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Immune dysfunction has been proposed to play a role in the pathophysiology behind the development and persistence of psychosis. Current immunophenotyping studies are limited by small sample sizes and the number of immune markers investigated. Pharmacological subtypes in schizophrenia based on antipsychotic response have been proposed, but few studies have investigated immunophenotypes in treatment-resistant schizophrenia. In this study, we perform comprehensive immunophenotyping on 196 subjects comprising 147 schizophrenia patients stratified by antipsychotic response (49 antipsychotic-responsive, 70 clozapine-responsive, 28 clozapine-resistant) and 49 healthy controls. We aim to identify significant immune cell populations associated with schizophrenia and increasing treatment resistance, as potential modulators of underlying psychosis and/or treatment response. Patients with schizophrenia were recruited and assessed on the Clinical Global Impression - Schizophrenia (CGI-SCH). Treatment response was defined as a rating of three (mild severity) or less on the CGI-SCH positive symptom item after at least 8 weeks of adequate antipsychotic or clozapine treatment. Peripheral blood mononuclear cells were collected and flow cytometry was performed to identify 66 immune cell populations. Differences in cell population proportions were compared between schizophrenia cases and controls, and across all 4 groups, with post-hoc pairwise comparisons. Mucosal-associated invariant T (MAIT) cells (specifically CD8 + and DN double-negative subsets), total, exhausted and memory CD8 + T cells, VD1 + ϒδ T cells, plasmablasts, IgG + B cells and conventional dendritic cells 2 (cDC2) were among the top cell populations downregulated in schizophrenia. We observed increased downregulation with increasing treatment resistance. Conversely, naïve and exhausted CD4 + T cells, CD4/CD8 ratio and CCR5 + CCR2 + HLA DR + Myeloid cells were found to be upregulated in schizophrenia - we observed increased upregulation with increasing treatment resistance. We show significant immunophenotypic differences between schizophrenia cases and healthy controls, and consistent trend differences across varying degrees of antipsychotic resistance. We also examined immune cell populations not previously reported in schizophrenia. Future studies may explore immune markers identified as potential biomarkers of treatment resistance, and clarify on the relationship between immunological changes and pharmacological subtypes in schizophrenia., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Natural killer/T cell lymphoma (NKTL) is an aggressive malignancy associated with poor prognosis. This is largely due to limited treatment options, especially for relapsed patients. Immunotherapies like immune checkpoint inhibitors (ICI) and anti-CD38 therapies have shown promising but variable clinical efficacies. Combining these therapies has been suggested to enhance efficacy., Methods: We conducted a case study on a relapsed NKTL patient treated sequentially with anti-CD38 followed by ICI (anti-PD1) using cytometry analyses., Results and Discussion: Our analysis showed an expected depletion of peripheral CD38+ B cells following anti-CD38 treatment. Further analysis indicated that circulating anti-CD38 retained their function for up to 13 weeks post-administration. Anti-PD1 treatment triggered re-activation and upregulation of CD38 on the T cells. Consequently, these anti-PD1-activated T cells were depleted by residual circulating anti-CD38, rendering the ICI treatment ineffective. Finally, a meta-analysis confirmed this counterproductive effect, showing a reduced efficacy in patients undergoing combination therapy. In conclusion, our findings demonstrate that sequential anti-CD38 followed by anti-PD1 therapy leads to a counterproductive outcome in NKTL patients. This suggests that the treatment sequence is antithetic and warrants re-evaluation for optimizing cancer immunotherapy strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lee, Lim, Tang, Tan, Koh, Puan, Wang, Lim, Tan, Chng, Lim, Ong and Rotzschke.)

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of an infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of host response to pathogens., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Trait-associated genetic variants affect complex phenotypes primarily via regulatory mechanisms on the transcriptome. To investigate the genetics of gene expression, we performed cis- and trans-expression quantitative trait locus (eQTL) analyses using blood-derived expression from 31,684 individuals through the eQTLGen Consortium. We detected cis-eQTL for 88% of genes, and these were replicable in numerous tissues. Distal trans-eQTL (detected for 37% of 10,317 trait-associated variants tested) showed lower replication rates, partially due to low replication power and confounding by cell type composition. However, replication analyses in single-cell RNA-seq data prioritized intracellular trans-eQTL. Trans-eQTL exerted their effects via several mechanisms, primarily through regulation by transcription factors. Expression of 13% of the genes correlated with polygenic scores for 1,263 phenotypes, pinpointing potential drivers for those traits. In summary, this work represents a large eQTL resource, and its results serve as a starting point for in-depth interpretation of complex phenotypes., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1β inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

Increasing evidence suggests an association between gastrointestinal (GI) disorders and susceptibility and progress of Parkinson's disease (PD). Gut-brain axis has been proposed to play important roles in the pathogenesis of PD, though the exact pathophysiologic mechanism has yet to be elucidated. Here, we discuss the common factors involved in both PD and GI disorders, including genes, altered gut microbiota, diet, environmental toxins, and altered mucosal immunity. Large-scale prospective clinical studies are needed to define the exact relationship between dietary factors, microbiome, and genetic factors in PD. Identification of early diagnostic markers and demonstration of the efficacy of diet modulation and regulation of gut microbiome through specific therapeutics can potentially change the treatment paradigm for PD., (Copyright © 2020 Chao, Gulam, Chia, Feng, Rotzschke and Tan.)

The human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. Influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. Here, we elucidated the global transcriptomic changes during H3N2 infection of human nasal epithelial cells from multiple individuals. Using RNA sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. We used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. Statistical analysis highlighted strong transcriptomic signatures significantly associated with 24 and 48 h after infection, but not at the earlier 8-h time point. In particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, B-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. These molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management.

Novel wide array blood biomarkers of multisystem dysregulation, compared to conventional clinical and blood biomarkers, are potentially able to provide more accurate prognostic information of long-term mortality risks. We identified biomarker signatures of all-cause and disease-specific mortality from a comprehensive range of analytes related to six major physiological functions: cytokine, chemokine, and growth factors; glucose metabolism regulators and adipokines; adhesion molecules; acute phase response; pathogen-specific antibodies; and bone remodeling. A total of 144 elderly were prospectively followed up on mortality for median 136 months. Plasma levels of 93 biomarkers measured by enzyme-linked immunosorbent assay, Luminex, FlowCytomix, DNA quantification, and recombinant DNA technology at baseline were compared among deceased and surviving elderly and in a referent group of 72 healthy young adults. The elderly, and especially deceased elderly, exhibited differential profiles of the composite index of each physiological function from young adults. In Cox regression, we identified and validated in an independent cohort of 357 elderly the specific and common biomarkers predicting all-cause, cardiovascular disease-related, neoplasm-related, and respiratory disease-related mortalities after controlling age, sex, and major comorbidities. These biomarkers had 74.3% correct classification for deceased elderly from surviving elderly. We reported for the first time, stem cell growth factor-β and gastric inhibitory polypeptide as specific biomarkers of mortality risk., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: We established an in vitro co-culture model involving H3N2-infection of human nasal epithelium with peripheral blood mononuclear cells (PBMC) to investigate their cross-talk during early H3N2 infection. Methods: Nasal epithelium was differentiated from human nasal epithelial stem/progenitor cells and cultured wtih fresh human PBMC. PBMC and supernatants were harvested after 24 and 48 h of co-culture with H3N2-infected nasal epithelium. We used flow cytometry and Luminex to characterize PBMC subpopulations, their activation and secretion of cytokine and chemokines. Results: H3N2 infection of the nasal epithelium associated with significant increase in interferons (IFN-α, IFN-γ, IL-29), pro-inflammatory cytokines (TNF-α, BDNF, IL-3) and viral-associated chemokines (IP-10, MCP-3, I-TAC, MIG), detectable already after 24 h. This translates into rapid activation of monocytes, NK-cells and innate T-cells (MAIT and γδ T cells), evident with CD38+ and/or CD69+ upregulation. Conclusions: This system may contribute to in vitro mechanistic immunological studies bridging systemic models and possibly enable the development of targeted immunomodulatory therapies.

Background: Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death and there is a huge unmet clinical need to identify disease biomarkers in peripheral blood. Compared to gene level differential expression approaches to identify gene signatures, network analyses provide a biologically intuitive approach which leverages the co-expression patterns in the transcriptome to identify modules of co-expressed genes., Methods: A weighted gene co-expression network analysis (WGCNA) was applied to peripheral blood transcriptome from 238 COPD subjects to discover co-expressed gene modules. We then determined the relationship between these modules and forced expiratory volume in 1 s (FEV 1 ). In a second, independent cohort of 381 subjects, we determined the preservation of these modules and their relationship with FEV 1 . For those modules that were significantly related to FEV 1 , we determined the biological processes as well as the blood cell-specific gene expression that were over-represented using additional external datasets., Results: Using WGCNA, we identified 17 modules of co-expressed genes in the discovery cohort. Three of these modules were significantly correlated with FEV 1 (FDR < 0.1). In the replication cohort, these modules were highly preserved and their FEV 1 associations were reproducible (P < 0.05). Two of the three modules were negatively related to FEV 1 and were enriched in IL8 and IL10 pathways and correlated with neutrophil-specific gene expression. The positively related module, on the other hand, was enriched in DNA transcription and translation and was strongly correlated to CD4+, CD8+ T cell-specific gene expression., Conclusions: Network based approaches are promising tools to identify potential biomarkers for COPD., Trial Registration: The ECLIPSE study was funded by GlaxoSmithKline, under ClinicalTrials.gov identifier NCT00292552 and GSK No. SCO104960.

Aim: To characterise infiltrating T cells in kidneys and circulating lymphocyte subsets of adult patients with primary/idiopathic minimal change disease., Methods: In a cohort of 9 adult patients with primary/idiopathic minimal change recruited consecutively at disease onset, we characterized (1) infiltrating immune cells in the kidneys using immunohistochemistry and (2) circulating lymphocyte subsets using flow cytometry. As an exploratory analysis, association of the numbers and percentages of both kidney-infiltrating immune cells and the circulating lymphocyte subsets with kidney outcomes including deterioration of kidney function and proteinuria, as well as time to complete clinical remission up to 48 months of follow-up, was investigated., Results: In the recruited patients with primary/idiopathic minimal change disease, we observed (a) a dominance of infiltrating T helper 17 cells and cytotoxic cells, comprising cytotoxic T cells and natural killer cells, over Foxp3+ Treg cells in the renal interstitium; (b) an increase in the circulating total CD8+ T cells in peripheral blood; and (c) an association of some of these parameters with kidney function and proteinuria., Conclusions: In primary/idiopathic minimal change disease, a relative numerical dominance of effector over regulatory T cells can be observed in kidney tissue and peripheral blood. However, larger confirmatory studies are necessary.

Monocytes play a central role in regulating inflammation in response to infection or injury, and during auto-inflammatory diseases. Human blood contains classical, intermediate and non-classical monocyte subsets that each express characteristic patterns of cell surface CD16 and CD14; each subset also has specific functional properties, but the mechanisms underlying many of their distinctive features are undefined. Of particular interest is how monocyte subsets regulate secretion of the apical pro-inflammatory cytokine IL-1β, which is central to the initiation of immune responses but is also implicated in the pathology of various auto-immune/auto-inflammatory conditions. Here we show that primary human non-classical monocytes, exposed to LPS or LPS + BzATP (3'-O-(4-benzoyl)benzyl-ATP, a P2X7R agonist), produce approx. 80% less IL-1β than intermediate or classical monocytes. Despite their low CD14 expression, LPS-sensing, caspase-1 activation and P2X7R activity were comparable in non-classical monocytes to other subsets: their diminished ability to produce IL-1β instead arose from 50% increased IL-1β mRNA decay rates, mediated by Hsp27. These findings identify the Hsp27 pathway as a novel therapeutic target for the management of conditions featuring dysregulated IL-1β production, and represent an advancement in understanding of both physiological inflammatory responses and the pathogenesis of inflammatory diseases involving monocyte-derived IL-1β.

Objective: Neuropeptide Y, a widely circulating neurotransmitter, plays a pivotal role in energy balance, immunomodulation and asthma, and several NPY polymorphisms are promising genetic risk factors for asthma and obesity. We explored the associations of candidate NPY gene polymorphisms with prevalent asthma and its relationship with obesity in young adult asthma patients free of other chronic medical morbidity., Methods: Five common gene variants of NPY (rs16147 (-399T/C), rs17149106 (-602G/T), rs16140 (+1000C/G), rs5573 (+1201A/G), rs5574 (+5327C/T)) previously validated to account for most of the NPY expression in vitro and in vivo were investigated in 126 physician-diagnosed asthma patients without other chronic medical morbidity and 182 healthy controls (21-35years). Plasma levels of NPY, adiponectin, and CRP were determined using ELISA, and IL-6 was measured by Luminex in a subgroup of 70 patients and 69 age- and sex-matched healthy controls., Results: In logistic regression models controlling for gender and obesity, the CT genotype of rs5574 (OR=0.54, 95%CI: 0.30-0.89) and the GT genotype of rs17149106 (OR=5.58, 95%CI: 1.09-28.54) were significantly associated with asthma. No significant interaction between NPY SNP polymorphisms and obesity were detected. Plasma NPY level was correlated with adiponectin levels (p<0.05). Compared with the healthy controls, patients with asthma had higher BMI (p<0.001), adiponectin (p<0.05), IL-6 (p=0.001) and CRP (p<0.001), and lower NPY levels (p<0.01)., Conclusions: The CT genotype of rs5574 and the GT genotype of rs17149106 are significantly associated with prevalent asthma., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Genetic polymorphism within the 9q32 locus is linked with increased risk of several diseases, including Crohn's disease (CD), primary biliary cholangitis (PBC) and leprosy. The most likely disease-causing gene within 9q32 is TNFSF15, which encodes the pro-inflammatory cytokine TNF super-family member 15, but it was unknown whether these disparate diseases were associated with the same genetic variance in 9q32, and how variance within this locus might contribute to pathology. Using genetic data from published studies on CD, PBC and leprosy we revealed that bearing a T allele at rs6478108/rs6478109 (r(2) = 1) or rs4979462 was significantly associated with increased risk of CD and decreased risk of leprosy, while the T allele at rs4979462 was associated with significantly increased risk of PBC. In vitro analyses showed that the rs6478109 genotype significantly affected TNFSF15 expression in cells from whole blood of controls, while functional annotation using publicly-available data revealed the broad cell type/tissue-specific regulatory potential of variance at rs6478109 or rs4979462. In summary, we provide evidence that variance within TNFSF15 has the potential to affect cytokine expression across a range of tissues and thereby contribute to protection from infectious diseases such as leprosy, while increasing the risk of immune-mediated diseases including CD and PBC.

Chronological aging and a variety of stressors are driving forces towards immunosenescence. While much attention was paid to the main T cell component, α/β T cells, few studies concentrate on the impact of age on γ/δ T cells' characteristics. The latter are important players of adaptive immunity but also have features associated with innate immunity. Vδ2+ are the main component of γ/δ while Vδ1+ T cells expand upon Cytomegalovirus (CMV) infection and with age. The Vδ2+ T cells are not influenced by persistent infections but do contribute to immunosurveillance against bacterial pathogens. Here, we focus on Vδ2+ T cells and report that their composition and functionality is not altered in older adults. We have performed a side-by-side comparison of α/β and Vδ2 cells by using two robust markers of T cell replicative history and cell differentiation (CD28 and CD27), and cytokine secretion (IFN-γ and TNF-α). Significant differences in Vδ2 versus α/β homeostasis, as well as phenotypic and functional changes emerged. However, the data strongly suggest a sustained functionality of the Vδ2 population with age, independently of the challenge. This suggests differential trajectories towards immunosenescence in α/β and Vδ2+ T cells, most likely explained by their intrinsic functions., Competing Interests: The authors have no conflict of interest.

Patients with pancreatic ductal adenocarcinoma (PDAC) face a clinically intractable disease with poor survival rates, attributed to exceptionally high levels of metastasis. Epithelial-to-mesenchymal transition (EMT) is pronounced at inflammatory foci within the tumor; however, the immunological mechanisms promoting tumor dissemination remain unclear. It is well established that tumors exhibit the Warburg effect, a preferential use of glycolysis for energy production, even in the presence of oxygen, to support rapid growth. We hypothesized that the metabolic pathways utilized by tumor-infiltrating macrophages are altered in PDAC, conferring a pro-metastatic phenotype. We generated tumor-conditioned macrophages in vitro, in which human peripheral blood monocytes were cultured with conditioned media generated from normal pancreatic or PDAC cell lines to obtain steady-state and tumor-associated macrophages (TAMs), respectively. Compared with steady-state macrophages, TAMs promoted vascular network formation, augmented extravasation of tumor cells out of blood vessels, and induced higher levels of EMT. TAMs exhibited a pronounced glycolytic signature in a metabolic flux assay, corresponding with elevated glycolytic gene transcript levels. Inhibiting glycolysis in TAMs with a competitive inhibitor to Hexokinase II (HK2), 2-deoxyglucose (2DG), was sufficient to disrupt this pro-metastatic phenotype, reversing the observed increases in TAM-supported angiogenesis, extravasation, and EMT. Our results indicate a key role for metabolic reprogramming of tumor-infiltrating macrophages in PDAC metastasis, and highlight the therapeutic potential of using pharmacologics to modulate these metabolic pathways.

Background: Allergic rhinitis (AR) and asthma are common allergic conditions with a shared genetic component to their cause. The 17q12-21 locus includes several genes that have been linked to asthma susceptibility, but the role of this locus in AR is unclear. Asthma and AR in adults of Chinese ethnicity in Singapore are predominately caused by sensitization against house dust mites with a nearly complete penetrance of the allergen, which presents a unique opportunity for accurately identifying genetic associations with allergic diseases., Objective: We sought to define the functional role of 17q12-21 in patients with AR and allergic asthma., Methods: We asked whether single nucleotide polymorphisms (SNPs) in the 17q12-21 locus were associated with AR or asthma in a cohort of 3460 ethnic Chinese subjects residing in Singapore (1435 in the discovery phase and 2025 in the validation phase). Full-blood mRNA gene expression data, plasma IgE levels, and immune cell frequencies in peripheral blood were tested against the tag SNP genotypes. Luciferase assays were used to measure the effect of putative promoter SNPs on expression of the asthma-associated orosomucoid-like 3 gene (ORMDL3)., Results: Within 17q12-21, only the tag SNP rs8076131 was significantly associated with asthma (P = 8.53 × 10(-10); odds ratio, 0.6715), and AR status was independent of SNPs in this region. C-A alleles at rs8076131 resulted in significantly increased ORMDL3 expression in HEK293 cells in vitro relative to T-G alleles. Moreover, subjects with the risk genotype AA exhibited significantly higher total IgE levels and higher blood eosinophil counts than those with the lower-risk genotypes., Conclusion: The 17q12-21 locus has a strong genetic association with allergic asthma but not with AR. The polymorphic effect of this locus is attributed to the linkage set tagged by rs8076131, which affects the expression of ORMDL3, protein phosphatase 1, regulatory inhibitor subunit 1B (PPP1R1B), zona pellucida binding protein 2 (ZPBP2), and gasdermin B (GSDMB) and is correlated with high IgE levels and eosinophil counts in subjects bearing the risk genotype., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Neutrophils are an abundant immune cell type involved in both antimicrobial defence and autoimmunity. The regulation of their gene expression, however, is still largely unknown. Here we report an eQTL study on isolated neutrophils from 114 healthy individuals of Chinese ethnicity, identifying 21,210 eQTLs on 832 unique genes. Unsupervised clustering analysis of these eQTLs confirms their role in inflammatory responses and immunological diseases but also indicates strong involvement in dermatological pathologies. One of the strongest eQTL identified (rs2058660) is also the tagSNP of a linkage block reported to affect leprosy and Crohn's disease in opposite directions. In a functional study, we can link the C allele with low expression of the β-chain of IL18-receptor (IL18RAP). In neutrophils, this results in a reduced responsiveness to IL-18, detected both on the RNA and protein level. Thus, the polymorphic regulation of human neutrophils can impact beneficial as well as pathological inflammatory responses.

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.

IgA nephropathy (IgAN) is one of the most common primary glomerulonephritis. Previously identified genome-wide association study (GWAS) loci explain only a fraction of disease risk. To identify novel susceptibility loci in Han Chinese, we conduct a four-stage GWAS comprising 8,313 cases and 19,680 controls. Here, we show novel associations at ST6GAL1 on 3q27.3 (rs7634389, odds ratio (OR)=1.13, P=7.27 × 10(-10)), ACCS on 11p11.2 (rs2074038, OR=1.14, P=3.93 × 10(-9)) and ODF1-KLF10 on 8q22.3 (rs2033562, OR=1.13, P=1.41 × 10(-9)), validate a recently reported association at ITGAX-ITGAM on 16p11.2 (rs7190997, OR=1.22, P=2.26 × 10(-19)), and identify three independent signals within the DEFA locus (rs2738058, P=1.15 × 10(-19); rs12716641, P=9.53 × 10(-9); rs9314614, P=4.25 × 10(-9), multivariate association). The risk variants on 3q27.3 and 11p11.2 show strong association with mRNA expression levels in blood cells while allele frequencies of the risk variants within ST6GAL1, ACCS and DEFA correlate with geographical variation in IgAN prevalence. Our findings expand our understanding on IgAN genetic susceptibility and provide novel biological insights into molecular mechanisms underlying IgAN.

Background: Brain-derived neurotrophic factor (BDNF) is a secretory protein that has been implicated in the pathogenesis of allergic rhinitis (AR), atopic asthma, and eczema, but it is currently unknown whether BDNF polymorphisms influence susceptibility to moderate-to-severe AR., Objective: We sought to identify disease associations and the functional effect of BDNF genetic variants in patients with moderate-to-severe AR., Methods: Tagging single nucleotide polymorphisms (SNPs) spanning the BDNF gene were selected from the human HapMap Han Chinese from Beijing (CHB) data set, and associations with moderate-to-severe AR were assessed in 2 independent cohorts of Chinese patients (2216 from Shandong province and 1239 living in Singapore). The functional effects of the BDNF genetic variants were determined by using both in vitro and ex vivo assays., Results: The tagging SNP rs10767664 was significantly associated with the risk of moderate-to-severe AR in both Singapore Chinese (P = .0017; odds ratio, 1.324) and Shandong Chinese populations (P = .039; odds ratio, 1.180). The coding nonsynonymous SNP rs6265 was in perfect linkage with rs10767664 and conferred increased BDNF protein secretion by a human cell line in vitro. Subjects bearing the AA genotype of rs10767664 exhibited increased risk of moderate-to-severe AR and displayed increased BDNF protein and total IgE levels in plasma. Using a large-scale expression quantitative trait locus study, we demonstrated that BDNF SNPs are significantly associated with altered BDNF concentrations in peripheral blood., Conclusion: A common genetic variant of the BDNF gene is associated with increased risk of moderate-to-severe AR, and the AA genotype is associated with increased BDNF mRNA levels in peripheral blood. Together, these data indicate that functional BDNF gene variants increase the risk of moderate-to-severe AR., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

Genome-wide association studies (GWAS) have led to the discovery of several susceptibility loci for leprosy with robust evidence, providing biological insight into the role of host genetic factors in mycobacterial infection. However, the identified loci only partially explain disease heritability, and additional genetic risk factors remain to be discovered. We performed a 3-stage GWAS of leprosy in the Chinese population using 8,313 cases and 16,017 controls. Besides confirming all previously published loci, we discovered six new susceptibility loci, and further gene prioritization analysis of these loci implicated BATF3, CCDC88B and CIITA-SOCS1 as new susceptibility genes for leprosy. A systematic evaluation of pleiotropic effects demonstrated a high tendency for leprosy susceptibility loci to show association with autoimmunity and inflammatory diseases. Further analysis suggests that molecular sensing of infection might have a similar pathogenic role across these diseases, whereas immune responses have discordant roles in infectious and inflammatory diseases.

Background/objective: Molecular-based allergy diagnostics are gaining popularity in clinical practice. Our aim was to evaluate their role in the tropics, given the inherent genetic and environmental differences., Methods: We recruited subjects with history of atopy and collected data on demographics and atopic symptoms using validated questionnaires. Subjects underwent a series of skin prick tests (SPT). Serum total and specific IgE levels were measured using ImmunoCAP FEIA and ImmunoCAP ISAC®, respectively. We describe their pattern of sensitization and agreement between test methods., Results: A total of 135 subjects were recruited; mean ± SD age of 31.18 ± 12.72 years, 52.7% female. Allergic rhinitis (AR) was the most prevalent clinical manifestation of atopy (70.7%), followed by atopic dermatitis (AD) (50.5%) and asthma (26.2%). Polysensitization was seen in 51.1% of subjects by both SPT and ISAC. House dust mites (HDM) were the dominant allergen, with sensitization in 67.8% and 62% of subjects on SPT and ISAC, respectively. A group of subjects with monosensitization to B. tropicalis was identified. HDM sensitization was strongly associated with AR, while AD and asthma were not associated with sensitization to any allergen. Agreement between SPT and ISAC was mostly suboptimal. Greatest agreement was documented for the measurement of HDM sensitization with both methods (κ = 0.64). Sensitization to the bulk of the remaining allergens in the ISAC panel was infrequent., Conclusion: Multiplex methods should not be used as a screening tool, especially in a population with lower rates of polysensitization and a dominant sensitizing allergen. There may be a role in adjusting the antigen spectrum in the ISAC panel to regional differences.

Aging is associated with an increased susceptibility to infections and diseases. It has also been associated with reduced functionality and altered distribution of immune cells, especially T cells. Whereas classical α/β T cells, especially CD8(+) T cells, were shown to be highly susceptible to aging, the effects of viral persistent stimulations on the fate of γ/δ T cells are much less documented. Healthy, elderly individuals of Chinese ethnical background were recruited under the aegis of SLAS-II. In this observational study, γ/δ T cell populations were characterized by flow cytometry and compared with the α/β CD4(+) and CD8(+) T cells in elderly and young controls. In our study, we identified a reduced frequency of γ/δ T cells but not α/β T cells with aging. The classical markers of α/β T cell aging, including CD28, CD27, and CD57, did not prove significant for γ/δ T cells. The extreme range of expression of these markers in γ/δ T cells was responsible for the lack of relationship between γ/δ T cell subsets, CD4/CD8 ratio, and anti-CMV titers that was significant for α/β T cells and, especially, CD8(+) T cells. Although markers of aging for γ/δ T cells are not clearly identified, our data collectively suggest that the presence of CD27 γ/δ T cells is associated with markers of α/β T cell aging., (© 2014 Society for Leukocyte Biology.)

Background: Extracellular ATP is a pro-inflammatory molecule released by damaged cells. Regulatory T cells (Treg) can suppress inflammation by hydrolysing this molecule via ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also termed as CD39. Multiple studies have reported differences in CD39+ Treg percentages in diseases such as multiple sclerosis, Hepatitis B and HIV-1. In addition, CD39 polymorphisms have been implicated in immune-phenotypes such as susceptibility to inflammatory bowel disease and AIDS progression. However none of the studies published so far has linked disease-associated variants with differences in CD39 Treg surface expression. This study aims at identifying variants affecting CD39 expression on Treg and at evaluating their association with allergic rhinitis, a disease characterized by a strong Treg involvement., Methods: Cohorts consisting of individuals of different ethnicities were employed to identify any association of CD39 variants to surface expression. Significant variant(s) were tested for disease association in a published GWAS cohort by one-locus and two-locus genetic analyses based on logistic models. Further functional characterization was performed using existing microarray data and quantitative RT-PCR on sorted cells., Results: Our study shows that rs7071836, a promoter SNP in the CD39 gene region, affects the cell surface expression on Treg cells but not on other CD39+ leukocyte subsets. Epistasis analysis revealed that, in conjunction with a SNP upstream of the FAM134B gene (rs257174), it increased the risk of allergic rhinitis (P = 1.98 × 10-6). As a promoter SNP, rs257174 controlled the expression of the gene in monocytes but, notably, not in Treg cells. Whole blood transcriptome data of three large cohorts indicated an inverse relation in the expression of the two proteins. While this observation was in line with the epistasis data, it also implied that a functional link must exist. Exposure of monocytes to extracellular ATP resulted in an up-regulation of FAM134B gene expression, suggesting that extracellular ATP released from damaged cells represents the connection for the biological interaction of CD39 on Treg cells with FAM134B on monocytes., Conclusions: The interplay between promoter SNPs of CD39 and FAM134B results in an intercellular epistasis which influences the risk of a complex inflammatory disease.

Background: ZMIZ1 has been shown to be associated with multiple autoimmune diseases and play a role in the development of melanocyte. The association of ZMIZ1 with vitiligo was also suggested, but the evidence did not reach genome-wide significance and has not been confirmed by independent studies., Methods: A fine mapping analysis of the ZMIZ1 locus was carried out in the dataset of 1117 vitiligo patients and 3437 controls through deep imputation. Ten suggestive SNPs were then analysed in an independent validation cohort of 7458 cases and 7542 controls. SNPs within ZMIZ1 locus were functionally annotated using the ENCODE and RegulomeDB databases and published eQTL dataset of primary immune cells., Results: A genome-wide significant association was discovered at rs1408944 (OR(combined)=1.18, p(combined)=1.38E-09) that locates at a DNAse hypersensitivity site and within a Myb&#95;1 motif carried by the binding sites of six overlapping transcription factors (TFs) within the region. Gene Relationships Across Implicated Loci (GRAIL) analysis revealed biological connectivity between ZMIZ1 and previously discovered susceptibility loci for vitiligo as well as the six TFs., Conclusions: Our study has confirmed ZMIZ1 as a novel susceptibility locus for vitiligo and further suggested rs1408944 to be the putative causal variant that potentially interrupts TF binding and thus the transcriptional regulation of ZMIZ1.

Background: Southeast Asian populations are increasingly affected by allergic airway diseases. Etiology and specific causes, however, are still unknown. The aim of this study is therefore to identify allergens and risk factors for the high prevalence of allergic airway disease in the tropical urban environment., Methods: Symptoms of allergic rhinitis (AR), asthma, and allergic dermatitis were recorded in two independent cohorts of 576 and 7373 ethnic Chinese individuals living in Singapore. Reactivity against common allergens was determined by skin prick tests (SPT); specific immunoglobulin E (sIgE) titers against 12 common allergens, as well as total serum IgE (tIgE), were measured in the smaller cohort., Results: Immunoglobulin E sensitization was almost exclusively directed against house dust mite (HDM) allergens. More than 80% of individuals were HDM-sIgE positive. Of these, less than 30% also had sIgE for other allergens, and similarly, few of the HDM-sIgE-negative individuals reacted to other allergens. Titers for HDM-sIgE were 8-30 times higher than other non-HDM allergen titers and correlated directly with total serum tIgE levels. Migrants from nontropical countries typically arrived with low or undetectable HDM-sIgE but developed substantial titers in a time-dependent fashion. Importantly, prolonged stay in Singapore also resulted in the manifestation of AR and asthma symptoms, contributing to some of the highest national prevalence rates worldwide., Conclusion: In a tropical urban environment, the allergic response is dominated by a single allergen class. The mono-specific IgE sensitization against HDM translates into increased prevalence of allergic airway diseases, which now impact a large proportion of the population in Singapore., (© 2014 The Authors. Allergy published by John Wiley & Sons Ltd.)

In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap-ESI-MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC-ESI-MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC-MS-MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell-cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The immune system is a highly balanced network of different cell types. This balance is disturbed in the setting of organ or stem cell transplantation, which can lead to graft rejection or "Graft versus host disease" (GvHD). Conventional pharmacological treatment by broad immune suppression is restricted by dose-limiting side effects. A novel strategy for prevention and control is cell therapy. This applies particularly to GvHD. A number of phase I trials have already been launched. The most appropriate cell type appears to be the regulatory T (Treg) cell as it is a natural "suppressor" of the immune system. Treg cells are able to inhibit various effector cells including CD4+ and CD8+ T cells, the main drivers of GvHD. Like other T cells, also Treg cells can be divided into naïve and memory-type cells. We have previously identified effector/memory Treg cells (T(REM)), the regulatory counterparts of CD4+ effector/memory T cells (T(EM)). T(REM) may be particularly suited to inhibit proinflammatory reactions in peripheral tissues as they express the chemokine receptor CCR6, a feature they share with proinflammatory Th17 cells. As specific marker, they also express CD39 but lack the expression of CD49d and CD127. We could show that a simple depletion of CD49d and CD127 expressing cells yields a population of "untouched" Treg cells that is highly pure and largely consist of highly suppressive T(REM) cells. Mouse models have confirmed the efficacy of Treg cells in controlling GvHD but the translation has been lagging. First clinical trials suggesting safety of adoptive Treg transfer increase the need for methods that allow obtaining clinical-grade Treg cells in sufficient amounts. The new approach may therefore provide a promising new alternative to facilitate a simple access to these cells.

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.

The existence of T-cell subsets naturally committed to perform immunoregulation has led to enthusiastic efforts to investigate their role in the immunopathogenesis of transplantation. Being able to modulate alloresponses, regulatory T cells could be used as an immunodiagnostic tool in clinical kidney transplantation. Thus, the measurement of Foxp3 transcripts, the presence of regulatory T cells in kidney biopsies, and the phenotypic characterisation of the T-cell infiltrate could aid in the diagnosis of rejection and the immune monitoring and prediction of outcomes in kidney transplantation. Interestingly, the adoptive transfer of regulatory T cells in animal models has been proven to downmodulate powerful alloresponses, igniting translational research on their potential use as an immunomodulatory therapy. For busy transplant clinicians, the vast amount of information in the literature on regulatory T cells can be overwhelming. This paper aims to highlight the most applicable research findings on the use of regulatory T cells in the immune diagnosis and potential immunomodulatory therapy of kidney transplant patients. However, can we yet rely on differential regulatory T-cell profiles for the identification of rejection or to tailor patient's immunosuppression? Are we ready to administer regulatory T cells as inductive or adjunctive therapy for kidney transplantation?

Prenodal lymph is generated from the interstitial fluid that surrounds organs, and thus contains products of organ metabolism and catabolism. New proteomic analyses of lymph have identified proteins and peptides that are derived from capillary extravasation and tissue-specific proteins. Many of these peptides are detected at nanomolar concentrations in the lymph before passage through a regional lymph node. Before entering the node and once inside, proteins and processed peptides are filtered from the lymph by circulating immature dendritic cells (DCs) or non-activated nodal antigen-presenting cells (APCs) (macrophages, B cells and immature DCs). Here, we suggest that this process ensures organ-specific self-antigens are displayed to circulating and nodal APCs, thus contributing to the maintenance of peripheral tolerance., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The mapping between genotype and phenotype is encoded in the complex web of epistatic interaction between genetic loci. In this rugged fitness landscape, recombination processes, which tend to increase variation in the population, compete with selection processes that tend to reduce genetic variation. Here, we show that the Bose-Einstein distribution describe the multiple stationary states of a diploid population under this multiloci evolutionary dynamics. Moreover, the evolutionary process might undergo an interesting condensation phase transition in the universality class of a Bose-Einstein condensation when a finite fraction of pairs of linked loci is fixed into given allelic states. Below this phase transition the genetic variation within a species is significantly reduced and only maintained by the remaining polymorphic loci.

Despite tremendous improvements in short-term renal allograft survival, many patients still have chronic rejection or side effects of nonspecific immunosuppression. The discovery of Foxp3(+) regulatory T cells (Tregs) has revolutionized the concepts in immunoregulation and offers perspectives for overcoming rejection. Recently, a subset of Foxp3(+)CD39(+) effector/memory-like Tregs (T(REM)) was identified. The role of CD39(+) Tregs in immunoregulation is supported by the occurrence of alopecia areata and experimental autoimmune encephalomyelitis in CD39-deficient mice and by the failure of CD39(-) Tregs to suppress contact hypersensitivity. In humans, CD39 polymorphisms have been associated with diabetes and nephropathy, and multiple sclerosis patients have reduced numbers of blood CD39(+) Tregs. Preliminary experiments in a murine transplantation model showed that CD39(+) Tregs can determine allograft outcome. CD39 degrades the extracellular adenosine triphosphate (ATP) released during tissue injury, which otherwise would trigger inflammation. Currently, our groups are assessing the role of CD39(+) Tregs and extracellular ATP metabolism in clinical transplantation and whether tolerogenic Treg profiles possess immunopredictive value, envisioning the development of clinical trials using CD39(+) Treg-based vaccination for autoimmunity or transplantation. This is a comprehensive review on the fundamentals of Treg biology, the potential role of ATP metabolism in immunoregulation, and the potential use of Treg-based immunotherapy in transplantation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

Hydrogen bonds (H-bonds) are crucial for the stability of the peptide-major histocompatibility complex (MHC) complex. In particular, the H-bonds formed between the peptide ligand and the MHC class II binding site appear to have a great influence on the half-life of the complex. Here we show that functional groups with the capacity to disrupt hydrogen bonds (e.g. -OH) can efficiently catalyze ligand exchange reactions on HLA-DR molecules. In conjunction with simple carrier molecules (such as propyl or benzyl residues), they trigger the release of low affinity ligands, which permits the rapid binding of peptides with higher affinity. Similar to HLA-DM, these compounds are able to influence the MHC class II ligand repertoire. In contrast to HLA-DM, however, these simple small molecules are still active at neutral pH. Under physiological conditions, they increase the number of "peptide-receptive" MHC class II molecules and facilitate exogenous peptide loading of dendritic cells. The drastic acceleration of the ligand exchange on these antigen presenting cells suggests that, in general, availability of H-bond donors in the extracellular milieu controls the rate of MHC class II ligand exchange reactions on the cell surface. These molecules may therefore be extremely useful for the loading of antigens onto dendritic cells for therapeutic purposes.