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Summary: Visium HD by 10X Genomics is the first commercially available platform capable of capturing full scale transcriptomic data paired with a reference morphology image from archived FFPE blocks at sub-cellular resolution. However, aggregation of capture regions to single cells poses challenges. Bin2cell reconstructs cells from the highest resolution data (2 μm bins) by leveraging morphology image segmentation and gene expression information. It is compatible with established Python single cell and spatial transcriptomics software, and operates efficiently in a matter of minutes without requiring a GPU. We demonstrate improvements in downstream analysis when using the reconstructed cells over default 8 μm bins on mouse brain and human colorectal cancer data., Availability and Implementation: Bin2cell is available at https://github.com/Teichlab/bin2cell, along with documentation and usage examples, and can be installed from pip. Probe design functionality is available at https://github.com/Teichlab/gene2probe., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Tumor-associated macrophages (TAMs) are associated with unfavorable patient prognosis in many cancer types. However, TAMs are a heterogeneous cell population and subsets have been shown to activate tumor-infiltrating T cells and confer a good patient prognosis. Data on the prognostic value of TAMs in colorectal cancer are conflicting. We investigated the prognostic effect of TAMs in relation to tumor-infiltrating T cells in colorectal cancers., Methods: The TAM markers CD68 and CD163 were analyzed by multiplex fluorescence immunohistochemistry and digital image analysis on tissue microarrays of 1720 primary colorectal cancers. TAM density in the tumor stroma was scored in relation to T cell density (stromal CD3 + and epithelial CD8 + cells) and analyzed in Cox proportional hazards models of 5-year relapse-free survival. Multivariable survival models included clinicopathological factors, MSI status and BRAF V600E mutation status., Results: High TAM density was associated with a favorable 5-year relapse-free survival in a multivariable model of patients with stage I-III tumors (p = 0.004, hazard ratio 0.94, 95% confidence interval 0.90-0.98). However, the prognostic effect was dependent on tumoral T-cell density. High TAM density was associated with a good prognosis in patients who also had high T-cell levels in their tumors, while high TAM density was associated with poorer prognosis in patients with low T-cell levels (p interaction  = 0.0006). This prognostic heterogeneity was found for microsatellite stable tumors separately., Conclusions: This study supported a phenotypic heterogeneity of TAMs in colorectal cancer, and showed that combined tumor immunophenotyping of multiple immune cell types improved the prediction of patient prognosis., (© 2024. The Author(s).)

Background & Aims: Durable remissions of Crohn's Disease (CD) have followed myeloablative conditioning therapy and allogeneic marrow transplantation. For patients with treatment-refractory disease, we used reduced-intensity conditioning to minimize toxicity, marrow from donors with low Polygenic Risk Scores for CD as cell sources, and protracted immune suppression to lower the risk of graft-versus-host disease (GVHD). Our aim was to achieve durable CD remissions while minimizing transplant-related complications., Methods: DNA from patients and their HLA-matched unrelated donors was genotyped and Polygenic Risk Scores calculated. Donor marrow was infused following non-myeloablative conditioning. Patient symptoms and endoscopic findings were documented at intervals after transplant., Results: We screened 807 patients, 143 of whom met eligibility criteria; 2 patients received allografts. Patient 1 had multiple complications and died at day 332 from respiratory failure. Patient 2 had resolution of CD symptoms until day 178 when CD recurred, associated with persistent host chimerism in both peripheral blood and intestinal mucosa. Withdrawal of immune suppression was followed by dominant donor immune chimerism in peripheral blood and resolution of CD findings. Over time, mucosal T-cells became donor-dominant. At 5 years after allografting, Patient 2 remained off all medications but had mild symptoms related to a jejunal stricture that required stricturoplasty at 6 years. At 8 years, she remains stable off medications., Conclusions: The kinetics of immunologic chimerism after allogeneic marrow transplantation for CD patients depends on the intensity of the conditioning regimen and the magnitude of immune suppression. One patient achieved durable improvement of her previously refractory CD only after establishing donor immunologic chimerism in intestinal mucosa. Her course provides proof-of-principal for allografting as a potential treatment for refractory CD, but an immunoablative conditioning regimen should be considered for future studies.(ClinicalTrials.gov, NCT01570348)., Competing Interests: None of the authors has a conflict of interest related to the content of this work., (© 2024 The Authors. Published by Elsevier Ltd.)

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges., (© 2023. Springer Nature Limited.)

Dermatitis herpetiformis (DH) is an inflammatory skin disorder often considered as an extra intestinal manifestation of celiac disease (CeD). Hallmarks of CeD and DH are auto-antibodies to transglutaminase 2 (TG2) and transglutaminase 3 (TG3), respectively. DH patients have auto-antibodies reactive with both transglutaminase enzymes. Here it is reported that in DH both gut plasma cells and serum auto-antibodies are specific for either TG2 or TG3 with no TG2-TG3 cross reactivity. By generating monoclonal antibodies from TG3-specific duodenal plasma cells of DH patients, three conformational epitope groups are defined. Both TG2-specific and TG3-specific gut plasma cells have few immunoglobulin (Ig) mutations, and the two transglutaminase-reactive populations show distinct selection of certain heavy and light chain V-genes. Mass spectrometry analysis of TG3-specific serum IgA corroborates preferential usage of IGHV2-5 in combination with IGKV4-1. Collectively, these results demonstrate parallel induction of anti-TG2 and anti-TG3 auto-antibody responses involving separate B-cell populations in DH patients., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background: Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited., Objective: We performed a detailed characterization of Foxp3 + CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions., Methods: Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed., Results: SI Foxp3 + CD4 T cells were CD45RA - CD127 - CTLA-4 + and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3 + Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold., Conclusion: The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Celiac disease is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine leading to destruction of the lining epithelium. Current treatment focusses on lifelong adherence to a gluten-free diet. Gluten-specific CD4 + T cells and cytotoxic intraepithelial CD8 + T cells have been proposed to be central in disease pathogenesis. Here we use unbiased single-cell RNA-sequencing and explore the heterogeneity of CD45 + immune cells in the human small intestine. We show altered myeloid cell transcriptomes present in active celiac lesions. CD4 + and CD8 + T cells transcriptomes show extensive changes and we define a natural intraepithelial lymphocyte population that is reduced in celiac disease. We show that the immune landscape in Celiac patients on a gluten-free diet is only partially restored compared to control samples. Altogether, we provide a single cell transcriptomic resource that can inform the immune landscape of the small intestine during Celiac disease., (© 2022. The Author(s).)

Macrophages are a heterogeneous population of cells involved in tissue homeostasis, inflammation, and cancer. Although macrophages are densely distributed throughout the human intestine, our understanding of how gut macrophages maintain tissue homeostasis is limited. Here we show that colonic lamina propria macrophages (LpMs) and muscularis macrophages (MMs) consist of monocyte-like cells that differentiate into multiple transcriptionally distinct subsets. LpMs comprise subsets with proinflammatory properties and subsets with high antigen-presenting and phagocytic capacity. The latter are strategically positioned close to the surface epithelium. Most MMs differentiate along two trajectories: one that upregulates genes associated with immune activation and angiogenesis, and one that upregulates genes associated with neuronal homeostasis. Importantly, MMs are located adjacent to neurons and vessels. Cell-cell interaction and gene network analysis indicated that survival, migration, transcriptional reprogramming, and niche-specific localization of LpMs and MMs are controlled by an extensive interaction with tissue-resident cells and a few key transcription factors., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2022 Domanska et al.)

Studies in mice and humans have shown that CD8 + T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4 + T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4 + T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4 + T cells were still donor-derived one year after transplantation. In contrast to memory CD4 + T cells in peripheral blood, intestinal CD4 + T RM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4 + T RM cells were very potent cytokine producers; the vast majority being polyfunctional T H 1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4 + T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4 + T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67+) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ + IL-2 + TNF-α + ) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders., (© 2019 Bartolomé-Casado et al.)

Insulin deficiency in type 1 diabetes (T1D) is generally considered a consequence of immune-mediated specific beta-cell loss. Since healthy pancreatic islets consist of ~65% beta cells, this would lead to reduced islet size, while the number of islets per pancreas volume (islet density) would not be affected. In this study, we compared the islet density, size, and size distribution in biopsies from subjects with recent-onset or long-standing T1D, with that in matched non-diabetic subjects. The results presented show preserved islet size and islet size distribution, but a marked reduction in islet density in subjects with recent onset T1D compared with non-diabetic subjects. No further reduction in islet density occurred with increased disease duration. Insulin-negative islets in T1D subjects were dominated by glucagon-positive cells that often had lost the alpha-cell transcription factor ARX while instead expressing PDX1, normally only expressed in beta cells within the islets. Based on our findings, we propose that failure to establish a sufficient islet number to reach the beta-cell mass needed to cope with episodes of increased insulin demand contributes to T1D susceptibility. Exhaustion induced by relative lack of beta cells could then potentially drive beta-cell dedifferentiation to alpha-cells, explaining the preserved islet size observed in T1D compared to controls., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Background & Aims: Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4 + T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response., Methods: We generated monoclonal antibodies (mAbs) specific for the peptide-MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease)., Results: Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls., Conclusions: A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Activated T helper type 2 (Th2) cells are believed to play a pivotal role in allergic airway inflammation, but which cells attract and activate Th2 cells locally have not been fully determined. Recently, it was shown in an experimental human model of allergic rhinitis (AR) that activated monocytes rapidly accumulate in the nasal mucosa after local allergen challenge, where they promote recruitment of Th2 cells and eosinophils., Objective: To investigate whether monocytes are recruited to the lungs in paediatric asthma., Methods: Tissue samples obtained from children and adolescents with fatal asthma attack (n = 12), age-matched non-atopic controls (n = 9) and allergen-challenged AR patients (n = 8) were subjected to in situ immunostaining., Results: Monocytes, identified as CD68+S100A8/A9+ cells, were significantly increased in the lower airway mucosa and in the alveoli of fatal asthma patients compared with control individuals. Interestingly, cellular aggregates containing CD68+S100A8/A9+ monocytes obstructing the lumen of bronchioles were found in asthmatics (8 out of 12) but not in controls. Analysing tissue specimens from challenged AR patients, we confirmed that co-staining with CD68 and S100A8/A9 was a valid method to identify recently recruited monocytes. We also showed that the vast majority of accumulating monocytes both in the lungs and in the nasal mucosa expressed matrix metalloproteinase 10, suggesting that this protein may be involved in their migration within the tissue., Conclusions and Clinical Relevance: Monocytes accumulated in the lungs of children and adolescents with fatal asthma attack. This finding strongly suggests that monocytes are directly involved in the immunopathology of asthma and that these pro-inflammatory cells are potential targets for therapy., (© 2018 John Wiley & Sons Ltd.)

The conditionally essential amino acid glycine functions as inhibitory neurotransmitter in the mammalian central nervous system. Moreover, it has been shown to act as an anti-inflammatory compound in animal models of ischemic perfusion, post-operative inflammation, periodontal disease, arthritis and obesity. Glycine acts by binding to a glycine-gated chloride channel, which has been demonstrated on neurons and immune cells, including macrophages, polymorphonuclear neutrophils and lymphocytes. The present study aims to evaluate the effect of glycine on allergy development in a cow's milk allergy model. To this end, C3H/HeOuJ female mice were supplemented with glycine by oral gavage (50 or 100 mg/mouse) 4 hours prior to sensitization with cow's milk whey protein, using cholera toxin as adjuvant. Acute allergic skin responses and anaphylaxis were assessed after intradermal allergen challenge in the ears. Mouse mast cell protease-1 (mMCP-1) and whey specific IgE levels were detected in blood collected 30 minutes after an oral allergen challenge. Jejunum was dissected and evaluated for the presence of mMCP-1-positive cells by immunohistochemistry. Intake of glycine significantly inhibited allergy development in a concentration dependent manner as indicated by a reduction in; acute allergic skin response, anaphylaxis, serum mMCP-1 and serum levels of whey specific IgE. In addition, in-vitro experiments using rat basophilic leukemia cells (RBL), showed that free glycine inhibited cytokine release but not cellular degranulation. These findings support the hypothesis that the onset of cow's milk allergy is prevented by the oral intake of the amino acid glycine. An adequate intake of glycine might be important in the improvement of tolerance against whey allergy or protection against (whey-induced) allergy development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Disruptions to the gut microbiota have been associated with a variety of diseases. Understanding the underlying mechanisms that regulate the maintenance of a healthy microbiota may therefore have therapeutic implications. Secretory IgA play a unique role in immune-microbiota crosstalk by directly binding to bacteria in the gut lumen. Microbe-specific IgA responses co-develop with the assembly of the gut microbiota during infancy, and resemble those of adults by 2 years postnatally in the healthy host. We propose here that microbiota-specific IgA-producing gut plasma cells generated during infancy live for many decades and contribute to a stable microbiota community. We furthermore suggest that members of the microbiota that induce long-lasting IgA responses in the gut are putative targets for therapeutic interventions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Macrophages (Mfs) are instrumental in maintaining immune homeostasis in the intestine, yet studies on the origin and heterogeneity of human intestinal Mfs are scarce. Here, we identified four distinct Mf subpopulations in human small intestine (SI). Assessment of their turnover in duodenal transplants revealed that all Mf subsets were completely replaced over time; Mf1 and Mf2, phenotypically similar to peripheral blood monocytes (PBMos), were largely replaced within 3 wk, whereas two subsets with features of mature Mfs, Mf3 and Mf4, exhibited significantly slower replacement. Mf3 and Mf4 localized differently in SI; Mf3 formed a dense network in mucosal lamina propria, whereas Mf4 was enriched in submucosa. Transcriptional analysis showed that all Mf subsets were markedly distinct from PBMos and dendritic cells. Compared with PBMos, Mf subpopulations showed reduced responsiveness to proinflammatory stimuli but were proficient at endocytosis of particulate and soluble material. These data provide a comprehensive analysis of human SI Mf population and suggest a precursor-progeny relationship with PBMos., (© 2018 Bujko et al.)

Background: Thickening of reticular basement membrane, increased airway smooth muscle mass and eosinophilic inflammation are found in adult fatal asthma. At the present study the histopathology of fatal paediatric and adolescent asthma is evaluated., Methods: Post-mortem lung autopsies from 12 fatal asthma cases and 8 non-asthmatic control subjects were examined. Thickness of reticular basement membrane (RBM) and percentage of airway smooth muscle (ASM%) mass area were measured and inflammatory cells were counted. Patient records were reviewed for clinical history., Results: The age range of the cases was from 0.9 to 19.5 years, eight were males and five had received inhaled corticosteroids. Thickened RBM was detected in majority of the cases without any correlation to treatment delay, age at onset of symptoms or diagnosis. In the large airways ASM was clearly increased in one third of the cases whereas the median ASM% did not differ from that in healthy controls (14.0% vs. 14.0%). In small airways no increase of ASM was found, instead mucous plugs were seen in fatal asthma. The number of eosinophils, plasmacytoid dendritic cells, macrophages, and B-cells were significantly increased in fatal asthma cases compared with controls and the two latter correlated with the length of the fatal exacerbation., Conclusions: The findings highlight the strong presence of eosinophils and mucous plugs even in small airways in children and adolescents with fatal asthma. Thickened RBM was obvious in majority of the patients. Contrary to our hypothesis, increased ASM% was detected in only one third of the patients.

Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19 + PC subset was dynamically exchanged, whereas of two CD19 - PC subsets, CD45 + PCs exhibited little and CD45 - PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45 - PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19 - PC subsets support selection and maintenance of protective PCs for life in human intestine., (© 2017 Landsverk et al.)

Background: Alveolar macrophages (AMFs) are critical regulators of lung function, and may participate in graft rejection following lung transplantation. Recent studies in experimental animals suggest that most AMFs are self-maintaining cells of embryonic origin, but knowledge about the ontogeny and life span of human AMFs is scarce., Methods: To follow the origin and longevity of AMFs in patients with lung transplantation for more than 100 weeks, we obtained transbronchial biopsies from 10 gender-mismatched patients with lung transplantation. These were subjected to combined in situ hybridisation for X/Y chromosomes and immunofluorescence staining for macrophage markers. Moreover, development of AMFs in humanised mice reconstituted with CD34+ umbilical cord-derived cells was assessed., Results: The number of donor-derived AMFs was unchanged during the 2 year post-transplantation period. A fraction of the AMFs proliferated locally, demonstrating that at least a subset of human AMFs have the capacity to self-renew. Lungs of humanised mice were found to abundantly contain populations of human AMFs expressing markers compatible with a monocyte origin. Moreover, in patients with lung transplantation we found that recipient monocytes seeded the alveoli early after transplantation, and showed subsequent phenotypical changes consistent with differentiation into proliferating mature AMFs. This resulted in a stable mixed chimerism between donor and recipient AMFs throughout the 2-year period., Conclusions: The finding that human AMFs are maintained in the lung parenchyma for several years indicates that pulmonary macrophage transplantation can be a feasible therapeutic option for patients with diseases caused by dysfunctional AMFs. Moreover, in a lung transplantation setting, long-term persistence of donor AMFs may be important for the development of chronic graft rejection., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Conventional dendritic cells (cDCs) comprise a heterogeneous population of cells that are important regulators of immunity and homeostasis. CD1c+ cDCs are present in human blood and tissues, and found to efficiently activate naïve CD4+ T cells. While CD1c is thought to specifically identify this subset of human cDCs, we show here that also classical and intermediate monocytes express CD1c. Accordingly, the commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two distinct cell populations from blood: CD1c+CD14- cDCs and CD1c+CD14+ monocytes. CD1c+ cDCs and CD1c+ monocytes exhibited strikingly different properties, including their differential regulation of surface marker expression, their levels of cytokine production, and their ability to stimulate naïve CD4+ T cells. These results demonstrate that a commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two functionally different cell populations, which has important implications for the interpretation of previously generated data using this kit to characterize CD1c+ cDCs.

CD4+ T regulatory cells (Tregs) comprise a heterogeneous population of cells the regulate immune responses and prevent autoimmunity. Most reports on human Tregs are derived from studies of peripheral blood, although Tregs mainly exert their functions in the periphery. Here we performed a detailed analysis of Tregs in the human upper airway mucosa under non-inflammatory conditions, and found that 10% of all CD4+ T cells expressed the transcription factor FOXP3 and the memory marker CD45RO, as well as high levels of CTLA-4. The majority of FOXP3+CD4+ T cells co-expressed the transcription factor Helios and produced very little cytokines, compatible with being thymus-derived Tregs. FOXP3+Helios-CD4+ T cells were more heterogeneous. A mean of 24% produced the immunomodulatory cytokine IL-10, whereas a large fraction also produced IL-2, IFN-μ or IL-17. A significant population (6%) of FOXP3-negative T cells also produced IL-10, usually in combination with IFN-μ. Together, we found that CD4+ T cells in the upper airways differed functionally from their counterparts in peripheral blood, including higher expression of IL-10. Moreover, our findings suggest that several subsets of CD4+ T cells with functionally distinct regulatory properties reside in the upper airway mucosa which should be taken into account when targeting Tregs for therapy.

Background: Associations between early deficits of lung function, infant airway disease, and outcome at school age in symptomatic infants are still unclear., Objective: To report follow-up data on a unique cohort of children investigated invasively in infancy to determine predictive value of airway disease for school-aged respiratory outcomes., Methods: Fifty-three infants previously studied using bronchoscopy and airway conductance were approached at 8 years of age. Symptoms, lung volumes, and airway responsiveness were reassessed. Data on lifetime purchase of asthma medication were obtained. Lung function was compared with that of 63 healthy nonasthmatic children., Results: Forty-seven children were reevaluated. Physician-diagnosed asthma was present in 39 children (83%). Twenty-five children (53%) had current and 14 children (30%) had past asthma. No pathologic feature in infancy correlated with any outcome parameter. As expected, study children had significantly reduced lung function and increased airway responsiveness compared with healthy controls, and very early symptoms were risk factors for reduced lung function. Current asthma was associated with reduced infant lung function and parental asthma. Reduced lung function in infancy was associated with purchase of inhaled corticosteroids when 6 to 8 and 0 to 8 years of age., Conclusion: The lack of predictive value of any pathologic measure in infancy, reported here for the first time to our knowledge, demonstrates that pathologic processes determining the inception of asthma, which are as yet undescribed, are different from the eosinophilic inflammation associated with ongoing disease., (Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Thymic stromal lymphopoietin (TSLP) controls allergic TH2 inflammatory responses through induction of distinct activation programs in dendritic cells (DCs). However, knowledge about TSLP receptor expression and functional consequences of receptor activation by DCs residing in the human respiratory tract is limited., Objective: We wanted to identify TSLP-responding DC populations in the human upper airway mucosa and assess the TSLP-mediated effects on such DCs in allergic airway responses., Results: We found that the TSLP receptor was constitutively and preferentially expressed by myeloid CD1c(+) DCs in the human airway mucosa and that the density of this DC subset in nasal mucosa increased significantly after in vivo allergen challenge of patients with allergic rhinitis. In vitro, TSLP strongly enhanced the capacity of CD1c(+) DCs to activate allergen-specific memory CD4(+) T cells. Moreover, TSLP rapidly induced CCR7 expression on CD1c(+) DCs. However, TH2 cytokines attenuated TSLP-mediated CCR7 induction, thus inhibiting the TSLP-induced DC migration potential to the draining lymph nodes., Conclusion: Our results suggest that TSLP-mediated activation of human nasal mucosal CD1c(+) DCs triggers CCR7-dependent migration to the draining lymph nodes and enhances their capacity to initiate TH2 responses. However, the observation that TH2 cytokines abrogate the induction of CCR7 implies that during a TH2-mediated inflammatory reaction, TLSP-activated CD1c(+) DCs are retained in the inflamed tissue to further exacerbate local inflammation by activating local antigen-specific memory TH2 cells., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Objectives: In celiac disease (CD), gluten induces both adaptive and innate immune responses. Non-celiac gluten sensitivity (NCGS) is another form of gluten intolerance where the immune response is less characterized. The aim of our study was to explore and compare the early mucosal immunological events in CD and NCGS., Methods: We challenged 30 HLA-DQ2(+) NCGS and 15 CD patients, all on a gluten-free diet, with four slices of gluten-containing bread daily for 3 days. Duodenal biopsy specimens were collected before and after challenge. The specimens were examined for cytokine mRNA by quantitative reverse transcriptase-PCR and for MxA-expression and CD3(+) intraepithelial lymphocytes (IELs) by immunohistochemistry and compared with specimens from untreated CD patients and disease controls., Results: In CD patients, tumor necrosis factor alpha (P=0.02) and interleukin 8 (P=0.002) mRNA increased after in vivo gluten challenge. The interferon gamma (IFN-γ) level of treated CD patients was high both before and after challenge and did not increase significantly (P=0.06). Four IFN-γ-related genes increased significantly. Treated and untreated CD patients had comparable levels of IFN-γ. Increased expression of MxA in treated CD patients after challenge suggested that IFN-α was activated on gluten challenge. In NCGS patients only IFN-γ increased significantly (P=0.03). mRNA for heat shock protein (Hsp) 27 or Hsp70 did not change in any of the groups. Importantly, we found that the density of IELs was higher in NCGS patients compared with disease controls, independent of challenge, although lower than the level for treated CD patients., Conclusions: CD patients mounted a concomitant innate and adaptive immune response to gluten challenge. NCGS patients had increased density of intraepithelial CD3(+) T cells before challenge compared with disease controls and increased IFN-γ mRNA after challenge. Our results warrant further search for the pathogenic mechanisms for NCGS.

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality. Mice lacking SRF in epidermis displayed morphogenetic defects, including an eye-open-at-birth phenotype and lack of whiskers. SRF-null skin exhibited abnormal morphology, hyperplasia, aberrant expression of differentiation markers and transcriptional regulators, anomalous actin organization, enhanced inflammation, and retarded hair follicle (HF) development. Transcriptional profiling experiments uncovered profound molecular changes in SRF-null E17.5 epidermis and revealed that many previously identified SRF target CArG box-containing genes were markedly upregulated in SRF-null epidermis, indicating that SRF may function to repress transcription of a subset of its target genes in epidermis. Remarkably, when transplanted onto nude mice, engrafted SRF-null skin lacked hair but displayed normal epidermal architecture with proper expression of differentiation markers, suggesting that although keratinocyte SRF is essential for HF development, a cross-talk between SRF-null keratinocytes and the surrounding microenvironment is likely responsible for the barrier-deficient mutant epidermal phenotype.

In less than half a century, allergy, originally perceived as a rare disease, has become a major public health threat, today affecting the lives of more than 60 million people in Europe, and probably close to one billion worldwide, thereby heavily impacting the budgets of public health systems. More disturbingly, its prevalence and impact are on the rise, a development that has been associated with environmental and lifestyle changes accompanying the continuous process of urbanization and globalization. Therefore, there is an urgent need to prioritize and concert research efforts in the field of allergy, in order to achieve sustainable results on prevention, diagnosis and treatment of this most prevalent chronic disease of the 21st century.The European Academy of Allergy and Clinical Immunology (EAACI) is the leading professional organization in the field of allergy, promoting excellence in clinical care, education, training and basic and translational research, all with the ultimate goal of improving the health of allergic patients. The European Federation of Allergy and Airways Diseases Patients' Associations (EFA) is a non-profit network of allergy, asthma and Chronic Obstructive Pulmonary Disorder (COPD) patients' organizations. In support of their missions, the present EAACI Position Paper, in collaboration with EFA, highlights the most important research needs in the field of allergy to serve as key recommendations for future research funding at the national and European levels.Although allergies may involve almost every organ of the body and an array of diverse external factors act as triggers, there are several common themes that need to be prioritized in research efforts. As in many other chronic diseases, effective prevention, curative treatment and accurate, rapid diagnosis represent major unmet needs. Detailed phenotyping/endotyping stands out as widely required in order to arrange or re-categorize clinical syndromes into more coherent, uniform and treatment-responsive groups. Research efforts to unveil the basic pathophysiologic pathways and mechanisms, thus leading to the comprehension and resolution of the pathophysiologic complexity of allergies will allow for the design of novel patient-oriented diagnostic and treatment protocols. Several allergic diseases require well-controlled epidemiological description and surveillance, using disease registries, pharmacoeconomic evaluation, as well as large biobanks. Additionally, there is a need for extensive studies to bring promising new biotechnological innovations, such as biological agents, vaccines of modified allergen molecules and engineered components for allergy diagnosis, closer to clinical practice. Finally, particular attention should be paid to the difficult-to-manage, precarious and costly severe disease forms and/or exacerbations. Nonetheless, currently arising treatments, mainly in the fields of immunotherapy and biologicals, hold great promise for targeted and causal management of allergic conditions. Active involvement of all stakeholders, including Patient Organizations and policy makers are necessary to achieve the aims emphasized herein.

Innate and adaptive mucosal defense mechanisms ensure a homeostatic relationship with the large and complex mutualistic gut microbiota. Dimeric IgA and pentameric IgM are transported across the intestinal epithelium via the epithelial polymeric Ig receptor (pIgR) and provide a significant portion of the first line of natural or adaptive antibody-mediated immune defense of the intestinal mucosa. We found that colonic epithelial cells from pIgR KO mice differentially expressed (more than twofold change) more than 200 genes compared with cells from WT mice, and upregulated the expression of antimicrobial peptides in a commensal-dependent manner. Detailed profiling of microbial communities based on 16S rRNA genes revealed differences in the commensal microbiota between pIgR KO and WT mice. Furthermore, we found that pIgR KO mice showed increased susceptibility to dextran sulfate sodium-induced colitis, and that this was driven by their conventional intestinal microbiota. Thus, in the absence of pIgR, the stability of the commensal microbiota is disturbed, gut homeostasis is compromised, and the outcome of colitis is significantly worsened., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Ichthyosis prematurity syndrome (IPS) is classified as a syndromic congenital ichthyosis based on the presence of skin changes at birth, ultrastructural abnormalities in the epidermis, and extracutaneous manifestations. Recently, mutations in the fatty acid transporter protein 4 gene have been identified in patients with IPS., Objective: We sought to perform a detailed clinical evaluation of patients with IPS identified in Norway., Methods: Clinical examination and follow-up of all patients (n = 23) and light and electron microscopic examination of skin biopsy specimens were performed., Results: IPS was characterized prenatally by ultrasound findings of polyhydramnios, separation of membranes, echogenic amniotic fluid, and clear chorionic fluid. All patients were born prematurely with sometimes life-threatening neonatal asphyxia; this was likely caused by aspiration of corneocyte-containing amniotic fluid as postmortem examination of lung tissue in two patients revealed keratin debris filling the bronchial tree and alveoli. The skin appeared erythrodermic, swollen, and covered by a greasy, thick vernix caseosa-like "scale" at birth, and evolved rapidly to a mild chronic ichthyosis. Many patients subsequently had chronic, severe pruritus. Histopathologic and ultrastructural examination of skin biopsy specimens showed hyperkeratosis, acanthosis, dermal inflammation, and characteristic aggregates of curved lamellar structures in the upper epidermis. Peripheral blood eosinophilia was invariably present and most patients had increased serum immunoglobulin E levels. Over 70% of the patients had a history of respiratory allergy and/or food allergy., Limitations: The study included only 23 patients because of the rarity of the disease., Conclusion: IPS is characterized by defined genetic mutations, typical ultrastructural skin abnormalities, and distinct prenatal and postnatal clinical features., (Copyright © 2011 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Objectives: Investigation of uncertain celiac disease (CD) in patients already on a gluten-free diet (GFD) is difficult. We evaluated HLA-DQ2-gliadin tetramers for detection of gluten-specific T cells in peripheral blood and histological changes in the duodenum after a short gluten challenge as a diagnostic tool., Methods: HLA-DQ2+ individuals on a GFD for at least 4 weeks were investigated; 35 with uncertain diagnosis, 13 CD patients, and 2 disease controls. All participants had a challenge with four slices of gluten-containing white bread, daily for 3 days (d1-d3). An esophagogastroduodenoscopy with biopsy sampling was done on d0 and d4. Biopsies were scored according to revised Marsh criteria. Peripheral blood CD4+ T cells were isolated, stained with HLA-DQ2-gliadin peptide tetramers, and analyzed by flow cytometry on d0 and d6., Results: After challenge, a positive tetramer test was seen in 11/13 CD patients. Four of these subjects also showed typical histological changes on challenge. Of the 35 patients with uncertain diagnosis, 3 were diagnosed with CD. Two of these three patients had both positive tetramer staining and histological changes in biopsies after challenge., Conclusions: Tetramer staining for gluten-specific T cells is a sensitive method in detecting an immune response in CD patients after a short gluten challenge. The prevalence of CD in the group with self-prescribed GFD was about 10%.

Background: Childhood represents an immunological window of vulnerability in which individuals are at increased risk for both serious infections and development of allergic diseases, particularly affecting the airways. However, little is known about how the airway mucosal immune system is organised and functions during early age. Here, the organisation of immune cells in bronchial mucosa of children was characterised., Methods: Immunophenotyping was performed on mucosal samples obtained postmortem from nine children aged 2-15 years without any history of atopic manifestations or any signs of respiratory disease, who died from non-inflammatory causes., Results: In all nine cases, isolated lymphoid follicles (ILFs), interpreted as bronchus-associated lymphoid tissue (BALT), were found, constituting an average frequency of 60 ILFs/cm(2) of airway mucosal surface. Outside these ILFs, dense networks of CD11c(+) myeloid dendritic cells (DCs), CD68(+) macrophages and CD3(+)CD45RA(-) memory T cells were found. Plasmacytoid DCs occurred in low numbers. Importantly, intraepithelial antigen-presenting cells were found to extend cellular projections into the airway lumen., Conclusion: The density and location of antigen-presenting cells and T cells in this age group are similar to those observed in adults. However, in contrast to adults, BALT appears to be a normal feature of the airway mucosa throughout childhood, suggesting that these structures contribute to regional immunity and homeostasis. This indicates that the local immune system in the airways of children has unique features which should be taken into account, not only when studying airway immunology and immunopathology, but also in the development of mucosal vaccines.

Prospective studies tracking birth cohorts over periods of years indicate that the seeds for atopic asthma in adulthood are sewn during early life. The key events involve programming of functional phenotypes within the immune and respiratory systems which determine long-term responsiveness to ubiquitous environmental stimuli, particularly respiratory viruses and aeroallergens. A crucial component of asthma pathogenesis is early sensitization to aeroallergens stemming from a failure of mucosal tolerance mechanisms during the preschool years, which is associated with delayed postnatal maturation of a range of adaptive and innate immune functions. These maturational defects also increase risk for severe respiratory infections, and the combination of sensitization and infections maximizes risk for early development of the persistent asthma phenotype. Interactions between immunoinflammatory pathways stimulated by these agents also sustain the disease in later life as major triggers of asthma exacerbations. Recent studies on the nature of these interactions suggest the operation of an infection-associated lung:bone marrow axis involving upregulation of FcERlalpha on myeloid precursor populations prior to their migration to the airways, thus amplifying local inflammation via IgE-mediated recruitment of bystander atopic effector mechanisms. The key participants in the disease process are airway mucosal dendritic cells and adjacent epithelial cells, and transiting CD4(+) effector and regulatory T-cell populations, and increasingly detailed characterization of their roles at different stages of pathogenesis is opening up novel possibilities for therapeutic control of asthma. Of particular interest is the application of genomics-based approaches to drug target identification in cell populations of interest, exemplified by recent findings discussed below relating to the gene network(s) triggered by activation of Th2-memory cells from atopics., (2009 Elsevier Inc. All rights reserved.)

Objective: Naturally occurring regulatory T (T(R)) cells suppress autoreactive T cells whereas adaptive T(R) cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive T(R) cells express COX-2 and produce PGE(2) that suppress effector T cells in a manner that is reversed by COX-inhibitors., Methods and Results: Here we demonstrate that CRC patients have elevated levels of PGE(2) in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ T(R) cells. Depletion of T(R) cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity., Conclusion: We suggest that adaptive T(R) cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2-PGE(2)-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T(R) cells and the PGE(2)-cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.

The respiratory tract has an approximate surface area of 70 m2 in adult humans, which is in virtually direct contact with the outside environment. It contains a uniquely rich vascular bed containing a large pool of marginated T cells, and harbours a layer of single-cell-thick epithelial tissue through which re-oxygenation of blood must occur uninterrupted for survival. It is therefore not surprising that the respiratory tract is never more than a short step away from disaster. We have only a partial understanding of how immunological homeostasis is maintained in these tissues, but it is becoming clear that the immune system has evolved a range of specific mechanisms to deal with the unique problems encountered in this specialized microenvironment.

Dendritic cells (DCs) are believed to regulate T cell-mediated immunity primarily by directing differentiation of naive T cells. Here, we show that a large fraction of CD4(+) memory cells produce IL-10 within the first hours after interaction with plasmacytoid DCs (PDCs). In contrast, CD11c(+) DCs induce IFN-gamma and little IL-10. IL-10-secreting T cells isolated after 36 hours of culture with PDCs suppressed antigen-induced T-cell proliferation by an IL-10-dependent mechanism, but were distinct from natural and type 1 regulatory T cells. They proliferated strongly and continued to secrete IL-10 during expansion with PDCs, and after restimulation with immature monocyte-derived DCs or CD11c(+) DCs. The IL-10-producing T cells acquired the ability to secrete high levels of IFN-gamma after isolation and subsequent coculture with PDCs or CD11c(+) DCs. Compared to CD11c(+) DCs, PDCs were superior in their ability to selectively expand T cells that produced cytokines on repeated antigenic challenge. The DC-dependent differences in cytokine profiles were observed with viral recall antigen or staphylococcal enterotoxin B and were independent of extracellular type I interferon or IL-10. Our results show that DCs can regulate memory responses and that PDCs rapidly induce regulatory cytokines in effector T cells that can suppress bystander activity.

An increase in the tempo of local dendritic cell (DC)-mediated immune surveillance is a recognized feature of the response to acute inflammation at airway mucosal surfaces, and transient up-regulation of the APC functions of these DC preceding their emigration to regional lymph nodes has recently been identified as an important trigger for T cell-mediated airway tissue damage in diseases such as asthma. In this study, using a rat model, we demonstrate that the kinetics of the airway mucosal DC (AMDC) response to challenge with heat-killed bacteria is considerably more rapid and as a consequence more effectively compartmentalized than that in recall responses to soluble Ag. Notably, Ag-bearing AMDC expressing full APC activity reach regional lymph nodes within 30 min of cessation of microbial exposure, and in contrast to recall responses to nonpathogenic Ags, there is no evidence of local expression of APC activity within the airway mucosa preceding DC emigration. We additionally demonstrate that, analogous to that reported in the gut, a subset of airway intraepithelial DC extend their processes into the airway lumen. This function is constitutively expressed within the AMDC population, providing a mechanism for continuous immune surveillance of the airway luminal surface in the absence of "danger" signals.

Background & Aims: Celiac disease is a chronic inflammation of the duodenal mucosa driven by gluten-reactive T cells restricted by the disease-associated HLA-DQ2 molecule. The mechanisms that regulate the activation of mucosal T cells are, however, understood poorly. The aim of this study was to identify the antigen-presenting cells that are responsible for the activation of gluten-reactive T cells in the celiac lesion., Methods: Intestinal biopsy specimens obtained from untreated and treated celiac patients and normal controls were either snap-frozen directly or incubated for 24 hours with or without gluten peptides. Cryosections were subjected to multicolor immunofluorescence applying monoclonal antibodies to a range of antigen-presenting cell markers. Macrophages and dendritic cells were isolated from enzymatically digested small intestinal biopsies of untreated patients and incubated with gluten-reactive T-cell clones to measure their antigen-presenting capacity., Results: HLA-DQ2+ cells in the normal duodenal mucosa consisted of 2 distinct cell populations: about 80% were CD68+ DC-lysosome intercellular adhesion molecule-3-grabbing nonintegrin+ macrophages and 20% were CD11c+ dendritic cells. Importantly, the CD11c+ dendritic cells accumulated in the celiac lesion and revealed an activated phenotype expressing CD86 and DC-specific-associated membrane protein. Moreover, when isolated from challenged biopsy specimens, the CD11c+ dendritic cells efficiently activated gluten-reactive T cells., Conclusions: Our results suggest that a unique subset of dendritic cells are responsible for local activation of gluten-reactive T cells in the celiac lesion.

Expression of HLA-DO (DO) in cells that express HLA-DM (DM) results in an altered repertoire of MHC class II/peptide complexes, indicating that DO modulates DM function. Human and murine B cells and thymic epithelial cells express DO, while monocytes/macrophages do not. Monocyte-derived dendritic cells (DC) also have been found to be DO-negative, leading to the assumption that DC do not express DO. In this study, we report that, in fact, certain types of human primary DC express DO. These include Langerhans cells (LC) and some subtypes of circulating blood DC. Specifically, the majority of BDCA-3(+) DC, a small subset of uncertain function, are DO(+), while smaller proportions of CD11c(+), BDCA-1(+) (myeloid) DC, at most a minority of CD123(+)/BDCA-2(+) (plasmacytoid) DC, and no detectable CD16(+) (myeloid) DC, express DO. Immunohistochemistry of human tonsil sections demonstrates that tonsillar interdigitating DC are also DO(+). In a subset of immature LC with higher DO expression, an increased fraction of surface DR molecules carry CLIP peptides, indicating that DO functions as a DM inhibitor in these cells. LC expression of DO is down-regulated by maturation stimuli. DM levels also decrease under these conditions, but the DM:DO ratio generally increases. In the myeloid cell types tested, DO expression correlates with levels of DObeta, but not DOalpha, implying that modulation of DObeta regulates DO dimer abundance in these cells. The range of APC types shown to express DO suggests a broader role for DO in immune function than previously appreciated.

It has been suggested that human cytomegalovirus (HCMV) evades the immune system by infecting and paralyzing antigen-presenting cells. This view is based mainly on studies of dendritic cells (DCs) obtained after culture of monocytes (moDCs). It is contradicted by the asymptomatic course of HCMV infection in healthy persons, indicating that other key antigen-presenting cells induce an efficient immune response. Here we show that HCMV activates CD11c+ DCs and plasmacytoid DCs (PDCs). In contrast to moDCs, CD11c+ DCs and PDCs produced interferon (IFN) type 1 when exposed to HCMV. Autocrine IFN type 1 partially protected CD11c+ DCs against infection, whereas PDCs were resistant to HCMV even when IFN type 1 activity was inhibited. HCMV exposure induced the maturation of CD11c+ DCs by IFN type 1-dependent and -independent mechanisms. Importantly, CD11c+ DCs infected by inhibiting IFN type 1 activity retained full capacity to stimulate T cells. Renal transplant recipients receiving immunosuppressive treatment had lower frequencies of CD11c+ DCs and PDCs in blood than did healthy controls. The results show that HCMV activates the immune system by interacting with CD11c+ DCs and PDCs and that recipients of renal transplants have low frequencies of these cell types in blood.

CD11c+ dendritic cells (DC) and plasmacytoid DC (PDC) are the two major DC subsets in human peripheral blood. For the purpose of immunotherapy with DC, it is important to investigate the phagocytosis of killed tumor cells by different DC subsets. Using immature monocyte-derived DC (iMoDC) as reference, we have compared the ability of CD11c+ DC and PDC to phagocytose apoptotic and necrotic K562 leukemia cells. Freshly isolated CD11c+ DC phagocytosed apoptotic and necrotic K562 cells, whereas PDC did not show any evidence of uptake of dead cells. Blocking studies showed that CD36 is importantly involved in uptake of apoptotic and necrotic material. CD91 and CD11c were also involved. In addition, we found that beta5 integrin was expressed on CD11c+ DC but not in its classical association with alphaV. Uptake of apoptotic K562 cells by CD11c+ DC was increased following incubation with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, alone or in combination with transforming growth factor-beta1, to levels comparable with those observed for iMoDC. Phagocytosis of dead cellular material by the GM-CSF/IL-4-treated CD11c+ DC was largely restricted to a subset expressing low levels of human leukocyte antigen-DR and CD83. Thus, the relationship between phagocytosis of antigenic material and expression of maturation-related cell-surface molecules is similar for CD11c+ DC and MoDC. We conclude that CD11c+ DC in peripheral blood are precursor cells, which under the influence of cytokines, differentiate to cells with DC phenotype and function.

Background: Plasmacytoid dendritic cells (PDCs) accumulate in the nasal mucosa of allergic rhinitis patients, but their function in upper airway allergy has not been determined. CpG oligodeoxynucleotides, potent adjuvants in immunotherapeutic strategies in animal models, are especially effective at activating PDCs. These cells are therefore potential targets for immunomodulation in humans., Objective: In this study, PDCs were compared with CD11c+ dendritic cells (DCs), a very potent antigen-presenting cell type, for their capacity to induce allergen-dependent activation of TH2 memory cells. Then, we investigated whether CpG-activated PDCs were able to modulate the allergen-specific TH2 memory response., Methods: DCs were isolated from patients with upper airway allergy and cocultured with autologous CD4+ T cells with or without grass pollen extract and CpG. In some experiments cells were restimulated with allergen-pulsed monocyte-derived DCs. T-cell activation was measured by their proliferative response and cytokine production., Results: PDCs stimulated allergen-dependent T-cell proliferation and TH2 cytokine production as efficiently as CD11c+ DCs. CpG-activated PDCs inhibited allergen-dependent proliferation of TH2 memory cells and markedly increased IFN-gamma production in PDC/T-cell cocultures; the former effect depended on the CpG-induced IFN-alpha/beta production by the PDCs., Conclusion: Our results demonstrated that PDCs efficiently drive allergen-dependent TH2 memory responses, suggesting that they play an active role in the allergic reaction. However, in the presence of CpG, PDCs were responsible for increased production of the TH1-related cytokines IFN-alpha and IFN-gamma, indicating that mucosal PDCs may be targets for CpG-based immunotherapeutic strategies against airway allergy.

Professional antigen-presenting cells (APCs) constitute a heterogeneous leukocyte population that controls T cell induction. Experimental animal studies have delineated the principal APCs of the airway mucosa as a network of intraepithelial dendritic cells (DCs). Whether the situation is comparable in the human airways is unknown. Here we performed a detailed characterization of putative APCs residing in the normal upper airway mucosa, employing confocal microscopy of whole-mount preparations combined with immunophenotyping. A dense network of human leukocyte antigen-DR+ cells with dendritic morphology was found not only in the epithelium (median number, 573/mm2), but also in the lamina propria. In both compartments these cells could be divided into two main populations based on their phenotypic characteristics: the majority expressed a macrophage-like phenotype (CD11b+CD14+CD64+CD68+RFD7+), whereas the smaller population was predominantly constituted by CD1c+CD11c+ immature DCs intermingled with the former. These immature DCs corresponded to the lineage-negative human leukocyte antigen-DR+CD11c+ DC subset present in peripheral blood. Thus, the human upper airway mucosa, in contrast to the rodent counterpart, contains a heterogeneous dense network of dendritic APCs consisting of spatially closely related macrophages and DCs. How these two cell populations regulate the tone of the local adaptive immune system should be the focus of further studies.

Background: Pulmonary veno-occlusive disease is a rare and poorly understood condition that affects the postcapillary pulmonary vasculature, posing diagnostic problems and treatment dilemmas., Materials and Methods: We present a patient with veno-occlusive disease and give a short review on the disease., Results: The patient was a 54-year-old female with a history of the CREST variant of scleroderma. Admitted with dyspnoea, she was treated with epoprostenol in addition to oxygen, diuretics and warfarin. Epoprostenol improved her condition initially; her symptoms grew worse during further medical escalation. With an attempt to stop epoprostenol, however, she became even more dyspnoeic and tolerated best an intermediate dose. She died after three months of treatment with signs of progressive right heart failure., Interpretation: Veno-occlusive disease may be difficult to diagnose and treat. Clinical signs of pulmonary hypertension without evidence of left ventricular failure may give rise to suspicion of the disease, and high-resolution CT of the lungs with relatively specific findings can be helpful. The prognosis is poor and lung transplantation is the only form of effective treatment. Vasodilators as a bridge to transplantation must be used with caution because of the risk of intolerance and development of pulmonary oedema.

The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues.

The airway mucosal response to allergen in asthma involves influx of activated T helper type 2 cells and eosinophils, transient airflow obstruction, and airways hyperresponsiveness (AHR). The mechanism(s) underlying transient T cell activation during this inflammatory response is unclear. We present evidence that this response is regulated via bidirectional interactions between airway mucosal dendritic cells (AMDC) and T memory cells. After aerosol challenge, resident AMDC acquire antigen and rapidly mature into potent antigen-presenting cells (APCs) after cognate interactions with T memory cells. This process is restricted to dendritic cells (DCs) in the mucosae of the conducting airways, and is not seen in peripheral lung. Within 24 h, antigen-bearing mature DCs disappear from the airway wall, leaving in their wake activated interleukin 2R+ T cells and AHR. Antigen-bearing activated DCs appear in regional lymph nodes at 24 h, suggesting onward migration from the airway. Transient up-regulation of CD86 on AMDC accompanies this process, which can be reproduced by coculture of resting AMDC with T memory cells plus antigen. The APC activity of AMDC can be partially inhibited by anti-CD86, suggesting that CD86 may play an active role in this process and/or is a surrogate for other relevant costimulators. These findings provide a plausible model for local T cell activation at the lesional site in asthma, and for the transient nature of this inflammatory response.

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.