Immunology

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.

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

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.

Expression of the Csf1r gene in cells of the mononuclear phagocyte lineage is regulated by a conserved enhancer, the fms-intronic regulatory element (FIRE). In mice with a germ-line deletion of FIRE (Fireko) CSF1R expression is undetectable in bone marrow progenitors and classical monocytes. Fireko mice lack subpopulations of macrophages in the brain and periphery but develop normally. Here we show that loss of CSF1R expression in Fireko mice is partly overcome by CSF2 in vitro and inflammatory recruitment in vitro. Analysis of heterozygous mutant mice and deletion of the conserved AP1 motif in FIRE provide evidence that continuous receptor synthesis determines CSF1 responsiveness. The absence of macrophages in kidney and heart of Fireko mice was not associated with detectable loss of physiological function. In a model of renal injury macrophage recruitment and histopathology were similar in WT and Fireko mice. Tissue resident macrophages that were depleted in Fireko mice, including microglia, were replaced by donor-derived cells following intraperitoneal adoptive transfer of wild-type bone marrow at weaning. The Fireko mouse provides a novel platform to dissect the functions of tissue resident macrophages in development, homeostasis and pathology. Summary StatementThis study describes a unique model of selective tissue resident macrophage deficiency arising from dysregulated expression of the mouse Csf1r gene.

BackgroundHumans and mice display elevated levels of IL-27, an immunosuppressive cytokine shown to increase during neonatal bacterial sepsis and compromise survival. This study explores two hypotheses for regulation of IL-27 expression: 1) decreased DNA methylation in newborns that contributes to increased expression of IL-27 genes; 2) neonatal hormones regulate IL-27 expression through upstream hormone response elements (HREs). MethodsWhole genome methyl-seq analysis of neonatal and adult blood-derived macrophages identified differentially methylated regions (DMRs) at steady-state. Quantitative PCR (qPCR) measured expression of IL-27 genes (IL27p28 and EBI3) in human and murine neonatal macrophages stimulated in vitro with synthetic glucocorticoid or progesterone. Confocal microscopy and chromatin immunoprecipitation (ChIP) of glucocorticoid receptor (GR) assessed translocation into the nucleus and binding to the EBI3 promoter. ResultsThe IL-27p28 promoter contained DMRs that were increased in the neonatal cohort. The analysis did not identify DMRs within the EBI3 promoter. Dexamethasone stimulation increased EBI3 gene expression in human and murine neonatal macrophages. GR localized to the nucleus in response to dexamethasone and was enriched at the EBI3 upstream regulatory region. ConclusionThese data suggest glucocorticoid (GC) signaling increases EBI3 expression. This has importance in the context of antenatal GC administration that may increase IL-27 levels. Impact Statement{blacksquare} Elevated expression of IL-27 in early life impairs the host response to invasive bacterial infection in neonates. {blacksquare}Understanding the regulatory mechanisms contributing to increased IL-27 during the neonatal period is necessary to reduce susceptibility to infection in this vulnerable population. {blacksquare}The methylation status of the IL-27 genes in macrophages from neonatal and adult blood donors does not suggest regulation of differential expression with age. {blacksquare}Glucocorticoids are a signal that can induce EBI3 gene expression in a GR-dependent manner. {blacksquare}Glucocorticoid therapy for premature infants may increase IL-27 expression and promote enhanced susceptibility to infection.

To the editorO_ST_ABSBackgroundC_ST_ABSAnaphylaxis is an acute and potentially life-threatening hypersensitivity reaction often involving the cardiovascular system. Circulating microRNAs (miRNAs/miR), including those carried by extracellular vesicles (EVs), are emerging biomarkers that display regulatory functions in allergy. This study aims to investigate the role of miR-29a in anaphylaxis. MethodsMiR-29a (3p and 5p) levels were assessed by qPCR from acute and baseline samples of serum and EVs from 70 patients with food- and drug-mediated anaphylaxis. EVs purification was confirmed by Western blot, electron microscopy, and NanoSight. MiR-29a-3p target genes were studied in silico using systems biology analysis (SBA). Moreover, miR-29a levels were evaluated in vitro in endothelial cells (ECs) exposed to anaphylactic mediators. Additionally, a panel of endothelial glycocalyx (eGCX)-associated mRNA was analyzed after transfection with a miR-29a-3p inhibitor. ResultsPatients with food-induced anaphylaxis exhibited reduced miR-29a-3p levels in both serum and EVs during the acute reaction. In contrast, miR-29a-5p levels were decreased in serum but not in EVs. No significant modulation of either miRNA was observed in drug-induced anaphylaxis. SBA of miR-29a-3p identified molecular pathways, biological processes and functional networks associated with eGCX remodelling. Intracellular levels of miR-29a-3p were modulated in vitro in ECs following exposure to anaphylactic mediators. Inhibition of miR-29a-3p significantly reduced ESM1 expression. ConclusionsThe miR-29a-3p levels are decreased in serum and EVs from patients with acute food-induced anaphylaxis, suggesting its potential as a promising biomarker. Moreover, a role for miR-29a-3p in eGCX integrity under anaphylactic conditions was demonstrated, potentially regulating ESM1. Key MessageMiR-29a-3p is selectively reduced in serum and extracellular vesicles during acute food-induced anaphylaxis and may regulate endothelial glycocalyx-related pathways, which supports its potential as a novel biomarker and molecular mediator of vascular involvement in anaphylactic reactions.

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

Mycetoma is a neglected tropical disease caused by various bacterial and fungal pathogens that has a significant health impact across a broad geographically defined "mycetoma belt" spanning South America, Africa and Asia. Histologically, mycetoma is characterised by invasive and destructive granuloma development in the skin, deep tissues and bone, leading to tissue destruction, deformities and high morbidity. The presence of macroscopic, highly compacted pathogen microcolonies, or "grains," is a key diagnostic feature, and the formation of grains supports pathogen persistence and disease chronicity. However, there is a paucity of information on immune responses in mycetoma patients and on the relative importance of phylogeny and/or grains in establishing the local immune landscape. Here, we used spatial proteomics to examine the distribution of 43 immune-related proteins in surgical biopsies from 11 patients with mycetoma of bacterial (Actinomycetoma; Actinomadura pelletierii and Streptomyces somaliensis; n=6) and fungal (Eumycetoma; Madurella mycetomatis; n=5) origin. Using mixed-effects modelling, an exploratory analysis across species and pathogen classes revealed few significant differences in immune marker expression. In contrast, and independently of pathogen class, the cellular infiltrate closest to grain boundaries had higher per-cell expression of CD66b+, ARG1, and VISTA. The preferential accumulation of CD66b+ARG1+VISTA+ cells at grain boundaries was confirmed by quantitative immunofluorescence analysis. Hence, the local tissue microenvironment surrounding the mycetoma grain represents a specialised immunosuppressive niche, with parallels to the tumour microenvironment.

BackgroundIn World War 1 (WW1) outbreaks of measles were associated with high case fatality rates amongst soldiers. Recent studies have shown that survivors of acute measles can also develop immune amnesia, increasing their susceptibility to other infections. However, the impact of prior measles infection on infectious diseases during WWI remains unclear. MethodsHere, we create a searchable database documenting the medical history of 1,569 individuals from the Australian, New Zealand, and Canadian forces during WW1. ResultsWe use this novel database to show that a recent measles hospitalisation was associated with a higher chance of death for infectious diseases (excluding pandemic influenza like illness), consistent with immune amnesia. Surprisingly, a prior measles infection was associated with a significant reduction in hospitalisations duration from pandemic influenza like illness. ConclusionThese findings highlight the unique interaction between measles and pandemic influenza, contrasting with other infectious diseases, and underscore the significant health burden measles placed on young adults during WW1.

ImportanceDigital Phenotyping (DP) utilizes digital technologies to assess observable phenotypic traits, enhancing our understanding of various disease states. It aims to support equitable interventions through diverse digital data, fostering inclusivity by integrating various digital devices. Central to DP is identifying digital biomarkers (DBMs), which offer real-time health monitoring and personalized insights into disease conditions. Applying machine learning (ML) techniques on non-invasive signals sets the ideal platform for precision medicine application, especially in rare diseases (RDs). People living with rare diseases (PLWRD) often face significant challenges in receiving timely and accurate diagnoses, leading to what is known as a diagnostic odyssey. Digital phenotyping (DP) offers a promising solution by leveraging advanced technology, such as 3D facial photography, to capture unique digital signatures associated with various rare diseases. This innovative approach not only aids in the identification of these conditions but also facilitates the detection of digital biomarkers (DBM). These biomarkers enable healthcare providers to monitor the progression of the disease over time, enhancing patient care and potentially shortening the duration of the diagnostic odyssey. By utilizing DP and DBMs, we can improve both the diagnosis and management of RDs, ultimately leading to better health outcomes for affected participants. ObjectiveTo identify whether DBMs can be identified by DP utilizing 3D facial imaging techniques in outpatient settings in participants with RDs. The primary objective of this study was to determine if specific facial measurements in participants with RD who experience transient episodes of facial swelling (oedema) differ from established ethnically matched norms. The secondary objective was to assess peri-orbital and/or facial swelling as a potential biomarker for identifying flare-ups in hereditary angioedema (HAE). Design, setting, and participantsThis multicentre observational study was conducted in 3 hospitals in Singapore. The eligible participants were male and female RD participants of various age groups. The study duration was 4 years and 8 months. Interventions: Twenty participants of Chinese genetic ancestry were photographed using a 3D camera. Additionally, two participants with hereditary angioedema (HAE) were photographed during acute stages of disease flare-ups. Main outcomes and measuresThe obtained facial scans of participants (that included participants with HAE in non-acute phase) were plotted using Artificial Intelligence-powered software - Cliniface. The growth curves and facial landmarks obtained were compared against the growth curves of normal RD-unaffected individuals of Chinese genetic ancestry. The two participants with HAE were photographed qualitatively over a longer period of time, and their scans were plotted, yielding growth curves. ResultsDistinct facial markers such as periorbital swelling were identified in two qualitatively assessed HAE participants during flare-up stages. This provides an opportunity to explore and validate further if these facial signatures in a disease condition can be assigned as DBM for HAE. Conclusions and relevanceThis study explores the utility of 3D facial analysis as a DBM in rare diseases such as HAE. Applying non-invasive signals coupled with AI may open new vistas for precision medicine in real-world settings. The individual measurements that yielded small p-values demonstrate significant relevance and potential utility. These findings offer preliminary objective evidence that supports existing subjective reports of facial features in the literature. Additionally, while DPs diagnostic capabilities may be limited, it successfully identified DBM, which could facilitate disease monitoring in conditions such as HAE. Author SummaryRare diseases pose a significant challenge to all stakeholders, including clinicians, patients families, care providers, and the healthcare system. Diagnostic delays are integral and impose a massive financial and emotional burden on everyone involved in care delivery, beyond the patients themselves. A universally acceptable, scalable, and replicable non-invasive mechanism to detect distinct biomarkers associated with a rare disease can help identify the diseases signs and symptoms far earlier and ease the burden. An easy-to-deploy approach could be 3D facial imaging of patients with rare diseases, which are associated with distinct or subtle facial changes at different stages of disease progression. A rare disease, such as hereditary angioedema, which is known to exhibit facial swelling in patients during the acute disease state, is a prime example. The facial changes can be identified and assigned as specific disease markers, also known as facial biomarkers. These facial biomarkers can be identified and measured using 3D facial imaging when patients present to the clinic. Subsequently, these initial signs can be correlated clinically to establish a firm diagnosis earlier than traditional approaches.

BackgroundThis study investigates the role of the pioneer transcription factor FOXA1 as a master gene in sustaining epithelial cell polarization in early-stage lung adenocarcinoma. The partial loss of FOXA1 is explored to determine if it will affect plasticity and progression of lung adenocarcinoma. The study also addresses the transcriptional circuitry that links polarity defects to lysosome homeostasis. MethodsA multiomics approach was used to define the status of the chromatin in epithelial and mesenchymal states of A549 adenocarcinoma cells obtained with a newly synthetized TGF-{beta} receptor inhibitor or TGF-{beta} respectively. The study leveraged ATAC-seq, RNA sequencing, Cut&Tag sequencing of FOXA1 and histone marks profiling. The functional impact of FOXA1 was examined by partial silencing in vitro and by heterozygous FOXA1 deletion in a KrasG12D mouse model. Three-dimensional organoid culture, high-resolution electron microscopy, spatial transcriptomics and multiplex immunohistochemistry assessed carcinoma cell polarity, proliferation, the tumor microenvironment and organelle content. Group differences were evaluated with two-tailed t tests or one-way analysis of variance. ResultsFOXA1 binding and expression were highest in cells harboring an epithelial phenotype. In mouse KrasG12D LUAD tumors FOXA1 marked polarized, CDH1-positive cells; heterozygous loss diminished CDH1, disrupted apical-basal architecture, lowered organoid-forming efficiency and remodeled the immune microenvironment. Spatial transcriptomics and ultrastructural analyses showed that FOXA1-deficient carcinoma cells accumulated lysosomes, down-regulated vesicle fusion genes of the SNARE family and activated the lysosomal CLEAR gene network. FOXA1 occupied enhancers of lysosome-associated genes and competed with the transcription factor TFE3, thereby suppressing transcription of cathepsin B and cathepsin C and restricting lysosome biogenesis. ConclusionsFOXA1 is a central regulator that preserves epithelial cell polarity and limits lysosome formation in lung adenocarcinoma. Targeting the FOXA1-TFE3-lysosome axis may affect tumor plasticity and provide new therapeutic opportunities.

BackgroundThis study aims to examine the independent relationships between individual components of metabolic syndrome (MetS) and two key clinical outcomes in patients with Crohns disease (CD): disease activity, as quantified by the Crohns Disease Activity Index (CDAI), and the occurrence of complications. MethodsThis retrospective cross-sectional study included 376 adults with newly diagnosed Crohns disease. Multiple linear regression was used to examine associations between metabolic parameters and CDAI scores, while multivariate logistic regression assessed links to complications. Analyses were also based on clinical CDAI cut-offs. Predictive nomograms were developed and internally validated via bootstrap resampling. ResultsMultiple linear regression indicated that higher CDAI scores were independently associated with lower BMI (B = -5.866, P < 0.001), lower HDL-C levels (B = -81.770, P < 0.001), higher triglycerides (B = 15.618, P = 0.001), and lower ESR (B = -0.375, P = 0.03). Multivariate logistic regression established low HDL-C (OR = 0.042, P < 0.001), low BMI (OR = 0.915, P = 0.034), and high triglycerides (OR = 1.792, P = 0.007) as significant independent risk factors for complications. The developed nomograms demonstrated strong predictive performance, with an adjusted R2 of 0.207 for the CDAI model and an AUC of 0.765 for the complication model. For both predictive tasks, the model incorporating separate TG and HDL-C measurements significantly outperformed the TG/HDL-C ratio model. ConclusionMetabolic disturbances demonstrate a significant association with increased disease severity and a higher risk of complication development in Crohns disease. Core tipO_LIDual-outcome study reveals HDL-C and TG differentially link to CD inflammation and complications, pointing to distinct mechanisms. C_LIO_LILow HDL-C is the strongest independent predictor for CD complications, underscoring its protective role beyond cholesterol transport. C_LIO_LIIndividual TG and HDL-C metrics outperform their ratio in prediction, challenging its use and suggesting independent pathways in CD. C_LIO_LILow BMI independently associates with both adverse outcomes, refining the "obesity paradox" and highlighting malnutritions key role. C_LIO_LIA practical, validated nomogram (AUC=0.765) integrates HDL-C, TG, and BMI to stratify complication risk, aiding clinical decision-making. C_LI

BackgroundCutaneous melanoma (CM) is an aggressive skin cancer with rising incidence, representing a growing public health concern. Despite the remarkable success of immune-checkpoint inhibitors (ICIs) in the management of advanced disease, mortality remains high due to therapy resistance. Identifying reliable prognostic and predictive biomarkers is therefore essential to improve patient stratification, optimize treatment selection, and minimize unnecessary toxicity. MethodsWe comprehensively profiled the circulating immune landscape of 54 treatment-naive CM patients by integrating flow cytometry immunophenotyping with clinicopathological data, and performed tumor gene expression analysis in a subset of 26 patients. ResultsElevated HLA-DR and CD69 expression on circulating CD4+ T cells, together with reduced circulating CD8+ T cell frequency, emerged as candidate prognostic biomarkers associated with improved survival. Prognostic models combining these immune variables with clinical covariates accurately stratified patients by overall survival (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression/recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001). In a subset of 43 patients subsequently treated with ICIs, elevated baseline HLA-DR and CD69 expression on circulating CD4+ T cells was also associated with therapeutic benefit. A predictive model integrating these markers with clinical covariates achieved good discriminatory performance (65.2% sensitivity, 88.9% specificity; AUC = 0.775, p = 0.0027). Tumor gene expression profiling supported the role of IFN-{gamma}-related signatures, previously linked to ICI response, as complementary prognostic and predictive tools. ConclusionThese findings highlight systemic CD4+ T cell activation status as a promising, easily measurable biomarker in CM, laying the foundation for future strategies to refine patient stratification and guiding immunotherapy decisions.

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.

BackgroundThe tumour microenvironment (TME) influences breast cancer progression and treatment response. We investigated whether TME composition predicts tamoxifen benefit in postmenopausal women with oestrogen receptor-positive, HER2-negative (ER+HER2-) breast cancer. MethodsThis study included 513 patients from the Stockholm Tamoxifen (STO-3) trial, which randomised postmenopausal, lymph node-negative women to tamoxifen or no endocrine therapy. Bulk tumour transcriptomes were deconvoluted with the ConsensusTME algorithm to estimate the relative abundance of 18 immune and stromal cell types. A summary score of combined immune cells was created on a per patient basis and evaluated alongside fibroblast and endothelial stromal compartments. Patients were categorised into immune and stromal tertiles on the basis of these scores. Associations between TME composition and tumour characteristics were evaluated using Spearman correlations and Fishers exact test. Tamoxifen benefit was analysed by univariable Kaplan-Meier (log-rank) and multivariable Cox proportional hazards adjusting for age, tumour size, grade, progesterone receptor, Ki-67, and radiotherapy. Differential expression was assessed with limma and pathway enrichment with fgsea using Hallmark gene sets from MSigDB. ResultsLow immune abundance was significantly associated with higher ER expression (Fishers exact test p < 0.001). Among tamoxifen-treated patients, those with low immune scores showed improved distant recurrence-free interval (DRFI) relative to untreated patients (log-rank p < 0.001). Similarly, intermediate endothelial (p < 0.001) and low/intermediate fibroblast abundances (p = 0.042, p = 0.009) were associated with favourable DRFI. In multivariable models, low immune (aHR = 0.17, 95% CI 0.08-0.40), intermediate endothelial (aHR = 0.21, 95% CI 0.09-0.51), and low/intermediate fibroblast tertiles (aHR = 0.50, 95% CI 0.27-0.93; aHR = 0.36, 95% CI 0.17-0.77) retained significance. Transcriptomic analysis revealed enrichment of oestrogen-response, MYC-target, and oxidative-phosphorylation pathways in low-immune and low-fibroblast tumours, while interferon-{gamma} response and allograft rejection pathways were downregulated. ConclusionsTME composition modulates tamoxifen benefit in postmenopausal ER+HER2-breast cancer. Low immune, intermediate endothelial, and low/intermediate fibroblast abundances are associated with improved benefit from tamoxifen, suggesting that both immune and stromal compartments influence endocrine treatment efficacy.

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.

Purpose/ObjectiveThis study aimed to design and computationally evaluate a synthetic GluN1-mimetic peptide as a decoy to bind and neutralize pathogenic autoantibodies in anti-NMDA receptor (NMDAR) encephalitis, a severe autoimmune neurological disorder affecting approximately 1.5 per million individuals annually. MethodsKey GluN1 epitope residues (351-390 of the amino-terminal domain) were identified from crystallographic evidence and patient-derived antibody binding studies. Multiple peptide variants were rationally designed to mimic the antibody-binding interface. AlphaFold2 was used to predict peptide structures. Rigid-body docking simulations were conducted with HADDOCK 2.4 to model peptide-antibody complexes, and binding affinities were quantified using PRODIGY. A scrambled peptide control was included to establish docking specificity. ResultsThe top-performing peptide demonstrated favorable predicted binding ({Delta}G = -21.5 kcal/mol, Kd = 1.7 x 10-{superscript 1} M) with an average pLDDT score of 90%, a buried surface area of 3,255.5 [A]{superscript 2}, and 18 intermolecular hydrogen bonds. Relative to the scrambled control ({Delta}G = -8.3 kcal/mol), the designed peptide showed substantially stronger predicted binding. Conclusion/ImplicationsThese results support the validity of an epitope-mimicry design strategy and establish a scalable computational framework for prioritizing peptide decoy candidates applicable to other antibody-mediated autoimmune disorders. Experimental validation remains necessary to confirm real-world efficacy.

BackgroundThe tumor immune microenvironment likely plays a central role in progression of thyroid cancer. As for most other solid tumors, it is unknown if immune dysregulation contributes to earlier, subclinical stages of thyroid tumor development, or whether thyroid tumor heterogeneity might involve differential expression of pro-inflammatory mediators. MethodsThe time course of tumor-associated inflammation was studied in Tg-CreERT2;Braf CA/+ mice representing a model of BRAFV600E-driven papillary thyroid carcinoma (PTC). Tumor growth was estimated by histological examination and magnetic resonance imaging. Cytokine expression was monitored by quantitative RT-PCR, RNAScope and Western blot analyses. ResultsBased on spontaneous BrafCA activation due to leaky Cre activity in a minority of targeted cells tumors developed within a preserved thyroid tissue architecture to multifocal papillary thyroid carcinoma (PTC) over a period of 12 months. Tumorigenesis was accompanied by a gradually increased mRNA and protein expression of interleukin-1beta (IL-1{beta}), interleukin-6 and tumor necrosis factor-alpha (TNF-) starting already before Braf mutant cells commenced neoplastic growth. RNAScope revealed that both follicular cells and stromal cells expressed Il1b whereas Il6 and Tnfa transcripts were mostly confined to neoplastic epithelia. Early cytokine expression was associated with oncogene-induced senescence, whereas during tumor development (3-6 months) and in advanced tumor stages (at 12 months) the cytokine expression pattern differed among glands and tumor foci of the same gland accompanied by a highly variable locoregional lymphocytic infiltration. Oral treatment of mutant mice for 1 month with PLX4720, a vemurafenib prodrug, partially reduced cytokine expression along with inhibited tumor growth and redifferentiation of thyroid function. The magnitude of reduced cytokine expression differed much between glands and among mice of both sexes. ConclusionsThese findings indicate that oncogenic BRAFV600E targeted to the thyroid both stimulates endogenous production of IL-1{beta}, IL-6 and TNF- and recruits inflammatory cells to foci of early tumor development. PTCs of different clonal origin are distinguished by differential expression of pro-inflammatory cytokines. The anti-inflammatory effect of mutant Braf kinase inhibition varies presumably related to heterogeneous tumor development, which evolves from stochastic BrafCA activation suggesting there are clonally different probabilities of acquiring drug resistance among Braf mutant thyroid follicular cells.

BackgroundIn the course of testing mAb-based therapies to eradicate the persistent reservoir of HIV infection, we investigated the efficacy and mode of killing of HIV-infected cells by two categories of cytotoxic immunoconjugates (CICs) targeted by the same mAb, an immunotoxin (IT) and antibody-drug conjugate (ADC). MethodsWe performed metabolic and transcriptional analyses of treatment effects on the persistently-infected cell line H9/NL4-3. Cells were treated with CICs consisting of the anti-gp41 mAb 7B2 conjugated to either deglycosylated ricin A chain (dgA) or to the highly cytotoxic anthracycline derivative PNU-159682. At intervals up to 24 hr, intracellular metabolites were quantified by 1H nuclear magnetic resonance spectroscopy, and the transcriptome analyzed by RNA-Seq. ResultsSix hr post treatment, 7B2-dgA elicited both metabolic and transcriptional alterations, whereas 7B2-PNU treated cells did not differ from untreated cells. 7B2-dgA treated cells exhibited elevated intracellular levels of many amino acids, and activation of gene pathways for apoptosis, intracellular signaling, and immune activation. By 24 hr, both 7B2-dgA and 7B2-PNU treated cells differed markedly from untreated. Many of the changes observed following 7B2-PNU treatment at 24 hr were similar to those observed at 6hr following 7B2-dgA, likely indicating processes involved in cell death, but a number of alterations were unique to either IT or ADC treated cells. ConclusionsAn IT and ADC showed both similarities and differences in their cytotoxic effects. These results raise the question of whether the mode of cell killing could be a determinant of clinical efficacy. Although these studies were aimed at targeting the persistent reservoir of HIV infection, they have relevance for the design of CICs to treat cancer and other conditions. SUMMARYThe use of cytotoxic immunoconjugates, wherein an antibody is attached to a cellular poison, is effective in the treatment of cancer and other conditions. We seek to extend these results to treating HIV and other chronic viral infections. We analyzed the molecular mechanisms of cell killing when the same antibody was attached to different toxic structures. We report that each immunoconjugate induced both common and distinct patterns of killing. Such differences may have clinical relevance.

BACKGROUNDMyocarditis is an inflammatory cardiac disease in which Th17-driven immune responses contribute to progression toward dilated cardiomyopathy and heart failure. Current therapies mainly rely on corticosteroids but lack specificity, while the role of miR-721, synthesized by Th17 cells, remains largely unexplored in disease pathogenesis. METHODSWe characterized the presence of mmu-miR-721 and its human homolog hsa-RNA-Chr8:96 in extracellular vesicles (EVs) secreted by Th17 cells from IL-17eGFP mice with experimental autoimmune myocarditis (EAM) and myocarditis patients. MxCre-Ppargfl/fl mice and luciferase reporter assays were used to validate the target genes of miR-721 and hsa-RNA-Chr8:96, respectively. The functional role of miR-721 in EAM was investigated by lentiviral vectors overexpression and inhibition using miRNA sponge molecules. Th17 responses and heart inflammation were assessed and echocardiography was performed after in vivo blockade of mmu-miR-721 in EAM mice. RESULTSBoth mmu-miR-721 and hsa-RNA-Chr8:96 were encapsulated in EVs and secreted by Th17 cells of mice and patients with myocarditis. Overexpression of mmu-miR-721 in draining-lymph node cells from EAM mice inhibited Pparg transcription, leading to increased ROR{gamma}t and IL-17 expression and promoting Th17 differentiation. In contrast, in the absence of Pparg, a target of miR-721, no differences in ROR{gamma}t expression were observed, indicating that miR-721 promotes Th17 responses through repression of Pparg. Human PPARG was validated as a target gene of hsa-RNA-Chr8:96 and its overexpression in peripheral blood leukocytes downregulated PPARG mRNA levels, suggesting similar pathways involved in human pathology. In vivo blockade of mmu-miR-721 increased Pparg expression, reducing ROR{gamma}t and IL-17 activation in T cells and leading to decreased leukocyte infiltration in the heart and improved cardiac function. CONCLUSIONSmiR-721 is released by Th17 cells in EVs and promotes Th17 responses during myocarditis through repression of PPAR{gamma}, identifying this miRNA as both a mechanistic driver of disease and a potential therapeutic target. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/713340v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1a69953org.highwire.dtl.DTLVardef@9c36bdorg.highwire.dtl.DTLVardef@1cdce4dorg.highwire.dtl.DTLVardef@a34715_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and significanceO_ST_ABSWhat is known?C_ST_ABSO_LImiR-721 and its human homolog are upregulated in the plasma of mice and humans with myocarditis C_LIO_LITh17 cells synthesize miR-721 C_LIO_LIMmu-miR-721 targets Pparg mRNA C_LI What new information does this article contribute?O_LImiR-721 is sorted into extracellular vesicles in the context of acute myocarditis C_LIO_LImiR-721 enhances Th17 differentiation via the Pparg/Rorc double inhibitory axis. C_LIO_LIHsa-RNA-Chr8:96 targets human PPARG mRNA for degradation, inhibiting its expression C_LIO_LIBlockade of miR-721 dampens acute myocarditis development in vivo C_LI This study reveals a novel miRNA-based therapeutic strategy to inhibit Th17 responses and treat myocarditis. Using the experimental autoimmune myocarditis model, the authors unravel the mechanisms by which mmu-miR-721 can enhance Th17 responses and show how targeting this regulatory molecule could ameliorate the progression of the disease. Remarkably, this regulatory axis is suggested to be present in humans as well, since PPARG gene is validated as a target gene for hsa-RNA-Chr8:96. These findings highlights the potential of miR-721 not only as a diagnostic tool but also as a cell-specific therapeutic target to control Th17 responses in the clinical setting.