Genes & Immunity

Background Genome-wide association studies have identified >200 loci associated with IBD, yet the causal gene for most remains unknown. As multiple epithelial functions have been linked with susceptibility to IBD, there is a need to prioritize candidate causal genes for functional studies in this cellular context. Methods Using a standardized definition of regions implicated by index SNPs from three GWAS studies, we categorized regions as containing: (1) a known casual gene, (2) a single gene or (3) multiple genes. We then developed an IBD Priority Score to rank genes based on genetic, genomic and functional data. We next developed and applied an Epithelial Priority Score, based on expression patterns and quantitative traits, to prioritize genes for functional validation in epithelial models. Two candidate genes identified through this approach were tested for their impact on viral response pathways in HT-29 cells. Results The IBD Priority Score prioritized a single gene in 71 of the 104 regions containing multiple genes. The Epithelial Priority Score identified 31 epithelial candidates. Functional studies demonstrated that IRF6 enhanced, whereas IRF8 suppressed, antiviral responses in intestinal epithelial cells stimulated with Poly(I:C). Conclusions Combining multiple genetic, genomic, and functional data is a useful approach for prioritizing the most likely causal gene within IBD GWAS loci, and for prioritizing functional validation studies in epithelial cells and tissues. Moreover, we provide functional evidence for two IBD genes playing a role in the regulation of anti-viral responses in intestinal epithelial cells.

Introduction: Killer-cell immunoglobulin-like receptor (KIR) gene content shapes natural killer-cell responses and varies substantially among populations. We examined whether worldwide KIR diversity reflects regional structure, isolation by distance, serial founder effects, or latitudinal gradients. Data and methods: Carrier frequencies for 11 variable KIR genes were obtained from the Allele Frequency Net Database. The primary analysis included 189 locally sampled, non-diaspora, non-mixed populations from 70 countries, comprising 25,469 individuals; 161 populations had complete 11-gene profiles. Diversity indices, ordination, clustering, permutation-based variance decomposition, Mantel tests, and regression models were used to characterize geographic structure. Serial founder effects were evaluated using four distance measures from East Africa, both globally and within the Old World. Models of latitude incorporated spatial autocorrelation. Prespecified sensitivity analyses evaluated alternative population sets, gene panels, geographic classifications, weighting schemes, and multivariate outlier exclusion. Results: Geographic region explained 53.5% of variation in KIR profiles, substantially exceeding the permutation-null expectation of 6.9%, although multivariate dispersion differed among regions. Twenty-five of 28 pairwise regional comparisons remained significant after false-discovery-rate correction. Isolation by distance was modest (Mantel r=0.3003) and weakened after controlling for region (r=0.1557). KIR diversity did not decline with distance from East Africa globally but decreased strongly within the Old World (expected heterozygosity {beta}=-0.0066 and standardized gene content {beta}=-0.0789). B-content decreased nominally with absolute latitude, but latitude was collinear with region and the association survived neither spatial modelling nor sensitivity analyses. Regional differentiation remained robust across analytical specifications, whereas the Old World decline was carried by gene content once standard errors were clustered by country. Conclusion: Worldwide KIR gene-content variation shows pronounced regional differentiation, modest isolation by distance, and no independent latitudinal gradient. Diversity declines with distance from East Africa across the Old World, supporting serial founder effects, but not once the Americas and Oceania are included.

Background and objectivesMyasthenia gravis associated with antibodies against muscle-specific kinase (MuSK-MG) is a well-characterized IgG4-autoimmune disease, however, the mechanisms driving IgG4 predominance remain poorly understood. This study investigated whether promoter DNA methylation of cytokine genes involved in IgG4 class switching is associated with this immune response. MethodsPeripheral blood mononuclear cells were isolated from MuSK-MG patients (n=36), acetylcholine receptor myasthenia gravis (AChR-MG) patients as disease controls (n=7), and sex-matched healthy controls (n=12). Promoter DNA methylation of IL4, IL10, and IL13 was assessed by methylation-sensitive high-resolution melting and relative cytokine mRNA expression by qPCR. Associations with clinical variables, and antibody levels were subsequently evaluated. ResultsMuSK-MG patients showed lower median IL13 promoter methylation compared with healthy controls (p = 0.004). Median IL4 promoter methylation was also reduced in MuSK-MG compared with healthy controls (p < 0.001) and AChR-MG disease controls (p < 0.001), whereas no differences were observed for IL10 promoter methylation. Relative mRNA expression of IL4 (p = 0.0005), IL10 (p = 0.0462), and IL13 (p = 0.0002) was increased in MuSK-MG compared with AChR-MG. Compared with healthy controls, only IL4 expression remained significantly increased (p < 0.0001). Promoter methylation was inversely correlated with relative mRNA expression for IL4 (p < 0.0001), while IL13 showed a similar but non-significant trend (p = 0.054), no association was observed for IL10. Multivariable analysis demonstrated that treatment at sampling was independently associated with lower IL10 and IL13 promoter methylation, whereas no associations were observed with age, sex, disease phase, or disease duration. Promoter methylation did not correlate with total serum IgG4 or anti-MuSK IgG4 levels. DiscussionMuSK-MG is associated with selective hypomethylation of IL4 and IL13 promoters accompanied by increased cytokine gene expression, while IL10 promoter methylation remains unchanged. The association between treatment and IL10 and IL13 promoter methylation suggests that immunosuppressive therapy may influence epigenetic regulation in MuSK-MG. Together, these findings support a role for epigenetic dysregulation of Th2-associated cytokines in the immunological environment associated with IgG4 subclass switch. To our knowledge, this is the first study investigating IL4, IL10, and IL13 promoter DNA methylation in MuSK-MG.

APECED (Autoimmune PolyEndocrinopathy Candidiasis Ectodermal Dystrophy) is a rare syndrome of multi-organ autoimmunity driven by the presence of self-reactive T cells and autoantibodies caused by mutations in the gene Autoimmune regulator (AIRE). Compared to the well-defined role of AIRE in establishing and maintaining T cell central tolerance, less is known about how AIRE deficiency impacts B cell phenotypes that may contribute to a breakdown in peripheral B cell tolerance. Here we analyzed serum and peripheral blood cells from APECED patients and healthy donors using autoantibody profiling, proteomics, flow cytometry, scRNAseq based subset analysis, BCRseq, and autoantigen binding assays finding significant changes to the APECED B cell compartment. We show that while Naive and Transitional B cells are reduced, alternatively activated B cell subsets are expanded in APECED patients including IgM CD27+ and class switched Atypical B cells which exhibit BCR chain features prone to autoreactivity. Antibodies derived from either APECED or Healthy class switched atypical B cells bind autoantigens at significantly higher rates than control IgG B cells. Serum autoantibodies and proteomics highlight APECED common and patient variable changes. These results together show that APECED causes a compositional shift towards subsets that may promote broken B cell tolerance and autoimmunity.

Genetic ancestry composition has been used extensively as a predictor of clinical outcomes, multiomic biomarkers, and endophenotypes, but the models have most often used untransformed ancestry fractions as explanatory variables. Under a simplex constraint results from this type of model can be uninterpretable and misleading. We demonstrate the use of multiple transformed log ratio coordinates of ancestral proportions as predictors in a study of excreted myeloperoxidase (MPO), a neutrophilic enzyme used as a fecal biomarker to measure intestinal inflammation and enteropathy. We show how to convert these difficult-to-interpret effects into association results for shifts between two ancestries under an isoancestry constraint (holding an ancestry fraction constant) using post-estimation average marginal effects and explain their geometric interpretation. We found that increasing proportion of African ancestry was associated with increased levels of excreted MPO in low-income household infants in three Northeastern Brazil studies, with a smaller negative secondary association of European ancestry, all under a tri-continental ancestry composition (African, European, Amerindian). While the maximum log(MPO) outcome variance explained by differences in genetic ancestry was modest at a few percent, we show evidence that these differences in heritability were context-dependent on measurement of MPO under active enteric infection. The results were attenuated by socio-economic status, but a signal remained. Overall, we found that African genomes carry additional alleles that enable a more intensive neutrophil response to enteric infection relative to European and Amerindian genomes, illuminating a possible host genetic background role in modulating immune response to enteric diseases and pathogens.

Background: More than five years after the COVID-19 outbreak, the infection by the SARS-CoV-2 virus should still be considered as a global threat with highly heterogeneous clinical outcomes from asymptomatic to fatal depending on existing genetic and phenotypic inter-individual and inter-population variability. Our objective is to identify risk loci associated with COVID-19 in an admixed Latin American population. Methods: We performed a genome-wide association study in the framework of the Chilean C19-GenoNet team, analyzing a cohort of 3,417 individuals recruited from the northernmost to the southernmost regions of the country, who presented variations in the severity of their COVID-19 clinical manifestations. Results: Our investigation replicated the extensively reported association of several variants at the 3p21.31 locus with COVID-19 severity and found a suggestive association for a variant within the FOXP4 gene at 6p21.1 with COVID-19 hospitalization. Both associations appear to be shared with other populations. Importantly, our study identified three previously unreported loci associated with COVID-19 severity or hospitalization at 7q21.11, 16p13.12 and 6p25.3, and one additional locus at 4p13 associated with SARS-CoV-2 positivity, a proxy for infection susceptibility. Conclusions: these new risk loci may represent population-specific effects in Chilean and, potentially, other Latin American admixed groups. Our study highlights the importance of expanding efforts to study underrepresented and genetically different populations, which may provide new clues on the genetic basis of human diseases and may help to anticipate global responses to emerging viral infections and future pandemics.

Global incidence and outcomes of glioma have been found to vary significantly by region, however research into the disease continues to lack diversity. Here we investigated epigenetic patterns in glioma subtypes from cohorts collected from China and the USA. We retrospectively analysed the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) datasets following reclassification of glioma subtypes based on the WHO 2021 central nervous system (CNS) tumour classification. We used DNA methylation and transcriptomics data to identify methylation-driven cancer genes in the CGGA cohort, assessed their prognostic value and compared against the non-Hispanic White cohort in the TCGA database to consider ethnic influence. Furthermore, we used machine learning classification and clustering techniques to identify methylation patterns in glioma subgroups. Here, we showed that DNA methylation profiles of CGGA glioblastomas have a methylation signature more similar to TCGA high-grade astrocytomas: 58.1% of CGGA glioblastomas were identified as high-grade astrocytomas using classification modelling. Assessment of survival revealed that CGGA glioblastoma patients had a significantly better survival rate than non-Hispanic White glioblastoma patients (p = 0.037). Four key methylation-driven genes were identified in the CGGA glioblastoma samples: GLDN, PRKDC, S100A1 and NCAPH. Hypermethylation of GLDN significantly suppressed gene expression in all glioma subtypes in only the East Asian cohort; a gene that has not been previously described as a driver in gliomas. Together these data suggest alternative epigenetic mechanisms occurring in glioma subtypes of different ethnic populations, which is important for our understanding of glioma and strategies for personalized treatment.

Multiple sclerosis (MS) is approximately three times more common in females than in males, but sex-linked epigenetic mechanisms remain incompletely understood. We tested whether X-chromosome DNA methylation differs in females with MS and whether associated sites preferentially map to genes normally subject to X-chromosome inactivation (XCI). An X-chromosome-focused epigenome-wide association study was performed in whole blood from three datasets comprising 496 females with MS and 509 female controls, with independent evaluation in isolated CD4+ T cells (80 cases, 76 controls). At FDR < 0.01, 3 182 X-chromosome CpGs were hypomethylated in MS (mean {Delta}{beta} = -0.035). Hypomethylated CpGs were strongly enriched in non-escape genes normally subject to XCI relative to escape genes (OR 6.77; p = 1.55x10-57) and mapped to immune and chromatin-related processes. Exploratory cis-mQTL analysis identified strong local genetic effects at a small subset of loci, including WAS, IRAK1 and TLR7. In CD4+ T cells, 2 080 of the 3,182 whole-blood hypomethylated CpGs (65.4%) were also significantly hypomethylated. These findings identify a reproducible X-chromosome hypomethylation signature in female MS and support direct investigation of inactive-X regulation, XCI escape and X-linked immune gene dosage.

Background and Aims: Fibrostenotic stricture is a severe complication of Crohn's disease (CD) that affect up to 50% of patients over their lifetime, causing irreversible narrowing of the intestinal lumen and bowel obstruction, but the molecular programs that distinguish stenotic from non-stenotic inflammation and the relative contributions of fibrosis and smooth-muscle remodeling remain incompletely characterized. We aimed to dissect bowel-wall compartment-specific and histology-defined remodeling programs in fibrostenotic CD. Methods: Matched full-thickness terminal ileal tissues from 4 patients with fibrostenotic CD were sampled from stenotic stricture (SS), non-stenotic inflamed bowel (NS), and non-inflamed bowel (NI). Histomorphology-guided NanoString GeoMx whole-transcriptome profiling was performed across mucosa, muscularis mucosae, submucosa, and muscularis propria. GeoMx data were processed by standard software provided by GeoMx platform. DESeq2 models included patient identity as a blocking factor, and the analysis was performed in 74 histologically valid regions of interest (ROIs) representing full spectrum of CD histopathologies. DEGs were defined as adjusted P < .05 and |log2FC| >= 1.0. Results: Out of 18,677 genes analyzed in each of the ROIs, direct SS-versus-NS comparison identified 0 significant DEGs in mucosa, 18 in muscularis mucosae, 43 in submucosa, and 0 in muscularis propria. NS-versus-NI submucosa showed 132 DEGs (131 increased, 1 decreased), dominated by humoral/plasma-cell genes with a modest stromal-remodeling component. SS-versus-NI submucosa showed 144 DEGs (138 increased, 6 decreased) with coordinated contractile, extracellular-matrix/remodeling and cell-matrix adhesion programs. Muscularized versus fibrotic SS submucosa showed 219 DEGs, with marked enrichment of contractile and remodeling genes in muscularized regions. Excluding 5 follicle-containing submucosal ROIs markedly altered the NS comparison but not the core SS fibromuscular program. Exploratory follicular-niche analyses showed that lymphoid follicle-rich (LFR) submucosa was lymphoid/B-cell enriched relative to lymphoid follicle-null (LFN) areas; SS-LFR nevertheless acquired a stronger fibroblast/ECM program relative to NS-LFR, while CCL19 was the only significant LFR-versus-LFN DEG within SS. Conclusions: Fibrostenotic CD is characterized by spatially concentrated submucosal fibromuscular remodeled inflammatory program. Non-stenotic submucosa is heterogeneous and may already show regional stromal remodeling, whereas established stenosis displays a broader and more robust ECM-contractile-adhesion phenotype with molecularly distinct fibrotic and muscularized niches.

Human immune responses remain poorly characterized across Indigenous South American (ISA) populations, limiting our understanding of human immune variation. Here, we generated a single-cell transcriptional atlas of 1.4 million peripheral blood mononuclear cells from 345 individuals representing 13 South American populations, profiled at baseline and following stimulation with lipopolysaccharide, influenza A virus, or Trypanosoma cruzi. Genetic ancestry was associated with the expression of a large fraction of the transcriptome, often only after stimulation, and explained more variation than age or sex. On average, cis-eQTLs accounted for one-quarter of these ancestry-associated differences. Thousands of regulatory variants were common in ISA populations but rare or absent in European reference datasets. Integrating eQTLs with GWAS revealed stimulation-specific disease-associated regulatory variants and an enrichment of ancestry-associated expression differences among genes with colocalizing signals. Our findings highlight the importance of studying diverse populations across immune contexts to understand the genetic architecture of human immune responses.

The non-canonical NF-kappa B pathway, in which NFKB2 plays a central role, is critical for lymphoid organ development and immune homeostasis. Pathogenic NFKB2 variants have been strongly associated with a broad spectrum of human pathologies, notably immunodysregulation syndromes, characterized by recurrent infections, autoantibody production and endocrine abnormalities. The mechanisms underlying the resulting immune defects remain incompletely understood. In the present study, we characterized the immune phenotype of a novel rat model harboring a Nfkb2 non-sense mutation located in the c-terminal region of the gene (c.2602T>A; p.Tyr867*, Nfkb2p.Tyr867*). This variant has been identified in patients with a familial form of common variable immunodeficiency (CVID) presenting with autoimmunity manifestations. Using this model, we demonstrate that mutated NFKB2 resulted in profound alterations of the thymic microenvironment, including reduced K5+ medullary thymic epithelial cells and decreased AIRE expression, accompanied by altered thymocyte development and impaired thymic regulatory T populations. Peripheral lymphoid organization was also profoundly affected, with absence of lymph nodes, defects in the splenic marginal zone, and alterations of CD4+ and CD8+ T-cell homeostasis, including impaired Treg populations and reduced ICOS expression on CD4+ T cells. The B-cell compartment was markedly altered, with impaired B-cell maturation, characterized by accumulation of IgM+IgD- transitional cells and a marked reduction of IgD+ mature B cells. These abnormalities were associated with impaired humoral immunity and complete absence of circulating immunoglobulins. Collectively, our findings define a systemic combined immunodeficiency phenotype in the Nfkb2-mutated rat, highlighting its utility for studying the role of non-canonical NF-kB signaling in immune system organization and adaptative immunity.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination, neurodegeneration, and progressive neurological disability. Galanin, a neuropeptide with immunomodulatory properties, signals through G protein-coupled receptors, among which galanin receptor 2 (GALR2) has been implicated with neuroprotective and anti-inflammatory functions. A rare homozygous single nucleotide variant in GALR2 (rs61745847; p.W249L) has been identified in a patient diagnosed with relapsing-remitting MS, however, the biological relevance of this variant in neuroinflammation remains unknown. Here, we investigated the impact of the orthologous GALR2 W248L mutation using a knock-in mouse model and experimental autoimmune encephalomyelitis (EAE). GALR2 W248L knock-in (KI) mice exhibited a more severe clinical course of EAE, accompanied by enhanced inflammatory infiltration, exacerbated demyelination, and increased microglial activation in the spinal cord compared with wild-type (WT) mice. Despite comparable lymphoid and myeloid cell frequencies in the central nervous system, alterations in microglial density and morphology suggested an important contribution of the innate immune system to disease exacerbation in the KI mice. Ex vivo analyses revealed that bone marrow-derived macrophages from KI animals exhibited a pronounced shift toward a pro-inflammatory phenotype, characterized by enhanced M1 polarization, impaired M2-associated responses, and increased NLRP3 inflammasome activation. In parallel, live-cell imaging of primary hippocampal neurons demonstrated reduced galanin binding in mutant cells, consistent with impaired GALR2 functional availability at the plasma membrane. Together, these findings identify GALR2 as a modulator of the neuroinflammatory response and indicate that disruption of galanin-GALR2 signaling promotes sustained innate immune activation, highlighting the relevance of this pathway for MS pathogenesis and its potential as a therapeutic target in neuroinflammatory disorders.

The transcriptional and cell repertoire changes of lymph node (LN) T cells following influenza vaccination have been under-investigated across different ethnicities. Draining (dLN) and non-draining (ndLN) axillary LN cells from 17 participants with genotypically varied African or Asian ancestry (ISRCTN13657999) were collected by fine needle aspiration (FNA) before, 5 days and 42 days after adjuvanted influenza vaccination and analysed by single-cell RNA-sequencing (scRNA-seq). The early dLN transcriptional profile and cellular dynamics were distinct from the ndLN; BACH2 expressing T cells were highly induced, exhibiting stem-like and unique quiescence signatures. Proliferating T follicular helper cells (Tfh) were enriched in dLN and Th1-like Tfh predominated with highly induced transcriptional changes. The BACH2+ T cell state and Th1-like Tfh cells, transcriptionally distinct from germinal centre (GC) T cells, were co-localised with LAMP3+ dendritic cells in the T cell zone of publicly available LN datasets, a region that was also distinct from GC Tfh niches. Together, these findings indicate that the induction of a robust, proliferative, and transcriptionally diverse Tfh response, alongside a discrete subset of BACH2+ T cells, represents a marked feature of the early T cell zone immune response to adjuvanted influenza vaccine in the dLN.

Despite significant advancements in microsurgical techniques in recent years, the treatment and prognosis of craniopharyngiomas remain unsatisfactory. As a central nervous system tumor located adjacent to important brain structures such as the hypothalamus-pituitary axis and accompanied by a highly inflammatory microenvironment, the tumor heterogeneity and tumor microenvironment characteristics of papillary craniopharyngiomas (PCPs) remain unclear. In this study, we integrated multimodal single-cell and spatial profiling from PCP tissue and peripheral blood mononuclear cells (PBMCs) to elucidate the tumor heterogeneity and microenvironment characteristics of PCP. Our single-cell and spatial analyses defined four specific tumor cell states in PCP, representing specific transcriptional regulatory programs and spatial heterogeneity characteristics during tumor progression. By constructing a spatial niche composed of tumor, immune, and stromal cells, we analyzed the cellular and spatial ecosystem of PCP at multiple levels to further assess the communication relationships between different tumor cell states and microenvironment cells. This study established a multidimensional molecular atlas of PCP from the perspectives of cell state, spatial structure, and microenvironment interactions, providing a foundation for understanding its biological behavior and exploring new intervention strategies.

Background Fibromyalgia syndrome (FMS) affects 1 to 2 percent of the general population, with higher prevalence estimates in women. Recent evidence supports peripheral immune involvement in disease pathophysiology. Alternative splicing (AS) regulates immune cell function independently of transcript abundance and carries disease-relevant signal in related autoimmune conditions. No study has performed transcriptome-wide differential AS analysis directly in RNA-sequencing data from individuals with FMS. Methods We performed a secondary analysis of GSE221921, restricted to female participants (n = 91 FMS, n = 41 controls). Peripheral immune cell composition was estimated with ABIS, a deconvolution method specifically trained on peripheral blood mononuclear cell signatures. Differential AS was analysed with LeafCutter incorporating cell composition principal components as covariates. Differential gene expression was performed with DESeq2 under the same adjustment framework. Results FMS samples showed lower ABIS-inferred conventional monocyte estimates (Cliff's {delta} = -0.38, FDR = 0.008) and higher memory B-cell estimates (Cliff's {delta} = +0.32, FDR = 0.030). Composition-adjusted LeafCutter analysis identified 14 significant intron clusters, of which 9 were retained for biological interpretation. GNLY (granulysin) was the primary candidate, with a composition-adjusted 5' junction usage shift illustrated by pooled Sashimi visualisation. Composition adjustment substantially reduced significant differentially expressed genes from 10,803 to 3,186 (baseMean [&ge;] 10). Conclusions This first transcriptome-wide differential AS analysis in FMS identifies GNLY, encoding the cytolytic lymphocyte effector granulysin, as a splicing-specific candidate for functional follow-up. AS captures FMS-associated signal not detectable by gene-level expression analysis, and composition adjustment substantially altered the differential expression landscape.

Single-cell RNA sequencing technologies provide insights into gene expression at the cellular level, enabling detailed analysis of cellular heterogeneity. In this study, we systematically compared two scRNA-seq platforms,10X Genomics and Parse Biosciences, using human peripheral blood mononuclear cells (PBMCs) and terminally differentiated effector memory CD8+ T cells (TEMRAs). We identified significant differences in gene expression variability and platform-specific biases, such as ribosomal and mitochondrial gene capture. 10X has a bias for shorter genes, while Parse exhibited enhanced detection of longer transcripts. In CD8+ TEMRAs, the expression of key genes related to antimicrobial immune responses were underrepresented in Parse cells compared to 10X cells (e.g. GNLY, PRF1 and GZMB). These findings underscore the need for careful selection of scRNA-seq platforms based on specific research objectives, as platform-specific biases can influence cell type identification and as well as mechanistic insights derived from gene expression data. Our results provide insights for selection of scRNA-seq experimental platforms in immunological studies.

Background and aimsPerianal fistulizing Crohns disease (pCD) affects 20% of patients with Crohns disease (CD) and severely impacts quality of life. Current therapies fail to provide sustained relief, and the molecular underpinnings of pCD remain poorly understood. This study aimed to elucidate the molecular landscape of perianal fistulas through multiomic profiling. MethodsPaired fistula-tract and adjacent rectal mucosal biopsies were collected from 63 patients (48 with pCD, 15 with cryptoglandular fistula [CPTGL]). Longitudinal sampling generated 101 unique molecular profiles, comprising of both RNA sequencing (RNA-seq) and 16S rRNA sequencing from both tissue sites, followed by integrative multiomics and gene network analyses. ResultsUnsupervised clustering revealed three patient clusters primarily defined by host gene expression, with minimal contribution from microbial profiles. Fistulae in clusters 1 and 2 showed strong immune activation and epithelial-mesenchymal transition (EMT). In contrast, cluster 3 fistulae displayed keratinization and metabolic reprogramming resembling psoriatic skin, together with reduced JAK-STAT signalling. Cluster 3 was enriched for patients classified as TOpClass:2a, who are more suitable for surgical repair (p = 0.02). Conversely, cluster 2, characterized by rectal keratinization and EMT in both fistula and rectum, showed the highest MRI inflammatory-mass score (p = 0.02) and a greater risk of subsequent ileostomy (Kaplan-Meier; p = 0.008). An independent RNAseq dataset validated the keratinization signature in a subset of pCD fistulae. ConclusionIntegrated multiomic analysis identified distinct molecular subtypes of pCD with surgical and therapeutic relevance. These findings refine the molecular understanding of pCD and support a precision medicine approach. What You Need to Know?O_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSO_LIPerianal fistulas affect 1 in 5 Crohns disease patients and have a severe negative impact on the quality of life of the patients. C_LIO_LIMost advanced IBD therapies are not efficacious for perianal Crohns disease. C_LIO_LISurgical repair is not suitable for all patients. C_LI NEW FINDINGSO_LIThis study is the first to delineate molecularly defined patient subtypes in perianal fistulizing Crohns disease. It further characterizes a psoriasis-like keratinization program in a subset of fistulas. Finally, it identifies molecular features and histological indicators that may explain-and potentially help predict-favorable surgical outcomes in selected patients C_LI LIMITATIONSO_LIFunctional and mechanistic studies will be required to further dissect the drivers and dynamics of the epithelial remodeling identified. C_LI CLINICAL RESEARCH RELEVANCEO_LIRaises the possibility of refining of existing clinical stratification using molecular markers for improved therapeutic management and outcome. C_LI BASIC RESEARCH RELEVANCEO_LIProposes keratinization as an opposing molecular process to EMT within the perianal fistula tract. C_LIO_LIIdentifies robust gene signatures associated with EMT and keratinization in the fistula for further experimental and clinical studies. C_LI

BackgroundStrategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify "all monocytes" in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P<0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

Colorectal cancer (CRC) disproportionately affects Hispanic individuals, who remain underrepresented in genomic discovery and lack ancestry-matched functional resources. Many CRC risk variants are non-coding, and those in 3' untranslated regions (3' UTRs) can alter microRNA (miRNA) binding sites and reprogram post-transcriptional gene regulation. Here, we identify allele-specific miRNA-binding variants at CRC risk loci within the Hispanic population using a discovery pipeline anchored to Hispanic-relevant resources. CRC-associated 3' UTR variants were compiled from the GWAS Catalog, further verified in Hispanic/Latino individuals using the All of Us Research Program cohort (n = 453), expanded into proxy sets using Admixed American-specific linkage disequilibrium, annotated against Ensembl transcripts, and evaluated with TargetScan and RNAhybrid. Screening of 38 CRC-associated 3' UTR SNPs identified 19 variants that alter miRNA binding across nine genes, with each gene represented by a variant confirmed in the Hispanic cohort. Three oncogenic loci, DCBLD2 (lead SNP rs11552978), GREM1 (lead SNP rs10318), and SMC1B (rs3747239, prioritized as an AMR-specific proxy in near-complete LD with confirmed lead rs6007600), met all prioritization criteria. In each case, the risk allele disrupts a strong miRNA-binding site, while the gene shows increased tumor expression, weak correlation between expression and methylation, and an absence of local active transcriptional chromatin marks (cCREs/H3K27Ac). These results suggest that the variants promote gene overexpression through loss of miRNA-mediated repression rather than changes in promoter methylation. Overall, the study identifies non-coding CRC susceptibility variants in the Hispanic population and highlights DCBLD2, GREM1, and SMC1B as strong candidates for functional validation.

Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease defined by the production of autoantibodies directed against diverse self-antigens. Antinuclear antibodies (ANA) are central to SLE diagnosis, and anti-double-stranded DNA (anti-dsDNA) antibodies are clinically used to track disease activity. However, these biomarkers provide only partial insight into the full spectrum of autoimmune changes in patients. To expand the known landscape of SLE autoreactivity shared among patients and associated with disease activity, we performed an unbiased characterization of 1,268 SLE plasma samples from 695 SLE patients and 301 healthy controls using phage immunoprecipitation and sequencing (PhIP-seq). This technique preferentially displays linear epitopes, and thus provides a complementary set of autoreactivities to previously discovered autoreactive targets. Despite the marked clinical heterogeneity of SLE, machine learning applied to this data surfaced a shared, reproducible signature that robustly discriminates between SLE and healthy controls in two validation cohorts. Autoreactivities in SLE were significantly enriched for RNA-binding protein targets. Autoreactive proteins containing Basic Acidic Dipeptides (BAD) repeat domains, features associated with biomolecular condensate formation, distinguished patients with high disease activity (by SLEDAI score) from patients with no disease activity. Among all shared autoreactivities, a peptide derived from YTHDF2 emerged as the most prevalent target across SLE cohorts occurring in ~25% of SLE patients and displayed striking sequence similarity to a peptide derived from the Epstein Barr virus (EBV) lytic switch protein BZLF1. Competition assays demonstrated higher-affinity binding to the viral peptide, supporting a model in which antiviral humoral responses to EBV may drive secondary epitope spreading toward molecularly mimicked self-antigens. Remarkably, orthogonal validation using the Luminex bead platform containing a panel of only 10 peptides, including BZLF1, demonstrated strong discrimination between SLE and healthy controls (AUC = 0.95). Together, these findings identify a rich, greatly expanded landscape of autoreactivities in SLE, candidate biomarkers associated with high disease activity, and provide mechanistic insight linking EBV-driven immunity to epitope-specific autoimmunity in lupus.