Genes & Immunity

BackgroundPatients with Common Variable Immunodeficiency (CVID) exhibit diverse clinical manifestations, indicating heterogeneity in pathogenic mechanisms. Systematic application of standardised phenotyping in large cohorts is essential to dissect this heterogeneity. The Human Phenotype Ontology (HPO) provides a structured framework for capturing and comparing disease phenotypes. ObjectiveTo evaluate the implementation and outcomes of HPO-based phenotyping in CVID patients enrolled for whole-genome sequencing in a large national adult primary immunodeficiency cohort. MethodsWe developed a web-based Phenotype Capture Tool and delivered structured clinician training to standardise HPO annotation. Numerical laboratory parameters were mapped to corresponding HPO terms to enrich patient records. ResultsWe coded the phenotypes of 526 CVID patients across 11 UK centres. Clinician training increased phenotype granularity and improved phenotyping consistency between clinicians. We assigned 883 unique HPO terms across the cohort and applied logical rules to the terms to classify patients into an infection-only group and a complex phenotype group (42% vs 58%, respectively). Patients in the complex phenotype group were significantly more likely to have reduced switched memory and expanded CD21low B cells, as well as pathogenic variants in IUIS-listed genes overall and pathogenic NFKB1 variants specifically. Having a pathogenic variant in an IUIS-listed gene was associated with Autoimmune hemolytic anemia and having a pathogenic NFKB1 variant specifically was associated with Autoimmune neutropenia. ConclusionThis is the first study to systematically collect granular HPO-coded phenotypes in a large real-world CVID cohort, refining the CVID landscape and providing a comprehensive CVID HPO term set relevant for international research. Clinical ImplicationHPO allows systematic capture of CVID phenotypes with low inter-clinician variability and improves comparison of cohorts, enhancing identification of disease heterogeneity essential to support genotype-phenotype studies and targeted therapeutic strategies. Capsule summaryHPO-based phenotyping of 526 CVID patients improved annotation quality, identifying immunological and genetic associations with clinical manifestations, distinguishing infection-only from complex disease and refining clinical characterisation to support international collaboration.

Red blood cell (RBC) alloimmunization is a clinically significant complication in transfused patients whose immunological determinants remain incompletely understood. Type I interferon (IFN-I) signaling drives RBC alloimmunization in murine models, and systemic lupus erythematosus (SLE) is characterized by constitutive IFN-I hyperactivation alongside elevated alloimmunization rates. We analyzed three publicly available SLE RNA-seq cohorts (GSE72509, GSE112087, GSE122459; whole blood and PBMC; total n = 150 SLE) in a pre-specified discovery-replication-validation design. A 14-gene IFN-I signature score was computed per sample; differential expression, gene set enrichment analysis, and Spearman correlation were performed independently per cohort. IFN-I scores were significantly elevated in SLE versus healthy controls in all three cohorts (p < 0.01 each). IFN-high SLE patients showed 665 differentially expressed genes, with enrichment of alloimmunization-associated and plasmablast differentiation gene sets confirmed by GSEA. The alloimmunization signature score correlated significantly with IFN-I score across all three independent cohorts ({rho} = +0.77, +0.51, +0.60; all FDR q < 0.05); Tfh differentiation showed no association in any cohort. To our knowledge, this represents the first human transcriptomic evidence that IFN-I pathway activity in SLE is coupled to alloimmunization-associated immune programs in vivo. These findings identify IFN-I score as a candidate biomarker of alloimmunization susceptibility in SLE and provide translational rationale for prospective studies incorporating transfusion outcome data.

BackgroundEpidemiological studies have consistently documented an inverse association between atopic dermatitis (AD) and glioblastoma (GBM), yet the immunogenetic mechanisms underlying this paradox remain elusive. We hypothesized that distinct immune subsets driven by shared genetic variants exhibiting antagonistic pleiotropy may explain this relationship. ObjectiveTo dissect the immunogenetic basis underlying the inverse association between AD and GBM by integrating single-cell transcriptomics and clustered Mendelian randomization, and to identify shared immune subsets and genetic variants exhibiting antagonistic pleiotropy that may explain this epidemiological paradox. MethodsWe integrated single-cell RNA sequencing (scRNA-seq) of publicly available datasets from AD skin (GSE153760) and GBM tumors (GSE256490) with genome-wide association study (GWAS) summary statistics. Disease-specific immune cell subsets were identified, and pathway enrichment was conducted on marker genes. Clustered Mendelian randomization (MR-Clust) was applied to detect heterogeneous causal effects, followed by drug target enrichment analysis using the DGIdb database. ResultsscRNA-seq revealed that Th2A cells were the predominant pathogenic subset in AD lesions, whereas S100A9+HLA-low suppressive monocytes were enriched in the GBM microenvironment. Both subsets shared enrichment in the NF-{kappa}B and Fc{varepsilon}RI signaling pathways, revealing a common immunological framework linking peripheral Type 2 inflammation to central nervous system immunosuppression. MR-Clust identified a distinct genetic cluster (Cluster 2) comprising 32 genes (e.g., IL4R, JAK1, SYK, FCER1G) significantly overexpressed in these cell types. This cluster exhibited antagonistic pleiotropy: it was directionally associated with reduced AD risk (OR = 0.930, 95% CI 0.846-1.023, p = 0.137) but a non-significant risk trend for GBM (OR = 1.447, 95% CI 0.737-2.841, p = 0.283). Drug target analysis indicated that Cluster 2 genes are primary targets of approved AD therapies, including dupilumab (IL4R) and JAK inhibitors (JAK1). ConclusionOur integrative analysis uncovers an immune-genetic axis linking Th2A cells in AD to suppressive monocytes in GBM, providing a mechanistic basis for their inverse comorbidity. These findings highlight a potential therapeutic paradox, underscoring the need for pharmacovigilance regarding long-term cancer risk in AD patients receiving targeted immunomodulators.

UNC93B1 is a crucial chaperone protein for the trafficking of TLRs and regulates antigen presentation in dendritic cells (DCs), which activates downstream immune responses. Here, we identified a novel homozygous gain-of-function (GOF) UNC93B1 variant in an early-onset lupus patient. The patient presented with elevated level of inflammation and auto-antibody, and organ damage. The Unc93b1R95L/R95L transgenic mice also exhibited with autoimmune and autoinflammatory phenotypes. The transcriptional analysis revealed increased inflammation and elevated activation of DCs in the patients PBMCs and bone marrow-derived DCs (BMDCs) from Unc93b1R95L/R95L mice. In addition to the selected TLR7/8 activation in previously reported UNC93B1 GOF variants, the single-cell transcriptome and flow cytometry of splenocytes from Unc93b1R95L/R95L mice demonstrated increased phagocytosis activity and T helper cell differentiation with altered ICAM and MHC signaling in DCs and T cells, respectively. These results suggest UNC93B1 GOF variant enhances antigen presentation from DCs to T cells in the pathogenesis of immune dysregulation. Our study expands the pathogenic variants spectrum of UNC93B1 and offers insight into the underlying mechanism of antigen presentation in immune dysregulation caused by UNC93B1 beyond its trafficking function of TLRs.

Celiac disease (CD) is strongly associated with specific HLA DQ heterodimers, formed by HLA DQA1 and HLA DQB1 proteins. In particular DQ2.5 (DQB1*02 associated to DQA1*05) and DQ8 (DQB1*03:02 with DQA1*03) are present in virtually all celiac patients. HLA DQB1*02 is considered the main single genetic susceptibility marker and has been reported in 90 to 95% of CD patients. However, the distribution of these alleles may vary across populations, potentially impacting the performance of genetic screening strategies. In this study, we evaluated the prevalence of HLA DQ2.5 and DQ8 genotypes in celiac patients (n = 41) and an unbiased general population cohort (n = 60) from San Luis, Argentina, using a PCR-based genotyping approach. In addition, we assessed the feasibility of a simplified saliva direct PCR protocol for large scale testing. Overall, 95.1% of CD patients carried DQ2.5 and/or DQ8. Notably, 41.5% of patients were DQ8(+)/DQ2.5(-), and 36.6% lacked the DQB1*02 allele, indicating that DQB1*02 based screening alone would have reduced sensitivity in this population. In the general population, 53.3% of individuals carried CD associated genotypes, with a markedly higher prevalence of DQ8 compared to European cohorts. Genotype distributions deviated from Hardy Weinberg equilibrium in CD patients but not in the general population. We show that DQB1*03:02 is a reliable proxy for DQ8, allowing simplification of genotyping strategies, whereas DQA1*05 typing remains essential to discriminate DQ2.5 from other lower risk DQB1*02 carrying heterodimers. We also describe a saliva direct PCR approach showing a performance comparable to purified DNA based assays. These findings highlight the importance of population specific genetic data for optimizing CD screening strategies and foster the development of simplified, cost effective genotyping approaches for large scale applications.

Background: Human papillomaviruses (HPVs) pose a severe threat to global public health by driving nonmelanoma skin cancer (NMSC) and cervical cancer, with NMSC being one of the most common cancers worldwide. Epidermodysplasia verruciformis (EV) is an inborn error of immunity characterized by an increased susceptibility to persistent infection of cutaneous HPV and a high risk of NMSC. The genetic basis remains unknown in many patients with EV. Methods: We collected four unrelated pedigrees with EV. Genetic analysis identified five variants in JAK1 encoding the Janus kinase 1. Ex vivo models and patient-derived tissue were employed to evaluate the functional effects of JAK1 variants and delineate the pathogenic mechanisms. Results: We identified different variants in JAK1 in four pedigrees with dominant EV. Genetic analysis revealed five novel variants in JAK1, three of which resulted in nonsense-mediated mRNA decay (NMD). Functional assays identified a decreased phosphorylation of the signal transducers and activators of transcription (STATs), impaired interferon responses, and defective T cell activation. Immune dysregulation in patients, characterized by a reduced CD4/CD8 T cell ratio, decreased CD8 naive T cell proportion, and accumulated memory T cells, implies impaired antiviral immunity against HPV. Conclusions: Our findings confirm that JAK1 loss-of-function (LOF) variants underlie susceptibility to cutaneous HPV infection. [Funded by the National Natural Science Foundation of China (81788101, 81230015, 82394420, and 82394423), the National Key Research and Development Program of China (2022YFC2703900), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-018), and the Regione Lombardia, Italy (Innovative Research Project 1137-2010)].

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency caused by gain-of-function in CXCR4 chemokine receptor (CXCR4GOF) in response to its chemokine ligand CXCL12. The patients suffering from this syndrome display lymphopenia and neutropenia, and most of them show exacerbated susceptibility to human papillomavirus pathogenesis. In a mouse model harboring a WHIM-associated CXCR4 mutation and expressing HPV16 oncoproteins in keratinocytes, we previously reported reduced circulating plasmacytoid dendritic cells (pDCs), mirroring patients blood, and impaired dendritic cell (DC) trafficking from the skin to lymphoid organs, with the few migrating DCs displaying an overactivated phenotype. Given the promising results of CXCR4-targeted therapies in WHIM patients, we investigated whether and how the orally available CXCR4-specific antagonist, X4-136, affects DC localization, activation, and trafficking at the subset level, as well as skin immune landscape. CXCR4GOF inhibition corrected defects in circulating myeloid cells and pDCs, as well as in lymph node-resident DCs. Furthermore, it rescued skin DC migration to lymph nodes in WHIM mice, in a context- and subset-dependent manner, by promoting their activation and relocation within the dermis. Taken together, these findings indicate that inhibiting CXCR4GOF may restore skin immunity in WHIM syndrome by rescuing DC counts and functions. Key pointsO_LICXC R4 gain-of-function inhibition promotes subset-selective dermal dendritic cell migration to lymph nodes in a WHIM syndrome mouse model. C_LIO_LIInhibiting CXCR4 corrects migratory WHIM dendritic cell hyperactivation with subset-specific effects tied to the inflammatory context. C_LI

TAPBP is a key chaperone of the peptide-loading complex that facilitates peptide loading onto major histocompatibility complex class I (MHC I) molecules. This study characterized TAPBP alleles in Korean Native Chickens (KNCs), identified novel variants, and evaluated haplotypic associations with BF2. Thirty-six samples representing six KNC lines were genotyped using LEI0258 and the MHC-B SNP panel, and individuals homozygous at both markers were classified into 16 groups. The same samples were subjected to Sanger sequencing of TAPBP exons 3-8. Sequences were assembled and aligned against MHC-B reference haplotypes and the Red Junglefowl reference. Additional comparisons with "tapasin allele" datasets enabled the identification of novel variants. Six novel nucleotide variants were detected across exons 3-6, including one nonsynonymous substitution in exon 4 (D251H). This residue corresponds to position Q265 in human TAPBP and lies adjacent to residues involved in MHC I interaction, suggesting potential functional relevance. Furthermore, TAPBP exhibited high haplotype diversity (Hd = 0.93) and moderate nucleotide diversity ({pi} = 0.00892), with exon 5 showing the highest diversity ({pi} = 0.01). B9 was the most frequent haplotype at the nucleotide level, whereas B6/B24 predominated at the amino acid level. Comparison with BF2 data revealed haplotype-dependent pairing patterns: BF2-B9 consistently matched TAPBP-B9, whereas BF2-B6 was associated with distinct TAPBP nucleotide variants, indicating allelic diversification within a shared haplotypic background. Homozygosity at LEI0258 and the SNP panel corresponded with TAPBP homozygosity, supporting marker-based prediction. These findings highlight potential BF2-TAPBP associations and provide a foundation for understanding variation in MHC I peptide loading.

T cell development in the thymus is a tightly regulated process where epigenetic modifications, such as histone 3 lysine 27 acetylation (H3K27ac), play a crucial role in controlling the activation of genes. The epigenetic regulation of human mucosal-associated invariant T (MAIT) cell development is unknown; we mapped the regulatory chromatin landscape in the three developmental stages of thymic MAIT cells to identify the regulatory elements and enhancer activity involved in thymic maturation and analysed whether these chromatin dynamics are associated with the acquisition of effector programs in developing MAIT cells. Utilising cleavage under target and tagmentation (CUT&Tag), genome-wide H3K27ac profiles were generated and combined with transcriptome data from thymic MAIT cells, which revealed how developmental shifts in enhancer activity correspond to changes in gene expression. In total, 41,958 genomic regions with H3K27ac signal were identified in MAIT cells across the three development stages, of which 1,200 regions showed acetylation changes during differentiation from stage 1 to stage 3. At dynamic regions, the greatest differences were observed between stage 1 and stage 3, highlighting a progressive gain or loss of H3K27ac during MAIT cell development. Overall, MAIT cell maturation was associated with the gradual accumulation of H3K27ac at promoters and enhancers, which closely correlated with gene expression changes during development. Stage-specific enrichment of H3K27ac was observed at key transcription factor gene loci involved in MAIT cell development, including ZBTB16 (PLZF), EOMES, RUNX3, NFATC2, FOXO1, TGIF1, IRF1, and MAF genes. Epigenetic remodelling was also observed at cytokine and cytokine receptors (IL7R, IL18R1, IL23R, IFNG), chemokines and chemokine receptors (CCL4, CCL5, CCR5, CCR9, CXCR4, CXCR6), as well as several surface molecules with known immunological function. Our work reveals a previously uncharacterised epigenetic profile of human MAIT cells that regulates and inuences their development.

Abstract/SummaryInnate immune responses are crucial for host defence but vary markedly between individuals. Although determinants of this variation are well characterised in adults, data from healthy children remain scarce. We therefore profiled whole-blood cytokine responses to innate immune stimulation in 286 children aged approximately four years and examined genetic, host-intrinsic, and environmental correlates of response. Cytokine responses showed marked inter-individual heterogeneity and stimulus-specific patterns. The top 50 genetic variants explained a substantial proportion ([~]20-45%) of this variance across many stimulus conditions, including a biologically coherent association of the STING locus with cGAMP-induced cytokine production. In contrast, sex, age, adiposity, and perinatal variables showed limited or modest associations. Systemic inflammatory biomarkers of systemic inflammation (hsCRP, glycoprotein acetyls, granulocyte-to-lymphocyte ratio) were strongly positively associated with cytokine responses. Finally, seasonal population-level viral infection burden was positively associated with antiviral and inflammatory cytokine responses. Collectively, these findings advance our understanding of variation in early-life whole-blood cytokine responses, underscoring this developmental period as a critical window for understanding immune development trajectories relevant to long-term health.

Socioeconomic status (SES) is a potent determinant of immune variation, yet unbiased approaches to holistically map the effect of SES on the immune system are lacking. We developed a high-dimensional flow cytometry-based profiling approach to analyse 331 cell surface proteins across 33 immune cell subsets within an SES-stratified Senegalese cohort, alongside a European cohort (Netherlands). We identified 108 SES-related markers across the immune system, revealing that lower SES individuals exhibited downregulation of surface proteins, affecting in particular adhesins, chemokine and complement receptors. Conversely, lower SES was associated with hallmarks of chronic activation and exhaustion including upregulation of immune checkpoints. Metabolic profiling demonstrated that while lower SES individuals displayed elevated baseline RNA transcription, higher SES individuals exhibited superior protein translation rates. We validated these SES-related immune trends in an independent cohort and provide an interactive online resource for exploring this surface protein atlas on immune cell subsets. Taken together, these findings provide a global overview of how the cell surface proteome varies by SES, and identify molecular changes that can affect vaccine efficacy and disease outcomes.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are complex chronic conditions that often follow infectious triggers with overlapping clinical features but poorly defined pathophysiological relationships. This study aimed to identify disease-specific immune signatures through multiparameter immunophenotyping of monocytes, dendritic cells, and T-cell subsets. A total of 207 participants were included (ME/CFS: n = 103; long COVID: n = 63; healthy controls: n = 41). Peripheral blood mononuclear cells were analyzed using multiparameter flow cytometry. Statistical analyses included non-parametric testing, age-adjusted ANCOVA, correlation network analysis, and principal component analysis (PCA). Long COVID was characterized by increased M2-like monocyte polarization, elevated CD80 expression across monocyte subsets, expansion of dendritic cells, and reduced expression of activation markers, indicating persistent immune activation with features of immune exhaustion. In contrast, ME/CFS exhibited reduced costimulatory molecule expression, impaired CCR7-mediated immune cell trafficking, and less coordinated activation patterns, consistent with a state of immune suppression. Correlation network analysis revealed more extensive and integrated immune interactions in long COVID, while PCA identified distinct immunophenotypic components and enabled moderate discrimination between the two conditions. These findings demonstrate that ME/CFS and long COVID are characterized by distinct immune profiles, supporting the concept of divergent immunopathological mechanisms. The identified signatures may contribute to biomarker development and guide targeted therapeutic approaches.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system affecting 2.8 million people worldwide. Both genetic and environmental factors contribute to MS risk, with Epstein-Barr virus (EBV) infection being an important environmental factor. To better clarify the role of EBV in MS, we examined its impact on gene expression, chromatin accessibility, and transcription factor binding in primary B cells and EBV-transformed B cells derived from patients with MS and healthy controls. RNA-seq and ATAC-seq analyses revealed extensive MS-dependent gene expression and chromatin accessibility differences in EBV-transformed, but not in primary B cells. These changes are largely accounted for by the expression levels of EBNA2, an EBV-encoded transcriptional regulator previously implicated in MS. ChIP-seq analysis revealed that EBNA2 binding with its interacting human partners RBPJ, EBF1, and PU.1 is highly enriched at MS genetic risk loci, with extensive EBNA2 allelic binding and increased enrichment at MS genetic risk loci in MS-derived cells. Our findings demonstrate that enhanced EBNA2 activity in MS alters human gene expression, chromatin accessibility, and transcription factor binding in an MS-dependent manner. Collectively, this study provides new insights into the molecular mechanisms through which EBV, particularly EBNA2, interacts with host genetic risk to contribute to MS pathogenesis.

ObjectivesCD4+ T cells play key roles in regulating immune responses during pregnancy, therefore we aimed to understand the CD4+ T cell surface proteome and transcriptome during pregnancy. MethodsCD4+ T cells were analysed in blood and decidua from term-pregnancies (>37 weeks), and non-pregnant blood. >350 surface proteins were screened via flow cytometry, and transcriptomes were analysed using single-cell RNA sequencing with >130 CITE-seq barcoded antibodies. ResultsSurface protein screening identified changes to ILT4/CD85d, CD9, IFN-{gamma} receptor {beta}-chain, CX3CR1 and CCR5 in the pregnant blood and decidual CD4+ T cells. CX3CR1 and CCR5 had the highest expression on the effector-memory T cell (TEM) subset in the blood, with expression consistent across subsets in decidua. CD126/IL-6R was lower in pregnant blood and decidual CD4+ T cells, while scRNAseq identified enrichment in the IL-6R signalling pathway in naive CD4+ T cells in pregnant blood. Both sIL-6R and IL-6 concentrations were increased in plasma during pregnancy, suggesting perturbations to the IL-6/IL-6R signalling axis. Meanwhile, decidual CD4+ T cells had increased expression of transcription factor RUNX3 in the CD69+ tissue-resident-like subset. ConclusionsOur findings demonstrate altered molecular expression in CD4+ T cells during pregnancy. This provides important mechanistic insight of their adaptation and regulation during placental development, which may drive placental dysfunction or pregnancy complications including preeclampsia, fetal growth restriction and stillbirth. These new data may inform future studies that focus on determining the significance of differentially- expressed immune features in pregnancy to identify potential targets for immune modulation to treat pregnancy complications and infections.

PRV-101 is a multivalent formalin-inactivated Coxsackievirus B (CVB) vaccine developed to prevent CVB infections, which are associated with increased risk of islet autoimmunity. While PRV-101 induces robust neutralizing antibody responses, its T-cell immunogenicity is unknown. We analyzed peripheral blood mononuclear cells from 25 healthy adults receiving three high or low PRV-101 doses or placebo in a Phase I randomized, placebo-controlled trial. CVB-reactive CD8 T-cell responses were assessed using HLA Class I multimers, and CD4 and T follicular helper (Tfh) responses were measured by activation-induced marker assays following stimulation with a CVB peptide library. PRV-101 elicited minimal CVB-reactive CD8 T-cell responses but robust CD4 and Tfh responses, peaking at week 12 and persisting through week 32. Responses were observed in both seronegative and seropositive individuals, consistent with effective immune priming and boosting. Tfh frequencies correlated with neutralizing antibody titers. Female participants exhibited higher peak Tfh responses than males. We conclude that PRV-101 elicits a CVB-protective immune profile, dominated by Tfh responses supporting durable humoral immunity and devoid of potentially diabetogenic cytotoxic T-cell responses. This profile invites further investigations in vaccine trials for type 1 diabetes prevention.

Autoimmune diseases result from immune responses against self-antigens but exhibit marked phenotypic diversity shaped by genetic and environmental factors. Genome-wide association studies (GWAS) have identified susceptibility loci that inform polygenic scores (PGS) for risk prediction. This study integrates phenotypic and genetic data from the UK Biobank(UKB) to characterize disease overlap, genetic heterogeneity, and shared biological mechanisms across autoimmune conditions. Comorbidity patterns were further assessed using patient records from UKB and the TriNetX(TNX). Phenotypic data from 502,371 UKB participants were used to evaluate diagnostic overlap, with a subset of 104,544 individuals analyzed for PGS distributions. Significant variants were identified using genome-wide thresholds, allele frequency, and predicted impact, and shared genes were subsequently mapped to pathways using Hallmark gene sets. Comorbidity across rare and common autoimmune diseases was assessed in the UKB and TNX using ICD-10 codes, focusing on White individuals (71,069,654 in TNX; 502,371 in UKB). Odds ratios for 15 diseases were estimated, and cross-cohort comparisons evaluated reproducibility and cohort-specific differences. PGS analyses revealed both shared and distinct genetic architectures, indicating partial genetic overlap and supporting poly-autoimmunity. Integration of common, rare and impactful variants identified both known and novel gene associations, while pathway analysis highlighted systemic and tissue-specific immune dysregulation. Cross-dataset comparisons confirmed consistent comorbidity patterns but underscored the impact of dataset-specific factors, emphasizing the need for standardized approaches in autoimmune disease research.

DNA methylation is a stable epigenetic mark that critically influences the phenotype of immune cells. Identifying differentially methylated regions within immune cell lineages supports their phenotypic and functional characterization, leading to a better understanding of lineage-specific transcriptional regulation. Here, we performed a genome-wide methylation analysis of human innate lymphoid cells (ILCs), which allowed us to define specific epigenetic marker regions for ILC1, ILC2, and ILC3. These regions were associated with genes that have well-described functions in ILCs, such as TBX21 in ILC1, GATA3 and MAF in ILC2, RORC and IL23R in ILC3, but were also found in genetic loci that have not been previously associated with ILCs. In-depth analysis of ILC2-related marker regions within the HPGDS and NRROS gene loci confirmed their critical role in transcriptional regulation and suggested a novel role for NRROS in ILC2. Genome-wide methylation analysis of ILC2, derived from the blood of juvenile donors with atopy or asthma led to the identification of several disease-specific epigenetic regions associated with genes such as GIMAP4 and PTGS2. Together, our study not only provides novel epigenetic marker regions in human ILCs and confirms the functional role of ILC2-related markers, but also identifies promising markers for studying allergies in humans.

Coeliac Disease (CeD) is a chronic gastrointestinal inflammatory disease initiated by dietary gluten in genetically predisposed individuals. While the inflammatory processes which drive tissue destruction in the coeliac duodenum have been extensively characterised, an increased oxidative stress (OS) response has also been suggested to contribute to CeD pathogenesis. However, the precise mechanisms which regulate OS in the coeliac mucosa and whether they impact inflammation remain ill defined. The master anti-oxidant transcriptional regulator Nuclear factor erythroid 2-related factor 2 (Nrf2), and its inhibitor, Kelch like ECH-associated protein 1 (Keap1) have been implicated in chronic gastrointestinal inflammatory diseases, such as ulcerative colitis but have been largely unexplored in the context of CeD. To investigate redox balance in the CeD duodenum, we utilised single cell transcriptomics to assess overall OS and cytoprotective Nrf2 activation across cell subsets in duodenal biopsies from CeD patients. OS induced gene expression was broadly increased across multiple cell subsets in the CeD mucosa. Simultaneously, specific markers of Nrf2 activation were decreased in cell subtypes central to pathogenesis of CeD, including activated CD4+ T cells and intraepithelial T lymphocytes, indicating a distinct redox imbalance in these cells. Furthermore, pharmacological activation of Nrf2 significantly decreased gliadin induced IFNG expression in CeD duodenal biopsies. Taken together, our findings demonstrate that redox imbalance represents a therapeutic opportunity for the modulation of proinflammatory responses that drive the pathogenesis of CeD.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies. Immune dysregulation is believed to play an important role in ATC. Here, we aimed to characterize the systemic inflammation and the function of circulating immune cells of patients with ATC. First, we retrospectively assessed biochemical parameters of patients with ATC and observed that high systemic inflammation correlated with worse survival. Next, we prospectively investigated the inflammatory proteome, single-cell peripheral blood mononuclear cell transcriptome and epigenetic changes. Circulating concentrations of proinflammatory cytokines were increased in ATC patients. This proinflammatory profile was apparent at the level of gene transcription and chromatin accessibility, especially in monocytes. These findings were substantiated by an increased capacity of peripheral blood mononuclear cells of ATC patients to produce IL-6, IL-8 and lactate. As IL-6 is known to promote tumor cell survival, we assessed its capacity to influence ATC cell proliferation. Blocking IL-6/gp130/Jak/STAT3 pathway inhibited proliferation of ATC cell lines in vitro. In conclusion, these findings show that ATC is characterized by inappropriate systemic inflammation and epigenetic and transcriptional reprogramming of circulating monocytes. Proinflammatory cytokines released by monocytes support survival and proliferation of ATC tumor cells, suggesting a therapeutic potential of targeting this pathway in ATC patients.

Background: Accurate preoperative prediction of lymph node metastasis (LNM) in papillary thyroid carcinoma (PTC) remains challenging, particularly in clinically node-negative (cN0) patients, leading to potential overtreatment. We aimed to develop and validate a Transformer-based 2.5D deep learning model (ThyLNT) using preoperative computed tomography (CT) images for robust prediction of LNM and to explore its underlying biological basis through multi-omics analyses. Methods: A total of 1,560 PTC patients from six hospitals were retrospectively included. The Tongji Hospital cohort (n=1,010) was divided into training (70%) and internal validation (30%) sets, while five independent institutions served as external test cohorts. For each lesion, seven 2.5D slices were extracted and modeled using a DenseNet201 backbone. Slice-level features were integrated using a Transformer-based feature-level fusion strategy and compared with ensemble learning, multi-instance learning (MIL), and traditional radiomics approaches. Model performance was assessed using area under the receiver operating characteristic curve (AUC), calibration analysis, decision curve analysis (DCA), and precision-recall curves. Multi-omics analyses, including bulk RNA-seq, single-cell RNA-seq, spatial transcriptomics, and spatial metabolomics, were performed to investigate biological correlates. Results: The Transformer-based model consistently outperformed comparator models across cohorts. In the training and validation cohorts, ThyLNT achieved AUCs of 0.882 and 0.787, respectively, with external AUCs ranging from 0.772 to 0.827. Compared with ultrasound (US) and CT, ThyLNT showed superior predictive performance (all P < 0.001 in the validation cohort). Simulation analysis in cN0 patients suggested that ThyLNT could reduce unnecessary lymph node dissection (LND) from 52.16% to 4.88%. Transcriptomic analysis combined with WGCNA and correlation analysis identified VEGFA as the gene most strongly associated with ThyLNT prediction scores. Single-cell and spatial transcriptomic analyses suggested metastasis-related tumor microenvironment remodeling, while enrichment analysis of genes affected by virtual knockout of VEGFA indicated involvement of angiogenesis- and epithelial-mesenchymal transition (EMT)-related pathways. Spatial metabolomics further revealed coordinated lipid metabolic reprogramming in metastatic tissues. These findings suggest that ThyLNT provides robust predictive performance while capturing biologically relevant features associated with metastatic progression.