Breast cancer is the most common malignancy among women worldwide, causing >650,000 deaths annually. Despite advances in personalized therapies, many patients fail to obtain durable benefits from treatments tailored to their disease subtype. For instance, only some patients with PD-L1⁺ triple-negative breast cancer (TNBC) respond to chemotherapy plus immune checkpoint inhibitors. Moreover, while tumor-infiltrating lymphocyte levels correlate with improved survival in TNBC and HER2⁺ breast cancer, this is not true for HR⁺HER2- disease. Thus, other immunobiological features of the breast cancer microenvironment may hold clinically relevant prognostic or predictive value, especially in HR+HER2- tumors. Here, we investigated transcriptional signatures of immune cell infiltration across three public transcriptomic datasets from patients with breast cancer. The relative abundance and activation status of tumor-infiltrating mast cells were consistently associated with shifts in survival indicators amongst patients with HR+HER2- breast cancer. Specifically, infiltration by resting (but not activated) mast cells was associated with prolonged survival, correlating inversely with tumor infiltration by immune cells and proliferation markers in cancer cells, but positively with stromal richness. While based on retrospective transcriptional analyses, these findings suggest that interactions between mast cells and non-malignant components of the breast cancer microenvironment, particularly fibroblasts, may shape disease progression and treatment sensitivity.
Tumor-infiltrating lymphocytes (TILs) shape melanoma behavior and response to immunotherapy, but the links between immune regulation, TIL patterning, and outcomes remain unclear. We retrospectively studied 32 primary melanoma samples from patients treated with anti-PD-1 therapy. Histopathologic TILs were classified as brisk or non-brisk, and primary tumors underwent targeted immune transcriptomic profiling (NanoString nCounter Human Immunology panel). External validation was performed with 96 TCGA-SKCM primary tumors. Brisk tumors displayed broad upregulation of immune transcripts, with enrichment of adhesion pathways (directed global significance scores - DGSS = 2.15) and MHC class II antigen presentation (DGSS = 2.128). Cross-cohort comparison identified 22 shared differentially expressed genes, with ZAP70 remaining significant in both datasets. Brisk tumors also showed higher total TIL (p = 0.036), cytotoxic-cell (p = 0.0095), and Th1 (p = 0.027) scores. Response-associated genes differed by TIL pattern, and subgroup-specific gene scores predicted anti-PD-1 benefit in brisk (4-gene, Area under curve - AUC = 1.000) and non-brisk (3-gene, AUC = 0.852) tumors; the non-brisk score remained independently associated with response (p = 0.029). Higher scores were also associated with prolonged survival. Integrating histopathological TIL patterning with immune transcriptomics may refine prognostication and support immunotherapy stratification in melanoma.
Interleukin-33 (IL-33) is a central regulator of immune responses and inflammation, and genetic variation in IL33 and its receptor IL1RL1 (ST2) is strongly linked to disease susceptibility, notably asthma. Emerging evidence suggests that IL-33 also influences hematopoietic processes and platelet biology, indicating functions beyond canonical immunity. We investigated two missense variants identified in a patient with unexplained thrombocytopenia: a rare novel IL33 variant, c.385T>C (p.Tyr129His) and the common IL1RL1 variant c.1501_1502CA>AG (p.Gln501Arg), previously associated with reduced IL-33 signaling. Structural modeling revealed that IL-33 Y129H disrupts a conserved hydrogen bond within the IL-33/ST2/IL-1RAcP ternary complex, destabilizing receptor engagement. Functional assays confirmed markedly reduced binding affinity and biological activity, establishing Y129H as a loss-of-function variant. The IL1RL1 Q501R variant affects the Toll/IL-1 receptor (TIR) domain, critical for recruiting adapter proteins such as MyD88. Modeling revealed pronounced perturbation in a peripheral helix of the TIR domain, potentially impacting adapter recruitment and downstream signaling, providing a mechanistic basis for its protective association with asthma. Together, these findings provide structural and functional insights into clinically relevant IL33 and IL1RL1 variants. Their simultaneous occurrence in a patient with thrombocytopenia further supports a potential role for this pathway in platelet homeostasis and stress-responsive hematopoiesis.
Brain-Derived Neurotrophic Factor (BDNF), highly enriched in platelets, contributes to vascular integrity and thrombotic responses. The rs11030119 variant, located in a BDNF intronic enhancer, has been linked to stroke recovery, but its influence on peripheral BDNF dynamics and hemostasis remains unexplored. In this study, we aimed to understand the impact of rs11030119 on circulating levels of BDNF, and its subsequent effects on thrombus formation. Twenty-six healthy individuals homozygous for either the GG or AA genotype of rs11030119 were matched for age and sex. BDNF and proBDNF levels were quantified in serum, plasma, and platelets. Platelet aggregation, ATP secretion, thrombin generation, and clot firmness were assessed. Washed platelets were also tested with recombinant BDNF. AA carriers displayed significantly lower plasma and serum BDNF levels and diminished BDNF release upon activation, despite comparable platelet BDNF content. The expression of its receptor, TrkB, on the platelet surface was also reduced in AA carriers. However, platelet reactivity, thrombin generation, and viscoelastic clot properties were preserved across genotypes. In conclusion, rs11030119 modulates circulating and platelet-releasable BDNF without impairing hemostatic function, suggesting that BDNF bioavailability may not directly correlate with thrombotic potential. These findings uncover a novel genetic mechanism controlling platelet-derived neurotrophin output.
Cyclic neutropenia (CyN) is a rare haematological disorder most often caused by mutations in the neutrophil elastase gene (ELANE). We describe three novel ELANE mutations in CyN patients and link them to impaired neutrophil extracellular trap (NET) formation. ELANE sequencing was performed using the Sanger method, and AlphaFold2 was used to visualize the impact of mutations on the structure of neutrophil elastase (NE). NET formation was assessed by colocalization of MPO, NE, and DNA using fluorescence microscopy, and free DNA was quantified fluorometrically. We identified two missense substitutions (M66K, V133G) and one deletion (G192-G196del). These mutations did not significantly alter the overall NE structure but exhibited functional effects: M66K may disrupt substrate binding, V133G increases protein mobility, and the Deletion of G192-G196 may limit the opening of the catalytic pocket. NETosis was impaired in CyN patients compared with controls. Their NETs appeared less expanded, and DNA release into supernatants was reduced, reaching the lowest levels during neutropenia, even in patients with normal neutrophil counts. These abnormalities were accompanied by reduced NE activity. In sum, novel ELANE variants may contribute to altered NE function and defective NET formation in CyN, suggesting that impaired NETosis is partly independent of cyclic changes in neutrophil numbers.
Patients with Down Syndrome (DS) are characterized by dysfunction of several organs, including the liver, brain, heart defects, gastrointestinal anomalies, and lethal immune hypersensitivity. A person with DS is also susceptible to various inflammatory diseases, including hepatic autoimmune diseases. The Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is known to trigger the stimulator of interferon genes (STING) and downstream proinflammatory factors. In this work, we hypothesized that oxidative stress-associated DNA damage triggers activation of the cGAS-STING signaling pathway and promotes liver inflammation in DS. Here, we investigated the role of reactive oxygen species (ROS) associated DNA damage and the cGAS-STING signaling pathway in the pathogenesis of hepatic inflammation in the DS model. Our results showed that DS cells harbor excessive ROS and DNA damage in DS fibroblasts and DS mouse liver. Further, DS cells accumulate micronuclei that likely serve as a source of cytoplasmic DNA to stimulate cGAS-STING activation. In addition, RNA-seq analysis results showed enhanced expression of key type I interferon factors in cGAS-STING pathways in DS liver and inflammatory responses and elevated liver enzymes such as alanine transaminase (ALT) that indicate a hepatocellular liver injury in DS. The results of this study opened the opportunity to connect endogenous DNA damage triggers innate immune response, which may contribute to the upregulation of the cGAS-STING signaling to exacerbate hepatic inflammation in DS.
NRF2 modulates tumor immune microenvironment in several cancers. NRF2 is activated in about 50% of high-grade serous ovarian cancer (HGSOC), the most aggressive type of ovarian cancer. Through analyzing data from scRNA-seq (n = 7), bulk RNA-seq (n = 365), and tumor microarray (TMA) of human HGSOC (n = 240) samples, we demonstrated that NRF2 expression correlated with tumor immune microenvironment in HGSOC. Functional pathway enrichment analysis and transcription factors (TFs) prediction showed the functional relevance of NRF2 expression in shaping the immune phenotype of HGSOC. Pathways such as hedgehog and ROS signaling, and TFs including EGR1, ESRRA, SMAD proteins, and SP-family proteins, are implicated in the immune suppressive microenvironment of NRF2High tumors. Immune differentiation analysis showed patients with NRF2High tumors enriched with CD68 have lower survival (p = 0.038) than those with CD68Low tumors, whereas NRF2Low tumors enriched with immune-activated markers such as CD3E and CD80 exhibit a better prognosis. This study is the first that shows classification of HGSOC based on NRF2 levels, highlights new biomarkers, and suggests IHC-labeling and genomic evaluation of NRF2 and immune markers for better prognosis.
Complexity of the microenvironment of lung adenocarcinoma (LUAD) poses significant challenges in its clinical management. Systematic bioinformatic analysis of the Ras-association domain family (RASSF) identified RASSF2 as a potential tumour suppressor in LUAD. This study aims to investigate its clinical significance and functional mechanisms in LUAD. CCK-8, EdU, and colony formation assays were performed to investigate the impact of RASSF2 overexpression on the proliferative capacity of LUAD cells. Transwell and wound healing assays were performed to explore the significance of RASSF2 on LUAD cell migration and invasion. RNA sequencing analysis was conducted on three methylation-positive and three methylation-negative LUAD tissue samples to establish the underlying mechanism of RASSF2 methylation in LUAD. Functional studies indicated that RASSF2 overexpression significantly inhibited the proliferation, migration, and invasion of LUAD cells. Patients with RASSF2 promoter hypermethylation exhibited a significantly shorter overall survival than those without. RNA-seq of three pairs of LUAD tissue samples further demonstrated that RASSF2 methylation is associated with upregulation of the NF-κB signalling pathway and dysregulation of T-cell activation pathways. This study confirmed the tumour-suppressive role of RASSF2 in LUAD and suggests its potential as an immunotherapeutic target, thus providing new insights into LUAD treatment strategies.
Gastric cancer (GC) progression is linked to immune escape in the tumor microenvironment, yet the molecules regulating tumor-associated macrophage polarization and CD8+ T-cell exhaustion are unclear. This study analyzed TCGA data to examine SULF1 expression and its prognostic role. It used CRISPR/Cas9 and lentiviral methods in GC cells to test proliferation, invasion, and apoptosis, plus co-culture and flow cytometry to assess SULF1's impact on macrophages and CD8+ T-cells. STAT3 signaling was studied via immunoblotting and nuclear translocation assays, and a mouse model tested SULF1's therapeutic relevance. Results showed SULF1 was up-regulated in GC, tied to advanced stages and poor survival. SULF1 knockdown inhibited the malignant phenotypes of gastric cancer cells, including proliferation, migration, and invasion abilities, while promoting cell apoptosis; conversely, SULF1 overexpression enhanced these pro-tumor phenotypes. SULF1 activated macrophage STAT3, promoting M2 polarization and CD8+ T-cell dysfunction. In mice, SULF1 silencing reduced tumors and T-cell exhaustion, while supplementation reversed this. Conclusions: GC-secreted SULF1 creates an immunosuppressive microenvironment via STAT3-dependent pathways, and targeting SULF1-STAT3 may improve GC immunity.
Pregnant individuals are at increased risk of severe outcomes from COVID-19 infection, yet the immune responses elicited by vaccination during pregnancy remain poorly understood. In this study, longitudinal single-cell RNA sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells from three pregnant women at four time points: before vaccination, one and two weeks after the first dose of the Moderna mRNA vaccine, and after the second dose postpartum. Dynamic immune changes, including a transient increase in inflammatory classical monocytes (cM_1) after the first dose, marked by elevated expression of IL1B, NLRP3 and NFKB1, as well as moderate activation of interferon-stimulated genes were observed. CD4⁺ T cells exhibited activation and differentiation into memory subsets. By contrast, B cell responses were minimal after the first dose, and enhanced protein synthesis activity emerged only after the second dose, which suggested delayed humoral activation. Although limited by sample size, our findings indicate that vaccination in pregnancy induces a nonexcessive innate immune activation, coordinated T cell responses and attenuated B cell activity that likely reflect pregnancy-driven immunoregulatory adaptations. These findings reveal the immune dynamics following mRNA vaccination during pregnancy and highlight the importance of developing vaccination strategies tailored for pregnant individuals.
Sepsis severity is associated with sustained neutrophilia, yet the underlying heterogeneity remains unclear. By integrating single-cell and bulk RNA-seq of septic peripheral blood, we uncovered five neutrophils clusters. FOLR3+neutrophils were the predominant and terminally differentiated subgroup, which displayed hyper-inflammatory signatures and low HLA expression, especially in non-survivors of sepsis. Cell-chat analysis also showed these neutrophils could promote sepsis progression by recruiting platelet via RETN-CAP1 and NAMPT-ITGB1 axes. External validation found that higher FOLR3+neutrophils were associated with 28-day mortality of sepsis. Transcription-factor and pseudotime analyses identified HIF-1A as the key driver of FOLR3+neutrophils specification. In vitro experiments validated that FOLR3 expression and HIF-1A were also found to be higher in sepsis. Over-expression or knockout of HIF-1A in patient and mouse neutrophils confirmed direct control of HIF-1A on secretion of IL-1β, TNF-α, IL-8 and IL-6. In conclusion, FOLR3+neutrophils contribute to sepsis prognosis by exacerbating hyper-inflammation and FOLR3 may serve as a new promising prognostic biomarker for sepsis.
Myasthenia gravis (MG) is a T/B cell-driven autoimmune disease. The immunomodulatory mechanisms of the common immunosuppressant tacrolimus (TAC) on the immune repertoire are unclear. This study investigated TAC’s immunomodulatory effects via high-throughput sequencing of peripheral blood mononuclear cells from four MG patients pre- and post-four months of TAC monotherapy, revealing dynamic T-cell receptor (TCR) and B-cell receptor (BCR) repertoire remodeling. The immune repertoire of MG patients was characterized by a skewed usage of TRBV gene families compared to healthy controls, indicating an underlying immune dysfunction. Longitudinal analysis post-TAC therapy revealed potential downregulation of IGHV1-69 and IGHV3-43 gene frequencies (p < 0.05, FDR > 0.1), alongside non-significant trends toward shorter CDR3 lengths and reduced clonal diversity in both IGH and TRB (p > 0.05). The BCR repertoire underwent greater dynamic remodeling, while the TCR repertoire remained relatively stable, as evidenced by the persistence of dominant clones and overlapping TRB clones. BCR diversity and V or J gene usage in TCR and BCR showed potential associations with clinical severity. These data reveal skewed antigen recognition profiles in MG pathogenesis, with TAC orchestrating multimodal immunomodulation through peripheral immune repertoire reshaping.
Epitranscriptomic modifications are evolutionarily conserved changes to RNA and are present in all RNA types, including mRNA. Next to methylation, pseudouridine is also a prevalent modification found in mRNA and other RNA types. Pseudouridylation of RNA improves RNA stability, RNA stacking and translation fidelity. While m6A RNA methylation has been demonstrated to regulate the activation of various immune cell types, such as T cells, dendritic cells or NK cells, insight into the role of pseudouridine in the immune system is limited. Trub1 is one of 13 pseudouridine synthases (PUS) and has been suggested to co-install the majority of pseudouridine residues on mRNA alongside the activity of PUS7 and has been reported to install pseudouridine in tRNA. Against this background, we hypothesized that Trub1 may regulate immune cell development, homeostasis or functional differentiation. Using conditional and global Trub1-deficient mice, in vivo competition in mixed bone marrow chimaeras and high-parametric flow cytometry, we interrogated the role of Trub1 in T cells, B cells, NK cells and myeloid cells. Despite our rigorous analyses and the identification of Trub1-dependent pseudouridine residues in mitochondrial tRNAs, we did not identify a significant role for Trub1-mediated pseudouridylation in immune cells at steady state or during disease. We hence reason that the role of Trub1 in mRNA pseudouridylation has previously been overestimated in computational studies or that co-installations of pseudouridine at other positions in the same RNA molecules may compensate for the lack of Trub1-installed modifications.
The ameliorative effects of heparin on acute pancreatitis (AP) have received widespread attention. NLRP3 inflammasome-dependent pyroptosis has been reported to be a key factor contributing to AP development. However, it remains unclear whether heparin alleviates AP by modulating NLRP3 inflammasome-dependent pyroptosis. An AP model was established in vivo and in vitro using Sprague Dawley rats and AR42J cells. TUNEL and propidium iodide staining assessed cell death. Cellular pyroptosis was observed under a microscope. Intermolecular interactions were verified using Co-IP and RIP assays. The m6A level of KAT2B was assessed by MeRIP. Levels of pro-inflammatory factors were determined using ELISA. Our results showed that heparin attenuated AP in vivo by inhibiting NLRP3-mediated pyroptosis. VIRMA expression was increased in AP. Meanwhile, overexpression of VIRMA reversed the alleviating effect of heparin on cellular pyroptosis. KAT2B knockdown counteracted the promotion of pyroptosis by VIRMA. Mechanically, VIRMA promoted KAT2B mRNA stability in an m6A-ELAVL1-dependent manner, whereas KAT2B upregulation further promoted NLRP3 acetylation at K694. The wild-type NLRP3 reversed the inhibitory of heparin on pyroptosis, and KAT2B amplified this regulatory effect. Taken together, heparin prevented cellular pyroptosis by inhibiting the VIRMA-mediated m6A modification of KAT2B, which in turn inhibited NLRP3 acetylation at K694, ultimately attenuating AP.
Radiologically isolated syndrome (RIS) is a neurological condition in people with demyelinating lesions on brain and/or spinal cord magnetic resonance imaging (MRI) studies, but without clinical symptoms of disease. Elucidating the immune profile of people with RIS (pwRIS) who will display MRI or clinical features of advancing disease is critical to our understanding of disease pathogenesis. Our lab previously identified features of B cell dysregulation in people with clinically isolated syndrome (pwCIS), who have both demyelinating lesions and a first clinical event of disease. The goal of this study was to compare features of B cell dysregulation in pwRIS, pwCIS, and healthy controls (HC). The second goal was to determine if these features of B cell dysregulation would be evident in people who meet the new MS diagnostic criteria, particularly in the context of their disease course for 5 years post-sampling. Features of plasmablast responses that distinguish pwRIS from pwCIS include PB expansion, antigen-driven selection, VH4 and JH6 antibody gene over-usage, neuron reactivity by purified IgG and individually cloned antibodies. Furthermore, VH4:JH6 pairing in plasmablasts was higher in people with stable MS who do not show changes in MRI or clinical events from those with advancing disease activity.
Abnormal accumulation of prostaglandin E2 (PGE2) is a secondary effect of the presence of bacterial infection, and usually causes overproduction of proinflammatory cytokines, thereby leading to infiltration of neutrophils, serving as a key etiology of endometritis linking to infertility. Metastasis-associated protein 1 (MTA1), functioning as a master transcriptional coregulator, is increasingly recognized as an important influencer of inflammation, but its functional roles in endometritis, if any, remain unexplored. Herein, by investigating clinical correlations, in vitro assays, and uterus-specific Mta1 knockouts, we show that MTA1 expression was significantly downregulated in endometrial biopsies from patients with chronic endometritis (CE) and in human endometrial stromal cells (HESCs) challenged with clinically relevant pathogenic dosage of lipopolysaccharide (LPS). MTA1 deletion augmented endometrial neutrophil infiltration, exacerbated inflammatory phenotype and negatively affected the outcome of LPS-induced endometritis through modulation of PGE2 secretion. We have also identified cyclooxygenase-2 (COX2) as the key rate-limiting enzyme responsible for the deregulated PGE2 synthesis in MTA1 deficiency-exacerbated endometritis. Mechanistically, MTA1 negatively regulated the transcription of Cox2, likely in collaboration with HDAC2 (histone deacetylase 2), in LPS-challenged endometrial stromal cells (ESCs). In summary, MTA1 signaling may govern appropriate inflammatory response in ESCs against the pathogenesis of endometritis.
Head and neck squamous cell carcinoma (HNSCC) is a major health concern with considerable morbidity and mortality worldwide. Previous studies have applied single-cell sequencing to characterize the tumor microenvironment in HNSCC, providing insights into immune cell composition, stromal interactions, and malignant cell states. However, there remains a gap in analyzing lymph node metastasis (LM) and normal lymph node tissues (LN) by using single-cell RNA sequencing (scRNA-seq) analysis. We performed comparative scRNA-seq analysis on seven lymph node metastatic tissues of HNSCC patients and five non-metastatic tissues. We identified several cell types with significantly altered expression levels, such as CD8+ Tex cells, Macrophages, and Cancer-associated fibroblasts (CAFs). Tumor cells were classified into seven clusters, with cluster2 strongly linked to tumorigenesis and metastasis. Specific subsets such as CD4_Tfh_CXCL13, CD4_Treg_RPL26, CD8_teff_CREM, NK_GZMB, Macrophages_OLFML3 and macrophages_SPP1 cells were also associated with HNSCC progression and metastasis. Based on these findings, we constructed and validated a prognostic model for HNSCC using the expression of INHBA, SFRP2, SPP1, and IFI27. This model provides a tool for risk stratification and informs potential therapeutic strategies for HNSCC.
The prognosis for hepatocellular carcinoma remains grim. Combining radiotherapy with immune checkpoint blockade (ICB) has shown potential to enhance therapeutic outcomes, yet there is a pressing need for further advancements. Our previous research demonstrated that this combined approach suppresses ALKBH5 gene expression and increases m6A modification levels in hepatocellular carcinoma tissues. High-throughput sequencing and detailed molecular analysis revealed that inhibiting ALKBH5 amplifies CIITA m6A modifications post-therapy. This modulation triggers MHC II molecule expression in tumors, facilitating the presentation of tumor-associated antigens to CD4 + T lymphocytes and the recruitment of CD8 + T cells for an anti-tumor immune response. Building on these findings, we engineered a CIITA vector with a specific site mutation to confirm that the regulation of CIITA by the combined radiotherapy and immunotherapy is mediated through m6A methylation. Consequently, we established a comprehensive network involving ALKBH5, CIITA, MHC II, and CD4+ and CD8 + T cells. To elucidate the role and underlying molecular mechanisms of this combined therapy in reshaping the tumor immune microenvironment for hepatocellular carcinoma, we employed multi-omics approaches across in vitro, animal model, and clinical multi-dimensional studies, offering novel insights for enhancing treatment efficacy.
