Search

Contains fulltext : 237768.pdf (Publisher’s version ) (Open Access) The two most common chronic inflammatory skin diseases are atopic dermatitis (AD) and psoriasis. The underpinnings of the remarkable degree of clinical heterogeneity of AD and psoriasis are poorly understood and, as a consequence, disease onset and progression are unpredictable and the optimal type and time point for intervention are as yet unknown. The BIOMAP project is the first IMI (Innovative Medicines Initiative) project dedicated to investigating the causes and mechanisms of AD and psoriasis and to identify potential biomarkers responsible for the variation in disease outcome. The consortium includes 7 large pharmaceutical companies and 25 non-industry partners including academia. Since there is mounting evidence supporting an important role for microbial exposures and our microbiota as factors mediating immune polarization and AD and psoriasis pathogenesis, an entire work package is dedicated to the investigation of skin and gut microbiome linked to AD or psoriasis. The large collaborative BIOMAP project will enable the integration of patient cohorts, data and knowledge in unprecedented proportions. The project has a unique opportunity with a potential to bridge and fill the gaps between current problems and solutions. This review highlights the power and potential of the BIOMAP project in the investigation of microbe-host interplay in AD and psoriasis.

Structural, biochemical and cellular analyses show that bacterial antigens can mimic gliadin epitopes involved in celiac disease being presented by HLA-DQ2.5 and recognized by T cells derived from patients.The human leukocyte antigen (HLA) locus is strongly associated with T cell-mediated autoimmune disorders. HLA-DQ2.5-mediated celiac disease (CeD) is triggered by the ingestion of gluten, although the relative roles of genetic and environmental risk factors in CeD is unclear. Here we identify microbially derived mimics of gliadin epitopes and a parental bacterial protein that is naturally processed by antigen-presenting cells and activated gliadin reactive HLA-DQ2.5-restricted T cells derived from CeD patients. Crystal structures of T cell receptors in complex with HLA-DQ2.5 bound to two distinct bacterial peptides demonstrate that molecular mimicry underpins cross-reactivity toward the gliadin epitopes. Accordingly, gliadin reactive T cells involved in CeD pathogenesis cross-react with ubiquitous bacterial peptides, thereby suggesting microbial exposure as a potential environmental factor in CeD.

Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of gluten‑specific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease.

BackgroundCeliac disease (CeD) is a complex T cell–mediated enteropathy induced by gluten. Although genome-wide association studies have identified numerous genomic regions associated with CeD, it is difficult to accurately pinpoint which genes in these loci are most likely to cause CeD.ResultsWe used four different in silico approaches – Mendelian Randomization inverse variance weighting, COLOC, LD overlap and DEPICT – to integrate information gathered from a large transcriptomics dataset. This identified 118 prioritized genes across 50 CeD-associated regions. Co-expression and pathway analysis of these genes indicated an association with adaptive and innate cytokine signalling and T cell activation pathways. 51 of these genes are targets of known drug compounds and likely druggable genes, suggesting that our methods can be used to pinpoint potential therapeutic targets. In addition, we detected 172 gene-combinations that were affected by our CeD-prioritized genes in trans. Notably, 41 of these trans-mediated genes appear to be under control of one master regulator, TRAFD1, and were found to be involved in IFNγ signalling and MHC I antigen processing/presentation. Finally, we performed in vitro experiments that validated the role of TRAFD1 as an immune regulator acting in trans.ConclusionsOur strategy has confirmed the role of adaptive immunity in CeD and revealed a genetic link between CeD and the IFNγ signalling and MHC I antigen processing pathways, both major players of immune activation and CeD pathogenesis.

Controlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune signatures of malaria-naive Europeans and of Africans with lifelong malaria exposure using mass cytometry, RNA sequencing and data integration, before and 5 and 11 days after venous inoculation with Plasmodium falciparum sporozoites. We observed differences in immune cell populations, antigen-specific responses and gene expression profiles between Europeans and Africans and among Africans with differing degrees of immunity. Before inoculation, an activated/differentiated state of both innate and adaptive cells, including elevated CD161(+)CD4(+) T cells and interferon-gamma production, predicted Africans capable of controlling parasitemia. After inoculation, the rapidity of the transcriptional response and clusters of CD4(+) T cells, plasmacytoid dendritic cells and innate T cells were among the features distinguishing Africans capable of controlling parasitemia from susceptible individuals. These findings can guide the development of a vaccine effective in malaria-endemic regions.Malaria immunity can be acquired through natural infection, but the correlates of protection are still being determined. Yazdanbakhsh and colleagues combine experimental infection of volunteers with Plasmodium falciparum with systems analysis to throw light on the nature of protective immune responses.

During healthy pregnancy, a balanced microenvironment at the maternal-fetal interface with coordinated interaction between various immune cells is necessary to maintain immunological tolerance. While specific decidual immune cell subsets have been investigated, a system-wide unbiased approach is lacking. Here, mass cytometry was applied for data-driven, in-depth immune profiling of the total leukocyte population isolated from first, second, and third trimester decidua, as well as maternal peripheral blood at time of delivery. The maternal-fetal interface showed a unique composition of immune cells, different from peripheral blood, with significant differences between early and term pregnancy samples. Profiling revealed substantial heterogeneity in the decidual lymphoid and myeloid cell lineages that shape gestational-specific immune networks and putative differentiation trajectories over time during gestation. Uncovering the overall complexity at the maternal-fetal interface throughout pregnancy resulted in a human atlas that may serve as a foundation upon which comprehension of the immune microenvironment and alterations thereof in pregnancy complications can be built.

The lung niche provides important signals for proper functioning and interactions between epithelial and immune cells, and is suggested to be altered in emphysema in chronic obstructive pulmonary disease (COPD). Here, we determined whether high-dimensional mass cytometry (cytometry by time-of-flight; CyTOF) analysis is feasible to identify immune cell populations in the lung and can be used to delineate immune functions in human ‘healthy’ and diseased lungs. Therefore, we performed a first CyTOF analysis to compare immune cells derived from non-emphysematous and severe emphysematous lung tissue. We isolated single cells from fresh non-emphysematous lungs (n=5), emphysematous lungs (n=15) and paired blood samples and stored them at -80°C. We applied a CyTOF panel comprising 39 antibodies to obtain a global overview of the immune cell composition in the lung. Finally, we analyzed data with Hierarchical Stochastic Neighbor Embedding (HSNE) and dual t-SNE to identify tissue-specific cell clusters. CyTOF analysis revealed distinct immune cell populations in human lung tissue, which clustered differently from blood-derived cells. Importantly, tissue myeloid cells from emphysematous lungs clustered differently from cells derived from non-emphysematous controls. We showed that CyTOF analysis of lung tissue is feasible, and reveals distinct features of myeloid populations in emphysematous tissue. We are now further expanding the panel to detect lung-specific markers. Furthermore, we will apply imaging mass cytometry to allow the simultaneous detection of up to 40 markers with spatial information in a single lung tissue section, allowing examination of altered cellular interactions in emphysema.

Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantificationin situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at -20 degrees C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at -20 degrees C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4 degrees C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of alpha-smooth muscle actin (alpha-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality.

BackgroundAdenoviral vectors emerged as important platforms for cancer immunotherapy. Vaccination with adenoviral vectors is promising in this respect, however, their specific mechanisms of action are not fully understood. Here, we assessed the development and maintenance of vaccine-induced tumor-specific CD8+ T cells elicited upon immunization with adenoviral vectors.MethodsAdenoviral vaccine vectors encoding the full-length E7 protein from human papilloma virus (HPV) or the immunodominant epitope from E7 were generated, and mice were immunized intravenously with different quantities (107, 108 or 109 infectious units). The magnitude, kinetics and tumor protection capacity of the induced vaccine-specific T cell responses were evaluated.ResultsThe adenoviral vaccines elicited inflationary E7-specific memory CD8+ T cell responses in a dose-dependent manner. The magnitude of these vaccine-specific CD8+ T cells in the circulation related to the development of E7-specific CD8+ tissue-resident memory T (TRM) cells, which were maintained for months in multiple tissues after vaccination. The vaccine-specific CD8+ T cell responses conferred long-term protection against HPV-induced carcinomas in the skin and liver, and this protection required the induction and accumulation of CD8+ TRM cells. Moreover, the formation of CD8+ TRM cells could be enhanced by temporal targeting CD80/CD86 costimulatory interactions via CTLA-4 blockade early after immunization.ConclusionsTogether, these data show that adenoviral vector-induced CD8+ T cell inflation promotes protective TRM cell populations, and this can be enhanced by targeting CTLA-4.

Cancers are characterized by extensive heterogeneity that occurs intratumorally, between lesions, and across patients. To study cancer as a complex biological system, multidimensional analyses of the tumor microenvironment are paramount. Single-cell technologies such as flow cytometry, mass cytometry, or single-cell RNA-sequencing have revolutionized our ability to characterize individual cells in great detail and, with that, shed light on the complexity of cancer microenvironments. However, a key limitation of these single-cell technologies is the lack of information on spatial context and multicellular interactions. Investigating spatial contexts of cells requires the incorporation of tissue-based techniques such as multiparameter immunofluorescence, imaging mass cytometry, or in situ detection of transcripts. In this Review, we describe the rise of multidimensional single-cell technologies and provide an overview of their strengths and weaknesses. In addition, we discuss the integration of transcriptomic, genomic, epigenomic, proteomic, and spatially-resolved data in the context of human cancers. Lastly, we will deliberate on how the integration of multi-omics data will help to shed light on the complex role of cell types present within the human tumor microenvironment, and how such system-wide approaches may pave the way toward more effective therapies for the treatment of cancer.

Celiac disease (CD) affects a growing number of individuals worldwide. To elucidate the causes for this increase, future multidisciplinary collaboration is key to understanding the interactions between immunoreactive components in gluten-containing cereals and the human gastrointestinal tract and immune system and to devise strategies for CD prevention and treatment beyond the gluten-free diet. During the last meetings, the Working Group on Prolamin Analysis and Toxicity (Prolamin Working Group, PWG) discussed recent progress in the field together with key stakeholders from celiac disease societies, academia, industry and regulatory bodies. Based on the current state of knowledge, this perspective from the PWG members provides recommendations regarding clinical, analytical and legal aspects of CD. The selected key topics that require future multidisciplinary collaborative efforts in the clinical field are to collect robust data on the increasing prevalence of CD, to evaluate what is special about gluten-specific T cells, to study their kinetics and transcriptomics and to put some attention to the identification of the environmental agents that facilitate the breaking of tolerance to gluten. In the field of gluten analysis, the key topics are the precise assessment of gluten immunoreactive components in wheat, rye and barley to understand how these are affected by genetic and environmental factors, the comparison of different methods for compliance monitoring of gluten-free products and the development of improved reference materials for gluten analysis., Instituto de Estudios Inmunológicos y Fisiopatológicos

Li et al. apply mass cytometry to delineate the fetal gut innate lymphoid cell (ILC) population and utilize a t-SNE–based approach to predict potential differentiation trajectories. They identify an int-ILC subset that differentiates into NK cells or ILC3s in vitro., Innate lymphoid cells (ILCs) are abundant in mucosal tissues and involved in tissue homeostasis and barrier function. Although several ILC subsets have been identified, it is unknown if additional heterogeneity exists, and their differentiation pathways remain largely unclear. We applied mass cytometry to analyze ILCs in the human fetal intestine and distinguished 34 distinct clusters through a t-SNE–based analysis. A lineage (Lin)−CD7+CD127−CD45RO+CD56+ population clustered between the CD127+ ILC and natural killer (NK) cell subsets, and expressed diverse levels of Eomes, T-bet, GATA3, and RORγt. By visualizing the dynamics of the t-SNE computation, we identified smooth phenotypic transitions from cells within the Lin−CD7+CD127−CD45RO+CD56+ cluster to both the NK cells and CD127+ ILCs, revealing potential differentiation trajectories. In functional differentiation assays, the Lin−CD7+CD127−CD45RO+CD56+CD8a− cells could develop into CD45RA+ NK cells and CD127+RORγt+ ILC3-like cells. Thus, we identified a previously unknown intermediate innate subset that can differentiate into ILC3 and NK cells., Graphical Abstract

Background Local mesenchymal stromal cell (MSC)-therapy is approved for the treatment of Crohn’s disease-associated perianal fistulas. However, little is known about the working mechanism of local MSC-therapy. For the first time we evaluated engraftment and immunoregulatory effects of local MSC-therapy in patients with refractory proctitis. To do so, we analyzed biopsies and serum from patients with ulcerative proctitis before and after treatment with endoscopically injected MSCs in a phase IIa clinical trial (EudraCT number 2017-003524-75). Methods Thirteen therapy-refractory ulcerative proctitis patients were endoscopically injected bone marrow-derived allogeneic MSCs from healthy donors. Clinical efficacy was evaluated by the endoscopic and full Mayo score. Engraftment of the MSCs was investigated using fluorescence in-situ hybridization (FISH) of sex chromosomes on post-treatment biopsies. The presence of anti-HLA-antibodies against the MSC-donor was determined in the serum. Changes in immune cell subsets were evaluated using cytometry-by-time-of-flight (CyTOF) analysis. Results Thirteen patients with an endoscopic Mayo score of 2 (n=3) or 3 (n=10) of the rectum were treated with local MSC-therapy. Although complete remission was not achieved, full Mayo score was improved at week 6 (median 8 [IQR 6–10]) compared to baseline (median 11 [IQR 9.5–12]) (p=0.001). Preliminary data using FISH on the Y-chromosome, indicated the presence of MSCs in the rectum biopsies of female patients treated with male donor derived-MSCs at week 6. At baseline, HLA-antibodies were present in four patients. Six weeks after local injection of the MSCs, two out of thirteen patients developed new class I and II HLA-antibodies against the MSCs. Interestingly, in two patients pre-existing HLA-antibodies showed increased/boosted levels after local MSC-therapy, while one additionally developed new HLA-antibodies. CyTOF analysis of inflamed rectal biopsies 6 weeks after MSC treatment revealed significantly increased frequencies of several myeloid subsets (i.e. CD11b+CD14+CCR7+/-CD127+CD25+HLADR+ and CD14+HLA-DR-CD123-CCR7+) and a subset of CD4+ memory T cells with a more exhausted/regulated phenotype (PD-1+TIGIT+CD69+CD38+CD69). Conclusion Local MSC-therapy in patients with refractory proctitis changed the rectal immune profile characterised by a significant increase in a subset of effector memory CD4+ cells and several myeloid subsets, which might be associated with immune modulation. These results provide the basis for future studies on the mechanism of action of MSCs on rectal mucosa. New anti-HLA class antibodies developed in 2/13 patients after local administration. Whether these latter results have consequences for MSC-donor selection deserves further study.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ 3- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ 3- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ 3-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ 3- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

We read with interest the letter to the editor by Massier et al 1 and the accompanying response by Fasano.2 We would like to point out one critical issue that has not been addressed, namely, the relationship between AT1001, zonulin and prehaptoglobin-2. AT1001 is an octapeptide that was thought to represent the N-terminal sequence of zonulin.3 4 However, in all likelihood this octapeptide sequence (GGVLVQPG) derives from immunoglobulin. In their attempt to identify the human homolog of the Vibrio cholera zonula occludens …

Background Ulcerative proctitis (UP) causes a chronic rectal mucosal inflammation. A subset of patients does not respond to one or more medication options. Mesenchymal stromal cells (MSCs) show beneficial effects in animal models and patients with immune diseases. However, the effects of MSCs on the mucosal immune composition in patients with inflammatory bowel disease (IBD) are not unravelled yet. Methods Patients with UP (N = 7) were locally treated with allogeneic bone marrow-derived MSCs. A total of 20 × 106 MSCs was injected if the inflammation was restricted to 7 cm and 40 × 106 if inflammation exceeds 7 cm. Medication was stable in all patients from at least 2 weeks before treatment up to week 6 after treatment. Biopsies were taken from the affected and unaffected colon before (t = 0) and 6 weeks (t = 6) after treatment from the same locations and faecal calprotectin (FCP) was determined. Single-cell suspensions from the biopsies were stained with a 42 antibody panel and analysed with a mass cytometer to identify and characterise possible effects of MSCs on immune subsets. The generated dataset was analysed with Hierarchical SNE (HSNE) in the Cytosplore analysis and visualisation tool. Results FCP decreased significantly in four out of seven patients (419→191; 365→40; 1744→346; 3270→291), ‘responders’, while in three patients FCP increased, ‘non-responders’. Unbiased hierarchical clustering of the subsets showed no clear differences in the major immune lineages before and after treatment. In some patients; however, a prominent myeloid population was present in the affected colon, whereas the CD8+− and CD3−CD7+ populations in affected tissue were in general less frequent compared with unaffected tissue from the same patients. We then zoomed into every major lineage separately, and observed changes in the frequencies of two myeloid clusters in the affected tissue of ‘responders’ compared with the ‘non-responders’ (see figure, Cluster 1=CD11b+CD11c+CD66b+CD15intCD16+CD45RA−CD45RO+ cells, Cluster 2= CD11b+CD11c+CD66b+ CD15−CD16−CD69+CD45RA+CD45RO+ cells). Conclusion These preliminary data show differences between ‘responders’ and ‘non-responders’ in the myeloid compartment in the inflamed tissue after MSC therapy. Since CD11b+ and CD11c+ populations mediate the tolerogenic effect of MSC in mouse models, we will explore the regulatory potential of the observed populations in MSC-treated UP patients in future studies. The inclusion of patients in a second cohort (N = 7) is currently ongoing.

Celiac disease is caused by an abnormal intestinal T cell response to cereal gluten proteins. The disease has a strong human leukocyte antigen (HLA) association, and CD4(+) T cells recognizing gluten epitopes presented by disease-associated HLA-DQ allotypes are considered to be drivers of the disease. This paper provides an update of the currently known HLA-DQ restricted gluten T cell epitopes with their names and sequences.

Motivation High-dimensional mass cytometry (CyTOF) allows the simultaneous measurement of multiple cellular markers at single-cell level, providing a comprehensive view of cell compositions. However, the power of CyTOF to explore the full heterogeneity of a biological sample at the single-cell level is currently limited by the number of markers measured simultaneously on a single panel. Results To extend the number of markers per cell, we propose an in silico method to integrate CyTOF datasets measured using multiple panels that share a set of markers. Additionally, we present an approach to select the most informative markers from an existing CyTOF dataset to be used as a shared marker set between panels. We demonstrate the feasibility of our methods by evaluating the quality of clustering and neighborhood preservation of the integrated dataset, on two public CyTOF datasets. We illustrate that by computationally extending the number of markers we can further untangle the heterogeneity of mass cytometry data, including rare cell-population detection. Availability and implementation Implementation is available on GitHub (https://github.com/tabdelaal/CyTOFmerge). Supplementary information Supplementary data are available at Bioinformatics online.

International audience; Narcolepsy is a rare brain disorder that reflects a selective loss or dysfunction of orexin (also known as hypocretin) neurons of the lateral hypothalamus. Narcolepsy type 1 (NT1) is characterized by excessive daytime sleepiness and cataplexy, accompanied by sleep-wake symptoms, such as hallucinations, sleep paralysis and disturbed sleep. Diagnosis is based on these clinical features and supported by biomarkers: evidence of rapid eye movement sleep periods soon after sleep onset; cerebrospinal fluid orexin deficiency; and positivity for HLA-DQB1*06:02. Symptomatic treatment with stimulant and anticataplectic drugs is usually efficacious. This Review focuses on our current understanding of how genetic, environmental and immune-related factors contribute to a prominent (but not isolated) orexin signalling deficiency in patients with NT1. Data supporting the view of NT1 as a hypothalamic disorder affecting not only sleep-wake but also motor, psychiatric, emotional, cognitive, metabolic and autonomic functions are presented, along with uncertainties concerning the 'narcoleptic borderland', including narcolepsy type 2 (NT2). The limitations of current diagnostic criteria for narcolepsy are discussed, and a possible new classification system incorporating the borderland conditions is presented. Finally, advances and obstacles in the symptomatic and causal treatment of narcolepsy are reviewed.

Current international guidelines for the risk assessment of biotechnology-derived foods date back to 2003. We present new strategies and directions for assessing immune adverse reactions to novel food proteins. Understanding genetic factors involved in food allergy and the role of the gastrointestinal tract will streamline risk assessment strategies.

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology\ud between an H1N1 hemagglutinin and two hypocretin sequences has been reported.\ud T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched\ud healthy controls.\ud HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and\ud 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T\ud cell cross-reactivity was not detected.\ud These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin\ud peptides presented by HLA-DQ6 is involved in the development of narcolepsy.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA-sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging-mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

ObjectiveA comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.DesignThirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.ResultsWe discovered that a previously unappreciated innate lymphocyte population (Lin–CD7+CD127–CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.ConclusionThis work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these "extra-thymic AIRE expressing cells" (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of AIRE was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of AIRE to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that AIRE expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE.

The human fetal immune system must protect the infant against the sudden exposure to a large variety of pathogens upon birth. While it is known that the fetal immune system develops in sequential waves, relatively little is known about the composition of the innate and adaptive immune system in the tissues. Here, we applied high-dimensional mass cytometry to profile the immune system in human fetal liver, spleen, and intestine. With Hierarchical Stochastic Neighbor Embedding (HSNE) we distinguished 177 distinct immune cell clusters, including both previously identified and novel cell clusters. PCA analysis indicated substantial differences between the compositions of the immune system in the different organs. Through dual t-SNE we identified tissue-specific cell clusters, which were found both in the innate and adaptive compartment. To determine the spatial location of tissue-specific subsets we developed a 31-antibody panel to reveal both the immune compartment and surrounding stromal elements through analysis of snap-frozen tissue samples with imaging mass cytometry. Imaging mass cytometry reconstructed the tissue architecture and allowed both the characterization and determination of the location of the various immune cell clusters within the tissue context. Moreover, it further underpinned the distinctness of the immune system in the tissues. Thus, our results provide evidence for early compartmentalization of the adaptive and innate immune compartment in fetal spleen, liver, and intestine. Together, our data provide a unique and comprehensive overview of the composition and organization of the human fetal immune system in several tissues.

Background Ulcerative proctitis (UP) can be refractory to treatment, which calls for development of new (local) therapies. Local injection of mesenchymal stromal cells (MSCs) has shown beneficial effects in patients with fistulizing Crohn’s disease and promising results have been obtained when MSCs were locally injected in the bowel of mice with experimental colitis. Our primary aim was to determine the safety, feasibility and tolerability of endoscopically injected allogeneic bone marrow-derived MSCs (bmMSCs) in UP patients. Methods UP patients with endoscopic Mayo score (EMS) 2 or 3, who failed on both rectal 5-ASA and corticosteroids for at least 4 weeks, were eligible for inclusion (EudraCT number 2017-003524-75). Rectal therapies were stopped 2 weeks prior to baseline, but other medication was kept stable until at least 6 weeks after MSC injection. MSCs were locally injected in 4 quadrants of the inflamed rectal submucosa if the inflammation was limited to 7 cm. If the length of inflammation was >7 cm, MSCs were injected in another 4 quadrants more proximally as well. Patients in the first cohort (n=7) were treated with 5*106 MSCs/spot and in the second cohort (n=6) with 10*106MSCs/spot. Adverse events, full Mayo score, fecal calprotectin (FCP), histologic activity (Geboes score [GS]) and quality of life (sIBDQ), were assessed at week 0, 2, 6, 12, and 24, and evaluated by non-parametric paired statistical analyses. Results All reported adverse events were minor, no patients required interventions and no feasibility issues were reported. Median[interquartile range (IQR)] full Mayo score was 11[9.5-12] at baseline, 9[8-11] at week 2 (p=0.003), 8[6-10] at week 6 (p=0.001), and 4[1.5-7] at week 24 (p Conclusion Local administration of allogeneic bmMSCs appears safe, tolerable and feasible for the treatment of refractory UP, and shows encouraging indications of clinical efficacy. Further mechanistic and immunological analyses are currently being performed.

Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 induces B29-specific regulatory T cells, which suppressed experimental arthritis in mice by cross-recognition of their mammalian HSP70 homologs (1). The aim of this study is to characterize the B29 binding and specific CD4(+) T cell responses in the context of human MHC molecules (HLA). Binding of B29 peptide and its mammalian homologs to HLA molecules was examined with competitive binding assays. The effect of B29 immunization was assessed in proteoglycan-induced arthritis in HLA-DQ8 transgenic mice, followed by ex vivo restimulation with B29 to examine the T cell response. Human PBMC were used to investigate the presence of B29-specific T cells with immunoregulatory potential. We found a high to moderate binding affinity for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was functional, as B29 immunization resulted in suppression of arthritis and T cell responses in HLA-DQ8 transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4(+) T cells, which were cross-reactive with the mammalian homologs. With HLA-DR4(+) tetramers specific for B29 or mB29b we showed expansion of cross-reactive T cells, especially the human CD4(+) CD25(+) FoxP3(+) T cell population after in vitro stimulation with B29. These results demonstrated a conserved fine-specificity and functionality of the B29-induced regulatory T cell responses in the context of the human MHC. Based on these findings, a translational path of the B29 experimental findings into a clinical immunomodulatory therapeutic approach comes within reach. This article is protected by copyright. All rights reserved.

MotivationMass cytometry (CyTOF) is a valuable technology for high-dimensional analysis at the single cell level. Identification of different cell populations is an important task during the data analysis. Many clustering tools can perform this task, however, they are time consuming, often involve a manual step, and lack reproducibility when new data is included in the analysis. Learning cell types from an annotated set of cells solves these problems. However, currently available mass cytometry classifiers are either complex, dependent on prior knowledge of the cell type markers during the learning process, or can only identify canonical cell types.ResultsWe propose to use a Linear Discriminant Analysis (LDA) classifier to automatically identify cell populations in CyTOF data. LDA shows comparable results with two state-of-the-art algorithms on four benchmark datasets and also outperforms a non-linear classifier such as the k-nearest neighbour classifier. To illustrate its scalability to large datasets with deeply annotated cell subtypes, we apply LDA to a dataset of ~3.5 million cells representing 57 cell types. LDA has high performance on abundant cell types as well as the majority of rare cell types, and provides accurate estimates of cell type frequencies. Further incorporating a rejection option, based on the estimated posterior probabilities, allows LDA to identify cell types that were not encountered during training. Altogether, reproducible prediction of cell type compositions using LDA opens up possibilities to analyse large cohort studies based on mass cytometry data.AvailabilityImplementation is available on GitHub (https://github.com/tabdelaal/CyTOF-Linear-Classifier).Contacta.mahfouz@lumc.nl

Purpose: Human papillomavirus (HPV)–associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome. Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database. Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I–oriented tumor microenvironment, including high numbers of activated CD161+ T cells, CD103+ tissue-resident T cells, dendritic cells (DC), and DC-like macrophages. Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634–47. ©2017 AACR. See related commentary by Laban and Hoffmann, p. 505

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fc gamma receptors (Fc gamma R) and by activating the complement system. The aim of our study was to investigate whether activation of Fc gamma R and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four Fc gamma Rs (Fc.RI/II/III/IV-/-), only the inhibitory Fc gamma RIIb (Fc gamma RIIb(-/-)), only the central component of the complement system C3 (C3(-/-)), and mice lacking both Fc gamma Rs and C3 (Fc gamma RI/II/III/IV/C3(-/-)). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+ 142%, P

The relationship between human cytomegalovirus (HCMV) infections and accelerated immune senescence is controversial. Whereas some studies reported a CMV-associated impaired capacity to control heterologous infections at old age, other studies could not confirm this. We hypothesized that these discrepancies might relate to the variability in the infectious dose of CMV occurring in real life. Here, we investigated the influence of persistent CMV infection on immune perturbations and specifically addressed the role of the infectious dose on the contribution of CMV to accelerated immune senescence. We show in experimental mouse models that the degree of mouse CMV (MCMV)-specific memory CD8+ T cell accumulation and the phenotypic T cell profile are directly influenced by the infectious dose, and data on HCMV-specific T cells indicate a similar connection. Detailed cluster analysis of the memory CD8+ T cell development showed that high-dose infection causes a differentiation pathway that progresses faster throughout the life span of the host, suggesting a virus–host balance that is influenced by aging and infectious dose. Importantly, short-term MCMV infection in adult mice is not disadvantageous for heterologous superinfection with lymphocytic choriomeningitis virus (LCMV). However, following long-term CMV infection the strength of the CD8+ T cell immunity to LCMV superinfection was affected by the initial CMV infectious dose, wherein a high infectious dose was found to be a prerequisite for impaired heterologous immunity. Altogether our results underscore the importance of stratification based on the size and differentiation of the CMV-specific memory T cell pools for the impact on immune senescence, and indicate that reduction of the latent/lytic viral load can be beneficial to diminish CMV-associated immune senescence., Frontiers in Immunology, 8, ISSN:1664-3224

Objective Pathogenic immunoglobulins are produced during the development of obesity and contribute to the development of insulin resistance (IR). However, the mechanisms by which these antibodies affect IR are largely unknown. This study investigated whether Fc-receptors contribute to the development of diet-induced obesity and IR by studying FcRγ−/− mice that lack the γ-subunit necessary for signaling and cell surface expression of FcγR and FceRI. Methods FcRγ−/− and wild-type (WT) mice were fed a high-fat diet (HFD) to induce obesity. At 4 and 11 weeks, body weight and insulin sensitivity were measured, and adipose tissue (AT) inflammation was determined. Furthermore, intestinal triglyceride (TG) uptake and plasma TG clearance were determined, and gut microbiota composition was analyzed. Results FcRγ−/− mice gained less weight after 11 weeks of HFD. They had reduced adiposity, adipose tissue inflammation, and IR. Interestingly, FcRγ−/− mice had higher lean mass compared to WT mice, which was associated with increased energy expenditure. Intestinal TG absorption was increased whereas plasma TG clearance was not affected in FcRγ−/− mice. Gut microbial composition differed significantly and might therefore have added to the observed phenotype. Conclusions FcRγ-chain deficiency reduces the development of diet-induced obesity, as well as associated AT inflammation and IR at 11 weeks of HFD.

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.

In patients with celiac disease, gluten consumption causes inflammation of the duodenum, and, to a lesser extent, the proximal jejunum. Immune-dominant gluten peptides are modified by the enzyme TG2, leading to their high-affinity binding to HLA-DQ2 or HLA-DQ8 molecules, present in people with a predisposition to celiac disease. Gluten peptide−loaded HLA-DQ2 or HLA-DQ8 molecules are recognized by highly conserved receptors on CD4 + T cells in the lamina propria. B cells specific for TG2 and modified gluten peptides are also abundant in the lamina propria of patients with celiac disease. In the epithelium, interleukin-15 activates intraepithelial lymphocytes that promote destruction of epithelial cells. However, it is not clear how the immune responses in the lamina propria and the epithelium, separated by a basement membrane, are linked. We review the immune processes that occur in the lamina propria and their potential effects on epithelial pathology in celiac disease.

BACKGROUND: Aspergillus niger prolyl endoprotease (AN-PEP) efficiently degrades gluten molecules into non-immunogenic peptides in vitro. AIM: To assess the efficacy of AN-PEP on gluten degradation in a low and high calorie meal in healthy subjects. METHODS: In this randomised, double-blind, placebo-controlled, cross-over study 12 healthy volunteers attended to four test days. A liquid low or high calorie meal (4 g gluten) with AN-PEP or placebo was administered into the stomach. Via a triple-lumen catheter gastric and duodenal aspirates were sampled, and polyethylene glycol (PEG)-3350 was continuously infused. Acetaminophen in the meals tracked gastric emptying time. Gastric and duodenal samples were used to calculate 240-min area under the curve (AUC0-240 min ) of ?-gliadin concentrations. Absolute ?-gliadin AUC0-240 min was calculated using duodenal PEG-3350 concentrations. RESULTS: AN-PEP lowered alpha-gliadin concentration AUC0-240 min, compared to placebo, from low and high calorie meals in stomach (low: 35 vs. 389 mug x min/mL; high: 53 vs. 386 mug x min/mL; P < 0.001) and duodenum (low: 7 vs. 168 mug x min/mL; high: 4 vs. 32 mug x min/mL; P < 0.001) and absolute alpha-gliadin AUC0-240 min in the duodenum from low (2813 vs. 31 952 mug x min; P < 0.001) and high (2553 vs. 13 095 mug x min; P = 0.013) calorie meals. In the placebo group, the high compared to low calorie meal slowed gastric emptying and lowered the duodenal alpha-gliadin concentration AUC0-240 min (32 vs. 168 mug x min/mL; P = 0.001). CONCLUSIONS: AN-PEP significantly enhanced gluten digestion in the stomach of healthy volunteers. Increasing caloric density prolonged gastric residence time of the meal. Since AN-PEP already degraded most gluten from low calorie meals, no incremental effect was observed by increasing meal caloric density. ClinicalTrials.gov, Number: NCT01335503; www.trialregister.nl, Number: NTR2780.

In HLA-DQ8–associated celiac disease (CD), the pathogenic T cell response is directed toward an immunodominant α-gliadin–derived peptide (DQ8-glia-α1). However, our knowledge of TCR gene usage within the primary intestinal tissue of HLA-DQ8+ CD patients is limited. We identified two populations of HLA-DQ8-glia-α1 tetramer+ CD4+ T cells that were essentially undetectable in biopsy samples from patients on a gluten-free diet but expanded rapidly and specifically after antigenic stimulation. Distinguished by expression of TRBV9, both T cell populations displayed biased clonotypic repertoires and reacted similarly against HLA-DQ8-glia-α1. In particular, TRBV9 paired most often with TRAV26-2, whereas the majority of TRBV9− TCRs used TRBV6-1 with no clear TRAV gene preference. Strikingly, both tetramer+/TRBV9+ and tetramer+/TRBV9− T cells possessed a non–germline-encoded arginine residue in their CDR3α and CDR3β loops, respectively. Comparison of the crystal structures of three TRBV9+ TCRs and a TRBV9− TCR revealed that, as a result of distinct TCR docking modes, the HLA-DQ8-glia-α1 contacts mediated by the CDR3-encoded arginine were almost identical between TRBV9+ and TRBV9− TCRs. In all cases, this interaction centered on two hydrogen bonds with a specific serine residue in the bound peptide. Replacement of serine with alanine at this position abrogated TRBV9+ and TRBV9− clonal T cell proliferation in response to HLA-DQ8-glia-α1. Gluten-specific memory CD4+ T cells with structurally and functionally conserved TCRs therefore predominate in the disease-affected tissue of patients with HLA-DQ8–mediated CD.

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either Ho-165 or In-111) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (Ho-165-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (Ho-165), 3) compare in vivo biodistribution patterns obtained with ICP-MS (Ho-165) and radiodetection (In-111) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (Ho-165) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 +/- 141 nM and 245 +/- 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (Ho-165) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (Ho-165) and radiodetection (In-111) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/ radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods.

Wheat gluten contains peptide sequences, which activate specific T cells causing a chronic inflammation of the small intestine in celiac disease patients. It is well established that next to wheat gluten, the gluten-like proteins in barley and rye are similarly harmful to celiac disease patients whereas oat is generally considered safe. This study focuses on the development of an ELISA method for the detection of native and processed gluten proteins. The developed test utilizes a monoclonal antibody specific for the DQ2.5-glia-α3 T cell epitope present in the α-gliadins, which are part of wheat gluten that triggers celiac disease. The developed competitive ELISA uses a synthetic DQ2.5-glia-α3 peptide standard for calibration. The conversion from the measured DQ2.5-glia-α3 peptide concentration to gliadin content is achieved by using the experimentally determined multiplication factor of 250. The gluten content can be then calculated by multiplying the gliadin concentration by a factor of 2. A simple sample preparation method with 60% ethanol is used to extract the disease-causing proteins from cereals and processed foods. The assay was found to be specific for the detection of gluten from wheat, barley and rye with no cross-reaction with 8 tested oat varieties. The LOD and LOQ for gliadin were calculated based on the results obtained for 60 blank oats samples and they were 2.9 and 3.6 ppm, respectively. The assay could detect as little as 0.01% wheat gluten and gluten-like proteins from rye and barley in oats. The ELISA was also found to be applicable to the analysis of a range of processed food such as sauces, beers, soups and bread. In conclusion, the developed assay is a sensitive, specific and cost-effective tool for screening cereals and processed foods for the presence of harmful wheat gluten and gluten-like proteins from barley and rye.

Refractory celiac disease type II (RCDII) is a severe complication of celiac disease (CD) characterized by the presence of an enlarged clonal population of innate intraepithelial lymphocytes (IELs) lacking classical B-, T-, and natural killer (NK)-cell lineage markers (Lin-IELs) in the duodenum. In ∼50% of patients with RCDII, these Lin-IELs develop into a lymphoma for which no effective treatment is available. Current evidence indicates that the survival and expansion of these malignant Lin-IELs is driven by epithelial cell-derived IL-15. Like CD, RCDII is strongly associated with HLA-DQ2, suggesting the involvement of HLA-DQ2-restricted gluten-specific CD4+T cells. We now show that gluten-specific CD4+T cells isolated from CD duodenal biopsy specimens produce cytokines able to trigger proliferation of malignant Lin-IEL lines as powerfully as IL-15. Furthermore, we identify TNF, IL-2, and IL-21 as CD4+T-cell cytokines that synergistically mediate this effect. Like IL-15, these cytokines were found to increase the phosphorylation of STAT5 and Akt and transcription of antiapoptotic mediator bcl-xLSeveral small-molecule inhibitors targeting the JAK/STAT pathway blocked proliferation elicited by IL-2 and IL-15, but only an inhibitor targeting the PI3K/Akt/mTOR pathway blocked proliferation induced by IL-15 as well as the CD4+T-cell cytokines. Confirming and extending these findings, TNF, IL-2, and IL-21 also synergistically triggered the proliferation of freshly isolated Lin-IELs and CD3-CD56+IELs (NK-IELs) from RCDII as well as non-RCDII duodenal biopsy specimens. These data provide evidence implicating CD4+T-cell cytokines in the pathogenesis of RCDII. More broadly, they suggest that adaptive immune responses can contribute to innate IEL activation during mucosal inflammation.

Checkpoint blockade has revived the potential of immunotherapy for cancer treatment. For optimal application and development of cancer immunotherapies, a comprehensive understanding of the antitumor immune response is required. We unraveled local and systemic immune profiles of colorectal cancer by multidimensional mass cytometric analysis of 36 immune cell markers at the single-cell level in tumor tissues, tumor-associated lymph nodes, adjacent normal mucosa, and peripheral blood samples from CRC patients. We identified 218 phenotypically distinct immune cell clusters, including a previously neglected innate lymphoid cell (CD7+CD3-CD127-CD45RO+CD56+) population with cytotoxic potential. This subset demonstrated a tissue-resident (CD69+, CD103+) phenotype, and was most abundant in the immunogenic mismatch repair deficient (MMRd) cancers. Furthermore, tumor-resident immune cell populations were identified across the adaptive (CD4+ and CD8+) and innate (gammadelta) T-cell compartments showing a highly similar activated (HLA-DR+, CD38+, PD-1+) phenotype. PD-1 intermediate and PD-1 high CD8+ T-cell subsets represented distinct states of T-cell activation that further discriminated immunogenic from non-immunogenic colorectal cancers. Remarkably, activated gammadelta T-cells were specific for MMRd cancers, and their potential role in the response to PD-1 checkpoint blockade requires further clarification. The nonactivated counterparts of the tumor-resident CD103+PD-1+ cytotoxic and gammadelta T-cells were present in both tumor and healthy colorectal tissues. We did not detect any of the aforementioned tumor-resident immune cell populations in lymph node samples, with the exception of a tumor-positive lymph node. This indicates that the critical immune cell populations with antitumor activity reside in the colorectal mucosa, and that the role of lymph nodes in the antitumor immune response should be revisited. Finally, by applying imaging mass cytometry we demonstrated that the cytotoxic anti-tumor response in colorectal cancer is highly diverse and not restricted to cytotoxic T-cells, which opens new avenues for the management of this disease.The findings presented here advance the paradigm of antitumor immunity in colorectal cancer and provide a blueprint for the detailed characterization of the involved immune cell subsets. The coordinated action of innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumor properties in a therapeutic setting. Citation Format: Noel F. de Miranda, Natasja L. de Vries, Vincent van Unen, Tamim Abdelaal, Marieke E. Ijsselsteijn, Ruud van der Breggen, Arantza Farina-Sarasqueta, Koen C.M.J. Peeters, Thomas Höllt, Boudewijn P.F. Lelieveldt, Frits Koning. Multidimensional cytometric analysis of colorectal cancer reveals novel and diverse mediators of antitumor immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A063.