cells, B cells, dendritic cells, children, adults.

The development of immunological tolerance to orally fed antigens depends on the sampling, processing and transportation events followed in the intestinal epithelium. We present here a description of a "tolerosome": a supra-molecular, exosome-like structure assembled in and released from the small intestinal epithelial cell. The tolerosome is a approximately 40 nm large vesicular structure that carries MHC class II (MHC II) with bound antigenic peptides sampled from the gut lumen. Tolerosomes isolated from serum shortly after antigen feeding or from an in vitro pulsed intestinal epithelial cell line are fully capable of inducing antigen specific tolerance in naive recipient animals. Purified tolerosomes represent a structure by which fed antigens can be efficiently presented to the immune system. Removal of the tolerosomes from serum by ultracentrifugation or absorption of MHC II results in abrogated tolerance development.

CD1 cell surface glycoproteins represent a family of non-major histocompatibility complex (MHC) encoded antigen-presenting molecules. All members of the CD1 family appear to mediate the recognition of microbial or endogenous lipid and glycolipid antigens. The recognition of CD1d by a unique subset of natural killer (NK) T cells that leads to rapid production of large amounts of both type 1 and type 2 cytokines can be augmented by some synthetic glycolipids. Because of the proposed role of such CD1d-restricted T cells in immunoregulation, we hypothesized that CD1d molecules participate in mucosal immune responses in patients with gastrointestinal symptoms owing to food hypersensitivity. Patients of that category represent a heterogeneous group in which poorly defined immunological mechanisms are believed to contribute to disease pathogenesis. The expression of CD1 in duodenal biopsy samples from six patients with verified intolerance to cow's milk and six healthy controls was studied by immunoperoxidase staining of cryostat sections using a panel of mouse monoclonal antibodies (MoAbs) specific for CD1a, b, c, and d. Large numbers of CD1d positive cells were found in the lamina propria of all the patients, both during the symptomatic and the asymptomatic periods, whereas healthy controls were virtually devoid of CD1d expression in the duodenum. The localization of CD1d positive cells corresponded to areas where B cells, plasma cells and dendritic cells (DC) were present. A positive correlation was found between the numbers of CD1d(+) and CD19(+) cells in the lamina propria. In contrast to previous reports, no CD1d expression was found on the epithelial cells. Although less numerous than CD1d(+) the CD1c(+) cells were also present in all the patients and in five out of six controls. No staining for CD1a or CD1b was detected in the duodenal biopsy samples from any of the subjects. The exclusive presence of CD1d in the duodenal lamina propria of the patients with cow's milk hypersensitivity might suggest the participation of these molecules in the pathogenesis of allergic reactions to food.

In the present study we have investigated if transfer of serum from rats fed ovalbumin (OVA) leads to specific tolerance and bystander suppression in recipient animals. Rats that received serum from OVA-fed donors had a lower delayed-type hypersensitivity reaction (DTH) both against OVA and the bystander antigen, human serum albumin (HSA), compared with recipients given serum from control-fed animals. The in vitro proliferation of OVA- and HSA-stimulated spleen cells and the serum immunoglobulin G (IgG) antibody levels against OVA and HSA were also lower in the animals that received serum from OVA-fed animals compared with the controls. There was no reduction of the immune response to HSA if the recipient animals, given serum from OVA-fed donors were immunized with OVA and HSA at separate sites. Depletion of CD25-positive cells from spleen suspensions from rats receiving serum from OVA-fed animals, resulted in a significant increase in proliferation of OVA-stimulated cells in vitro compared with the controls. Tolerogenic activity could be demonstrated, both in a fraction from serum containing structures smaller than 100 000 MW and a fraction with components larger than 100 000 MW, compared with size-related serum fractions obtained from control-fed animals. This implies that the tolerogenic activity could be mediated by more than one serum component. The tolerogenic activity was most prominent in animals receiving the larger size fraction with a more pronounced suppression of the DTH reaction and lower levels of IgG anti-OVA antibodies in serum compared with controls. A novel finding in the present study was that the transfer of serum, collected from rats fed OVA, led to a reduction of the immune response to a bystander antigen in the recipients. This suggests that the induced tolerance is at least partly due to suppression. The suppression could have been mediated by CD25-positive cells since removal of these cells resulted in an increased in vitro proliferation against OVA.

We wanted to investigate the immunological events occurring in rats intestinally colonized from birth (neonatally) or at adult age with an ovalbumin (OVA)-producing Escherichia coli O6K13 strain, carrying type 1 pili. The neonatally colonized animals responded with lower delayed type hypersensitivity (DTH) against OVA and lower levels of IgG antibodies against OVA, O6 lipopolysaccharide (LPS) and type 1 pili compared to age-matched controls. The IgG antibody response against the bystander antigen, human serum albumin (HSA), was lower in the neonatally colonized animals than in the controls co-immunized with HSA and E. coli, indicating a release of suppressive factors induced by the bacterial antigens. The adult colonized animals showed an increased DTH and antibody response against OVA after immunization. They also had high pre-immunization levels of IgG anti-O6 LPS antibodies compared to controls. However, the relative increase in IgG anti-O6 LPS antibody levels after the immunization with dead E. coli was much lower in the adult colonized animals. The present results suggest that neonatal animals develop tolerance against antigen on bacterial colonizers of the intestine. In addition, this tolerance contains components of suppression.

Feeding a soluble antigen to an animal is known to cause a state of unresponsiveness against this antigen. If this antigen is given together with another antigen during the sensitization procedure, impairment of the response to the new antigen can also be seen, a phenomenon referred to as bystander suppression. The induction of tolerance against ovalbumin (OvA) and the effect of bystander suppression on the response to the hapten trimellitic anhydride (TMA), a cause of occupational asthma, were studied in Brown-Norway rats. Rats were fed either OvA-containing pellets or standard diet for 16 days before sensitization with the mixture of TMA and OvA. The animals were followed for 6 weeks after sensitization. Animals made tolerant to OvA showed a significantly suppressed delayed-type hypersensitivity (DTH) reaction against both OvA and TMA compared with the nontolerized control group at 5 weeks after sensitization, implying bystander suppression. By contrast, immunoglobulin (Ig)E and IgG antibody levels were suppressed only against OvA, whereas anti-TMA antibody levels were not affected. Airway eosinophilia after a single aerosol challenge at 6 weeks after sensitization using TMA conjugated to rat serum albumin, correlated with IgE anti-TMA levels in the group made tolerant to OvA and was not affected by OvA ingestion. In conclusion, suppressive factors released in ovalbumin-tolerant rats when they are challenged with ovalbumin, can suppress the response to trimellitic anhydride and this suppression is more pronounced for T-helper1-type responses.

Immunological tolerance normally develops early in life against foods to avoid that the immune system induces inflammatory reactions against food components. In an experimental model in rats they can be made allergic to ovalbumin (OA) by colonisation with a transgenic E. coli which produces OA. The sensitised animals have IgE and IgG antibodies as well as T cells directed against OA and may develop diarrhoea when fed OA. They also have IgE-carrying mast cells, eosinophils and goblet cells in their intestinal mucosa. In contrast animals which have been tolerised by feeding OA have few IgE-covered mast cells, eosinophils and goblet cells. Instead they have a CD4 + T cell population with CD25 (IL-2Rα-chain) centrally in the villi, but no signs of inflammatory reactivity. These CD25 + T cells are found in the draining lymph nodes of orally tolerised animals after parenteral immunisation with the antigen. These cells seem to effectively down-regulate both T and B cell responses to fed antigen but also to an unrelated antigen given simultaneously and handled by the same lymph node - so called "bystander tolerance". Recently we have shown that such regulatory T cells are also involved in the control of the response to the normal bacterial flora. This model may be used to study in detail the mechanisms behind the normal appearance of tolerance to food components and the commensal bacterial flora.

The aim of this study was to compare the local gut immune response in sensitized and orally tolerized experimental animals. The development of IgE/IgG antibodies and the DTH to OA was studied in rats made orally tolerant to OA and compared with sensitized control rats after colonization with an Escherichia coli genetically engineered to produce OA. At 3 weeks of age, pups were weaned onto a standard diet without OA or an OA-containing diet for 4 weeks and then switched to a standard diet without OA. Both groups of rats were parenterally immunized with a mixture of OA and human serum albumin (HSA) in Freund's complete adjuvant when they were 8 weeks old. After DTH measurement 2 weeks later, all rats were colonized with an E. coli producing OA for 5 days. The local immune response in the small intestine was assessed, using immunohistochemistry, as the expression of MHC class II molecules and IL-2 receptor (IL-2R) α-chain. The OA-tolerant rats showed the classical signs of oral tolerance, with a reduced IgE and IgG antibody and DTH response to OA before colonization. The difference between the two groups in the anti-OA antibody response became even more pronounced after colonization with the E. coli that produce OA. Rats orally tolerant to OA maintained a normal villus architecture after colonization, with a normal expression of MHC class II molecules similar to non-treated adult rats, but with a significantly higher (P=0.004) expression of IL-2R α-chain on T cells in the lamina propria of the villus core compared with sensitized control rats. The tolerant rats showed a very weak staining with the anti-IL-2R α-chain-specific antibody on a few goblet cells in only one out of seven rats. In the sensitized control rats, a marked local immune response was seen with an intense staining with a monoclonal anti-IL-2R α-chain-specific antibody on goblet cells in five out of seven rats (P=0.019) and also an increased expression of MHC class II molecules in the epithelial cells and cells in the lamina propria of all rats. Rats orally tolerant to OA maintained a normal villus architecture after colonization, but with a significantly higher (P=0.004) expression of IL-2R α-chain on T cells in the lamina propria of the villus core compared with sensitized control rats. The novel finding that goblet cells express IL-2R α-chain and the striking difference in expression of the receptor and the numbers of goblet cells between tolerant and sensitized rats may suggest a direct T cell regulation of the goblet cells. A possibility that oral tolerance might be maintained by the activated T cells expressing IL-2R α-chain in the lamina propria of the villus core is also discussed.

Oral tolerance was induced in 4-week-old (young) and 12-week-old (adult) rats by feeding ovalbumin (OvA)-containing pellets during 4 weeks. Seven weeks after removal of the OvA-pellets the rats were immunized with a mixture of OvA and human serum albumin (HSA) in Freund's complete adjuvant (FCA), and the following immune response was monitored. Both the young and adult groups of OvA-fed rats had significantly suppressed OvA-specific delayed-type hypersensitivity (DTH) responses and T-cell proliferation, reflecting a long-lasting T-cell tolerance to OvA both in vivo and in vitro. Furthermore, spleen cells from rats tolerized as adults were able to suppress the proliferation of primed T-cells from normal immunized rats, demonstrating the presence of antigen-specific suppressive cells. Accordingly, the adult rats showed bystander suppression of the response to HSA with respect to DTH-reaction, specific proliferation, and reduced enlargement of the draining lymph nodes after immunization. There was no evidence of active suppression in vitro or bystander tolerance in the orally tolerized young group, indicating that anergy rather than active suppression was prevalent in these rats. Furthermore, in the young group there was no suppression of the antibody response since the IgG and IgE anti-OvA antibody levels were indistinguishable from those of the controls. Contrary to the young rats, the adult fed group showed transiently elevated levels of IgG anti-OvA antibodies at 1 week post-immunization, followed by a subsequent significantly suppressed IgG antibody response. In conclusion, the results demonstrate that the induction of anergy or active suppression after antigen feeding can be determined by the age at which the antigen is introduced to the mucosal immune system.

Antigen presentation determines immunologic outcome, and by modifying the presentation of allergen to the host one can prevent an allergic response. Under certain conditions, covalent linkage, of ovalbumin to rat IgG, a molecule already tolerated by the host, can make a protein-IgG conjugate which down-regulates the immune response to this food allergen. The suppression is allergen specific. It affects both T and B cell immune responses. Administration of allergens linked to isologous IgG may provide a novel strategy for allergy prevention.

This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G-) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4(+) T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G- bacteria and may be relevant for the use of bacterial carriers in vaccine development.

Mononuclear phagocytes, that is, monocytes and macrophages, are central in the defense against mycobacteria. Mycobacterium abscessu s is an opportunistic mycobacterial species able to cause chronic pulmonary infections in patients with cystic fibrosis but also soft tissue infections in immunocompetent individuals. Pathogenic isolates of M. abscessus with rough colony morphology form cord-like aggregates. In this study, we investigated the in vitro response of human peripheral blood mononuclear cells (PBMCs) from healthy blood donors to cord-forming M. abscessus strains from cystic fibrosis patients with clinical lung infection. Microscopic examination revealed that the PBMCs produced a coarse fibrous meshwork containing DNA and histones, which surrounded the mycobacterial cords. Thus, the bacterial cord formations were entrapped by monocytes and lymphocytes aggregated onto the DNA-rich meshwork fibers. Mycobacterium abscessus strains with smooth colony morphology, which do not form cords and are readily phagocytosed, did not induce any meshwork formation in PBMCs. The chromatin meshwork may represent a defense mechanism against nondigestible invaders.

The influence of feeding ovalbumin (OA) on the development of IgE/IgG antibodies and delayed-type hypersensitivity (DTH) against OA was studied in rats colonized from birth with an Escherichia coli genetically manipulated to produce OA. At 21 days of age, colonized pups and pups with a normal intestinal flora were weaned onto either an OA-containing or a conventional diet without OA. At 2 months of age the colonized rats showed an increased DTH reaction to OA, but they did not have any anti-OA antibodies in serum. The rats were then immunized intracutaneously with OA in Freund’s complete adjuvant. After immunization the colonized rats fed the conventional diet had a significantly higher DTH reaction to OA and significantly higher serum levels of IgE anti-OA antibodies than the uncolonized rats on the same diet. The colonized rats eating the OA-containing diet showed a 73% decrease in the DTH reaction to OA and also significantly lower levels of IgE and IgG antibodies against OA compared with the colonized rats fed conventional diet. The dams colonized as adults by the OA-producing E. coli developed IgE anti-lipopolysaccharide antibodies in serum while the pups colonized via the dams at birth did not. Neonatal colonization with an E. coli strain producing OA resulted in increased DTH reactivity against OA and priming for secondary IgE anti-OA response. Feeding the animals an OA-containing diet from weaning abrogated this intestinally induced hypersensitivity and rendered the animals orally tolerant to OA.

The hygiene hypothesis suggests that lack of microbial stimulation in early infancy may lead to allergy, but it has been difficult to identify particular protective microbial exposures. We have observed that infants colonised in the first week(s) of life with Staphylococcus aureus have lower risk of developing food allergy. As many S. aureus strains produce superantigens with T-cell stimulating properties, we here investigate whether neonatal mucosal exposure to superantigen could influence the capacity to develop oral tolerance and reduce sensitisation and allergy. BALB/c mice were exposed to staphylococcal enterotoxin A (SEA) as neonates and fed with OVA as adults, prior to sensitisation and i.n. OVA challenge. Our results show that SEA pre-treated mice are more efficiently tolerised by OVA feeding, as shown by lower lung-cell infiltration and antigen-specific IgE response in the SEA pre-treated mice, compared with sham-treated mice. This was not due to deletion or anergy of lymphocytes by SEA treatment, because the SEA pre-treated mice that were fed with PBS showed similar inflammatory response as the sham-treated PBS-fed mice. Our results suggest that strong T-cell activation in infancy conditions the mucosal immune system and promotes development of oral tolerance.

Exosomes are nano-sized membrane vesicles which are released extracellularly after fusion of multivesicular endosomes with the cell membrane. Despite their characteristic composition of proteins compared to the cell membrane, no exosome-specific molecule has so far been characterized. Exosomes are found in bronchoalveolar lavage (BAL), urine, serum and breast milk, and are released from several cells implicated in allergy including mast cells, dendritic cells (DC), T cells and epithelial cells. Antigen-loaded exosomes have been shown to be highly immunogenic and we propose that exosomes could be a modulating factor in allergic responses. Allergen-presenting exosomes could transport allergen and stimulate allergen-specific T cells, and possibly also biasing T cell responses depending on the molecules present on the exosome surface. Furthermore, exosomes from mast cells, highly active in allergic reactions, have been found to induce DC maturation and also to be able to transport functional RNA to recipient cells, suggesting a new pathway for cell communication. Reversely, tolerizing exosomes e.g. tolerosomes, from gut or breast milk, could block an allergic response or prevent allergy development. A better understanding of the role of exosomes in allergies could make us understand how allergy can be prevented or lead to the development of more efficient treatments.