Immunology

Production of aberrant glycans by cancer cells constitutes a key immunosuppressive strategy to avoid destruction by immune cells. Although sialic acid-containing glycans are known to dampen the activation of lymphocytes, including Natural Killer (NK) cells, the role of fucose-containing glycans remains poorly characterized. In this work, we explored the role of Lewis X (LeX) in cancer cell-NK cell interactions. We induced ectopic expression of FUT9, an 1-3/4-fucosyltransferase, in two colorectal cancer cell lines and showed this enzyme only synthesized LeX structures but not sialyl-LeX. FUT9 introduction was not associated with altered MHC class I surface expression, nor with CD2 (which has been proposed as a receptor for LeX) binding to cancer cells. By inhibiting fucosylation we could demonstrate that CD2 binding was furthermore independent of surface fucosylated glycans in three independent cell lines. Lastly, FUT9/LeX had a limited role in cancer cell destruction and expression of activation markers by NK cells. Overall, our study suggests that, unlike sialylated glycans, 1-3/4-fucosylated glycans have limited impact on cancer cell evasion of NK cell-mediated destruction.

The human host defence peptide LL-37 mediates pleiotropic immunomodulatory functions that can be both pro- and anti-inflammatory. Under inflammatory conditions in the lungs, LL-37 is susceptible to the post-translational modification (PTM) citrullination. The impact of this PTM on LL-37's immunomodulatory functions is not fully understood. Therefore, we characterized the transcriptome and cytokine profile in response to LL-37 and citrullinated-LL-37 (citLL-37), in human bronchial epithelial cells (HBEC). Cells were stimulated with either LL-37 or citLL-37 (0.5 M), or a scrambled peptide (sLL-37). RNA after 4 hours (h) was analyzed with NanoString nCounter Host Response Panel, and 96 cytokines were examined (after 24 h) in tissue culture supernatants (Luminex platform). Genes and cytokines with a Log2 fold-change [&ge;]0.5 with p<0.05 compared to unstimulated cells were considered differentially expressed (DE). These studies revealed overlapping and distinct biosignatures. There were 118 DE genes with LL-37 and 71 with citLL-37, of which magnitude of change was significantly different for 32 genes. 16 out of 17 DE genes altered by both peptides were directly related to inflammation, and these were significantly less (by ~45%) with citLL-37 compared to LL-37. Similarly, inflammatory cytokines enhanced in response to LL-37 were significantly less (by 37-94%) with citLL-37. Overall, our findings indicate that citrullination of LL-37 does not abrogate cellular response in HBEC, instead dampens the inflammatory biosignature induced by LL-37.

Background: Patients receiving anti-TNF treatment for chronic inflammatory disease display impaired antibody responses, but it remains unclear how immune memory formation is affected. We evaluated antibody responses and memory B cells (Bmem) after COVID-19 booster vaccination in inflammatory bowel disease (IBD) patients receiving anti-TNF treatment. Methodology: Blood was sampled at baseline, 1, and 6 months after WH1/BA.5 bivalent or XBB.1.5 monovalent vaccination from 27 IBD patients receiving intravenous anti-TNF and 44 controls. Neutralizing antibodies were measured using an infectious virus assay. SARS-CoV-2 spike receptor binding domain (RBD)-specific serum IgG was quantified by ELISA, and RBD-specific Bmem were immunophenotyped by flow cytometry using recombinant proteins from ancestral, Omicron BA.1, BA.5, XBB.1.5, and JN.1 variants. Results: Serum IgG to vaccine RBD and neutralizing antibodies in patients increased pre to 1 month post-vaccination, but were lower than controls. Ancestral-, BA.5- and XBB.1.5-specific Bmem increased after vaccination but were significantly lower in patients than controls. Within RBD-specific Bmem, frequencies of recently activated CD21lo cells were increased after vaccination, and were higher in patients than controls. Fewer antigen-specific Bmem in patients expressed IgG4, and more expressed IgG3 or IgD following vaccination. Following vaccination, more RBD-specific Bmem recognized multiple viral variants. However, patients had fewer Bmem that could bind to subvariants than controls. Conclusion: Antibody and Bmem responses to COVID-19 booster vaccination in anti-TNF-treated IBD patients displayed reduced capacity, durability and cross-reactivity, suggesting impaired immune memory for protection against breakthrough infection. This supports the recommendation for annual booster vaccination to prevent severe disease and viral spread.

Background: Primary immune regulatory disorders (PIRDs) represent a rapidly growing group of inborn errors of immunity (IEIs) characterized by infection susceptibility, autoimmunity, inflammation, and lymphoproliferation. However, the core features of the gene-disease relationships underlying these conditions are often obscured by limitations in available published data. The Clinical Genome Resource (ClinGen) is an international collaborative effort that seeks to address this challenge for PIRDs and other monogenic diseases through a standardized framework for classifying the strength of gene-disease relationships. Objective: We sought to systematically evaluate the evidence for proposed PIRD gene-disease relationships and identify conserved phenotypic features across this heterogeneous group of disorders. Methods: Using the standardized ClinGen framework, the ClinGen PIRD Gene Curation Expert Panel (GCEP) identified potential gene-disease relationships for monogenic conditions characterized predominantly by immune dysregulation. We subsequently curated evidence relevant to these relationships, classified the strength of evidence for these gene-disease relationships, and evaluated clinical patterns among these conditions through standardized phenotyping using the Human Phenotype Ontology (HPO). Results: As of April 2026, the PIRD-GCEP has curated a total of 46 genes corresponding to 49 gene-disease relationships characterized by immune dysregulation. Of these, 28 were categorized as definitive, 2 as strong, 9 as moderate, 7 as limited, and 3 as disputed. Analysis of HPO-based phenotyping revealed 3 major phenotypic clusters corresponding to lymphoproliferation and systemic inflammation, atopic and gastrointestinal inflammation, and combined immune deficiency with autoimmunity. Conclusion: The PIRD-GCEP framework provides validated gene-disease classifications and identifies three distinct phenotypic clusters, facilitating improved diagnosis while revealing genes requiring further investigation to confirm their role in immune regulatory disorders.

Abstract Objective Fungal infections can represent opportunistic infections leading to the diagnosis of immunocompromise. Specific inborn errors of immunity are associated with aspergillus but it is unclear how sensitive aspergillus is for the subsequent diagnosis of those inborn errors of immunity. Methods The patient registry USIDNET was utilized to extract clinical conditions and laboratory values from patients with candida and aspergillus diagnosis codes. We restricted the diagnoses to deep infections and then identified the associated clinical conditions along with potential settings, such as bone marrow transplant. Results We identified 41 patients with aspergillus as a diagnosis code. The types of diagnoses associated with aspergillus included T cell disorders, antibody disorders, as well as the anticipated myeloid disorders. Bone marrow transplant was identified as a potential predisposing condition. 64. Patients were identified with candida as a diagnosis code. Similarly, T cell disorders, antibody disorders, and myeloid disorders were found. Conclusions Candida and aspergillus are generally considered sentinel infections for myeloid disorders and T cell disorders. However, we found these infections associated with a broad range of inborn errors of immunity. This suggests that diagnostic testing should be broadened when facing patients with these infections.

Extravillous trophoblasts (EVTs) are a trophoblast subpopulation critical for feto-maternal tolerance during early pregnancy, primarily using HLA-G to exert immunomodulatory effect, and possessing N-glycomic profiles distinct from other trophoblast subpopulations. However, whether the N-glycosylation confers distinct immunological properties to EVTs remains poorly understood. To investigate this, we employed JEG-3, a human choriocarcinoma cell line having the capacity to produce pregnancy-related hormones and expressing both HLA-C and HLA-G resembling placental EVTs, as an in vitro EVT model, alongside cell line JAR which exhibits villous trophoblast phenotypes distinct from JEG-3. Both cell lines were treated with kifunensine or swainsonine, inhibitors of -mannosidases, to remodel their N-glycosylation patterns. This led to significant remodelling of their N-glycomic profiles, with JEG-3 cells showing an increased level of polylactosamine chains and decreased levels of -2,6-sialylation and core -1,6-fucosylation. Western blot analysis showed that inhibiting -mannosidases altered only the composition of N-glycans on cell-surface HLA-G, without affecting the overall abundance of cell-surface HLA-G. In kifunensine-treated JEG-3 cells that predominantly express oligomannose type N-glycans, an intracellular accumulation of unfolded HLA-G fragments, increased hCG secretion, and down-regulations of EVT markers GATA3 and KRT7 were observed compared to untreated control, while swainsonine treatment did not impact N-glycan expression. Cytotoxicity assays using NK-92 as effector cells showed that the de-sialylation of JEG-3 by neuraminidase treatment led to increased NK-92 mediated killing. JEG-3 cell sustained its EVT immunological properties through generating bioequivalent N-glycans, exemplified by NK-92 cells pre-conditioned with used culture media of kifunensine-treated JEG-3 cells displaying reduced cytotoxicity toward NK-sensitive lymphoblast cell line K562, an effect not observed with swainsonine-treated JEG-3 cells. This model suggests that EVTs immunological properties are dependent on specific N-glycomic profiles that are maintained by unique N-glycosylation homeostasis, and overall improves our understanding of how EVTs maintain their immunomodulatory effect at the maternal-fetal interface.

Introduction: Mast cells are central effectors of IgE-mediated allergic responses and reside in barrier tissues, where they encounter diverse environmental compounds. How naturally occurring bioactive compounds influence mast cell function under resting versus allergic conditions remains poorly defined. Methods: We compared the effects of cholera toxin, aconitine, and sennoside A on viability, degranulation, and cytokine production in murine bone marrow-derived mast cells. We then examined the acute physiological effects of oral sennoside A in an ovalbumin-induced mouse model of food allergy. Results: None of the compounds reduced mast cell viability at the concentrations tested. Sennoside A, but not cholera toxin or aconitine, induced modest degranulation in resting mast cells without increasing TNF- or IL-6 secretion. In contrast, sennoside A markedly suppressed IgE-dependent antigen-induced degranulation, TNF- and IL-6 secretion, and Il1b expression. Acute oral sennoside A induced dose-dependent diarrhea and hypothermia and increased colonic Tnfa, Il1b, Il33, and Hdc expression. In allergic mice, sennoside A attenuated allergen-induced hypothermia, reduced duodenal Tpsab1 expression, and decreased circulating total IgE, OVA-specific IgE, and OVA-specific IgG1, while increasing Il5 expression. Conclusion: Sennoside A differentially regulates mast cell responses according to activation state and attenuates features of experimental food allergy. These findings identify a previously unrecognized immunomodulatory effect of sennoside A on IgE-driven mast cell activation. However, the mechanisms and the contribution of mast cells to the in vivo phenotype remain to be defined.

Genetic ablation approaches are routinely used to establish gene function, but these are not beyond limitations. The intracellularly expressed camelid single-domain antibodies (sdAbs), or nanobodies, can specifically disrupt molecular interactions and, therefore, help ascertain their role in the process of cellular differentiation. The approach has not been evaluated in differentiating T cells or the stimulating DCs during viral infection. We modified the intracellular response of Galectin-3 (Gal-3) using an -Gal-3 intrabody (IB) in antigen-specific CD8+ T cells and assessed their differentiation patterns during a resolving Influenza A virus encoding SIINFEKL epitope (IAV: WSN-SIINFEKL). The -Gal-3 IB expressors, compared to the cells genetically depleted of Gal-3, exhibit a significantly prolonged activation, enhanced proliferation, and elevated cytokine production following in vitro stimulation. The -Gal-3 IB expressing cells show enhanced functionality and generate an efficient effector response, favoring the generation of memory precursor effector cells (MPECs: CD127hiKLRG1lo) over short-lived effector cells (SLECs: CD127loKLRG1hi) in IAV-infected animals. Such a differentiation pattern results in improved differentiation of effector memory (CD44hiCD62LloCCR7lo) cells amongst the -Gal-3 IB-expressing cells. The generated memory cells are efficiently recalled upon challenge infection with MHV68-SIINFEKL and help control the infection better. Therefore, modifying the intracellular response of Gal-3 using a sdAb could improve the primary and memory response of antigen-specific CD8+ T cells.

Unconventional T cells recognize non-peptide antigens presented by molecules other than the major histocompatibility complex (MHC) proteins. Among unconventional T cells, natural killer T (NKT) cells, which recognize CD1d-lipid complexes, mucosal-associated invariant T (MAIT) cells, which recognize MR1-metabolite complexes, and {gamma}{delta} T cells, are thoroughly studied. CD1b presents self- and mycobacterial lipids to relatively understudied T cell subsets. Like MAIT cells and type I NKT cells, CD1b-specific cells include subpopulations with conserved TCRs. Consequent to their recognition of a nearly monomorphic antigen-presenting molecule, CD1b-specific T cells might share innate-like features with MAIT, type I NKT, and {gamma}{delta} T cells. Due to their low frequency in the peripheral blood, CD1a-, CD1b-, and CD1c-specific T cells have been studied predominantly as in vitro-expanded clones, so even basic information about their native ex vivo immunophenotypes is lacking. Here, we sort and transcriptionally profile ex vivo two T cell populations that recognize CD1b presenting mycobacterial mycolipids and compare them with conventional CD4 and CD8 T cells, {gamma}{delta} T cells, NK cells, MAIT cells, and NKT cells. We show that both the invariant TCR-expressing CD4+, CD1b-GMM-specific germline encoded mycolyl-reactive (GEM) T cells, as well as the diverse TCR-expressing CD1b-GMM-specific T cells, are transcriptionally closer to conventional T cells than to the innate-like T cell populations {gamma}{delta}, MAIT and type I NKT cells. Thus, despite their recognition of non-polymorphic antigen presenting molecules, CD1b-specific T cells show adaptive rather than innate-like transcriptional features.

Glycosylphosphatidylinositol (GPI) anchors are involved in the organization of membrane microdomains that support T cell receptor (TCR) signaling. However, their role in regulating expression of TCR-related proteins and downstream functional output remains unclear. This study aimed to characterize the effects of GPI-deficiency on TCR, cluster of differentiation 3 (CD3), and CD28 expression as well as interleukin-2 (IL-2) production using a GPI-deficient Jurkat T cell line (S12). Flow cytometry confirmed the complete loss of GPI anchors and GPI-anchored proteins (GPI-APs) in the S12 cell line. Compared to GPI-producing parental Jurkat, S12 had significantly higher expression of CD3 and TCR{beta} while CD28 had similar expression. IL-2 production by S12 was assessed following stimulation with anti-CD3/anti-CD28 beads and following stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Neither S12 nor parental Jurkat produced detectable IL-2 in response to anti-CD3/anti-CD28 bead-mediated stimulation. Both parental Jurkat and S12 produced IL-2 following PMA/ionomycin-mediated stimulation. No significant difference in IL-2 production was observed between S12 and parental Jurkat following PMA/ionomycin-mediated stimulation. These findings demonstrate that GPI-deficiency influences surface receptor expression but does not significantly impair downstream IL-2 production under PMA/ionomycin stimulation. This finding suggests that GPI anchors and GPI-APs contribute to proximal signaling organization but are not required for cytokine production when downstream pathways are directly activated.

Glycosylation of proteins is arguably the most diverse post-translational modification that is altered in almost all cancer types, which has been demonstrated to play a crucial role in creating an immunosuppressive microenvironment that promotes immune tolerance and evasion. However, the biosynthesis of N-linked glycan is mediated by a series of enzymatic reactions catalysed by glycosyltransferases and glycosidases in a template-independent manner, hindering our understanding of specific structure-function relationships and roles of specific glycans on specific proteins. Here we use HLA class I-negative cell line K562, a known reference target for NK-mediated cytolysis, to establish a model investigating how cell surface glycan dynamics influence its susceptibility to cytolysis mediated by NK-92 cells. Treatment of K562 cells with kifunensine, swainsonine, 2F-peracetyl-fucose, or 3Fax-peracetyl Neu5Ac, inhibitors of N-linked glycan processing, resulted in drastic alterations in cell surface carbohydrate phenotype, as could be shown by flow cytometric analysis of the lectiNbinding properties of the cells. Despite these clear changes in carbohydrate phenotype, only K562 cells treated with either kifunensine or 3Fax-peracetyl Neu5Ac exhibited higher susceptibility to the cytolysis medidated by NK-92 cells accompanied with an increased CD107a expression by NK-92 cells. Although K562 cells overexpressing gene MGAT3 exhibited a decreased NK-susceptibility, we further found that this decrease was not exclusively determined by the overexpression of gene MGAT3 product bisecting {beta}1,4-GlcNAc, because the treatment of 3Fax-peracetyl Neu5Ac reversed the resistance of K562 cell against NK-92 cell in despite of expressing higher levels of bisecting {beta}1,4-GlcNAc. Expressing HLA-G on cell surface as extravillous trophoblast did not change the NK-susceptibility of K562 cells, despite evidence that HLA-G molecules expressed by K562 cells can bind to inhibitory receptor ILT2 expressed on NK-92 cell surface. These findings suggest that the level of terminal sialylation, outweighing other components in N-linked glycan, determines the NK-susceptibility of K562 cell, offering a new strategy to weaken the resistance of cancer cells so that the immune system can maximise the elimination.

BackgroundPremature ovarian insufficiency (POI) is a condition affecting female fertility e.g., in the context of autoimmune disease. A recent proof-of-concept study showed that B-cell depletion with rituximab (RTX) temporarily restored ovarian function in patients with autoimmune POI. However, the underlying immunological mechanisms behind this response is unknown. ObjectivesTo characterize B- and T-cell phenotypes before and after RTX therapy in the above-mentioned patients and associations with autoantibody levels and treatment response. MethodsCryopreserved cell samples were available from six autoimmune patients with POI, four of whom were positive for autoantibodies against steroidogenic enzymes (21OH, 17OH and/or SCC). Patients received RTX and blood samples were collected at baseline, second infusion (2 weeks), 3, 8 and 12 months. Peripheral B- and T-cell phenotypes were analysed using spectral flow cytometry and correlated with autoantibody levels. ResultsAt baseline, patients displayed signs of B-cell activation by alteration in subset composition compared to matched controls. Upon closer analysis, patients positive for steroidogenic enzyme autoantibodies had significantly increased frequency of CXCR3+switched memory (SWM) B-cells than autoantibody-negative patients, which correlated positively with anti-17OH and-SCC autoantibody levels. In parallel, increased Th1 cell frequencies were also observed in the same patient group. Longitudinal analyses showed expected B-cell depletion followed by repopulation after RTX. Changes in T-cell subsets were observed at 8 months with reduced frequencies of Th1 and Tfh cells. ConclusionAutoimmune POI displayed altered B- and T-cell subsets implicating cell crosstalk, immune cell infiltration to sites of inflammation such as ovaries, and presence of Th1-driven immunopathogenesis. Key messagesO_LIIncreased CXCR3+SWM B cells and Th1 cells were found in patients with autoimmune POI who were positive for autoantibodies against steroidogenic enzymes. C_LIO_LICXCR3 expression on SWM B cells positively associated with anti-17OH and anti-SCC autoantibody levels. C_LIO_LIRTX induced rapid B-cell depletion followed by repopulation and a delayed T-cell effect at 8 months with reduced Th1 and Tfh cell frequencies. C_LI Capsule summaryRTX has recently been shown to restore fertility in autoimmune POI. Lymphocyte profiling identified Th1 bias accompanied by CXCR3+memory B cells that correlate with autoantibody levels. B-cell depletion appears to temporarily alleviate ovarian autoimmunity and ameliorate physiological function.

The sialyl-Tn (STn) glycan antigen is aberrantly expressed in a subset of triple-negative breast cancer (TNBC) and is associated with poor prognosis and immunosuppressive microenvironment. Tumour-derived extracellular vesicles (TDEVs) are emerging regulators of immune escape however the role of glycan-mediated mechanisms remains elusive. Aberrant glycosylation is a hallmark of cancer that extends to TDEVs, yet how tumour-associated glycans within EV cargo modulate cell function remains poorly understood. Here we used engineered MDA-MB-231 TNBC cells to overexpress the glycosyltransferase ST6GalNAc-I, generating STn-positive cells whose EVs were enriched in STn (STn+ EV). The STn+ EVs impaired the maturation of monocyte-derived dendritic cells (DCs), reduced antigen presentation, and diminished CD4{square} and CD8{square} T cell priming, alongside the expansion of regulatory T cells. DCs co-cultured with STn{square} EVs display STn at their cell surface. Notably, STn+ EVs transferred both STn antigen and the ST6GalNAc-I to recipient DCs. Enzymatic removal of terminal sialic acids from STn{square} EVs reversed the immunosuppressive effects, confirming the STn{square}dependent nature of DC dysfunction. These findings add STn to the extensive list of components of EVs molecular cargo that play a role in immune suppression and may contribute for developing precision medicine approaches in oncology.

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.

Advances in immunophenotyping of tumour-infiltrating lymphocytes (TILs) have improved our understanding of prognostic biomarkers and immune targets in human melanoma. However, whether the tumour-immune landscape in canine oral malignant melanoma (COMM) is concordant with that of human melanoma has not been properly defined. To address this gap, we developed a 16-colour spectral flow cytometry panel to characterise TIL phenotypic and functional profiles in COMM. Validation using mitogen-stimulated peripheral blood mononuclear cells from healthy dogs (n = 5) demonstrated robust identification of major T cell lineages, including regulatory T cells (Tregs) and memory subsets, and reliable evaluation of their activation and exhaustion status. COMM patients (n = 8) displayed a distinct protumour microenvironment, characterised by an increased proportion of Tregs, enrichment of tumour-specific exhausted-like T cells co-expressing programmed cell death protein 1 (PD-1) and tumour necrosis factor receptor 2 (TNFR2), and a reduction in cytotoxic CD8 and natural killer T (NKT) cell populations compared with tissue-resident (n = 4) and circulating (n = 8) lymphocytes. Analysis of additional solid tumours, including a mast cell tumour, nerve sheath tumour and adrenal cortical carcinoma, further supported the capability of the panel to identify similar patterns of immune dysregulation across diverse canine tumour landscapes. Collectively, this work describes the first detailed evaluation of canine TIL immunophenotyping using spectral flow cytometry and provides insights into the immunosuppressive mechanisms shaping the tumour microenvironment in COMM. These findings not only increase our understanding of canine tumour immunology but also identify potential immune targets and support ongoing comparative immuno-oncology efforts.

Background: Although immune checkpoint inhibitors (ICIs) cause grade 3 or higher immune-related adverse events (irAEs), validated biomarkers for identifying patients at risk of these events are lacking. Given the potential contribution of B-cells to irAE development, we evaluated whether early changes in circulating B-cell subsets after nivolumab plus ipilimumab were associated with subsequent severe irAEs. Materials and Methods: This prospective observational study included 12 patients who received nivolumab plus ipilimumab, and an exploratory cohort of five patients who received anti-PD-1 monotherapy. Blood was sampled before treatment, at week 1, and before cycle 2. Flow cytometry quantified CD21low B-cells, CD27+IgD- class-switched B-cells, plasmablasts, and CD19+CD138+ plasma cells. irAEs were assessed for 6 months, and fold changes at week 1 were compared between patients with and without severe irAEs. Paired immunoglobulin G B-cell receptor (BCR) repertoires were analyzed in four patients with grade 3 or higher irAEs. One patient developed myasthenia gravis, and anti-acetylcholine receptor (AChR) antibody was measured in serial serum samples. Results: In the nivolumab plus ipilimumab patients, grade 3 or higher irAEs occurred in five patients. Week-1 increases in all four activated B-cell subsets were significantly greater in these patients, whereas total CD19+ B-cell changes did not differ. Rarefied IgG BCR diversity generally increased in repertoires derived from peripheral blood mononuclear cells, while Gini coefficients decreased in all paired repertoires. In the myasthenia gravis case, anti-AChR antibody exceeded the reference range by Day 11, before symptoms and diagnosis. Conclusion: First-week expansion of activated peripheral B-cell subsets after nivolumab plus ipilimumab was associated with subsequent severe irAEs. These findings support future studies of early B-cell monitoring within integrated multimarker assessments of irAE risk.

Background: Approximately 30% of the North American population are affected by seasonal or perennial rhinitis. Allergens from cats are common triggers of allergic rhinitis. Importantly, cat allergic rhinitis (cat-AR) is a major risk factor for more severe allergic diseases, including asthma. Current therapies rarely achieve full symptom control and development of novel therapeutics is constrained by the lack of physiologically relevant preclinical models. Objective: Establishment of a clinically-informed mouse model of cat allergen exposure in humans. Methods: Controlled human exposure was performed using airborne natural cat dander (CD) in a validated environmental exposure facility. Nasal immune cells and IgE concentrations in serum and nasal samples were assessed. In mice, we established a 14-day intermittent intranasal sensitization and challenge protocol using the same CD allergens. Clinical signs in mice were objectively quantified using AI-based video detection. Cellular changes in the nose were assessed with level-specific, spatial resolution. Lastly, systemic sensitization was confirmed through serum IgE and intradermal ear challenge. Measurements and Main Results: Following CD exposure, cat-allergic participants displayed increased nasal symptoms and eosinophil influx. CD-sensitized mice recapitulated key clinical features, including increased nasal rubbing, elevated nasal immune cell infiltration, and systemic sensitization. Importantly, spatial investigation of the cellular changes revealed major changes in the turbinate and olfactory regions of the nose. Conclusions: This novel mouse model provides a translationally valuable platform for investigating local and systemic mechanisms of cat-AR. It will serve for mechanistic and therapeutic evaluations with the goal of improving clinical translation and therapeutic precision.

Background. Obesity has been associated with higher risk of dengue virus (DENV) infection and disease, yet its influence on antibody responses to DENV remains undefined. Methods. We evaluated whether nutritional status -- based on BMI z-score (BMIz) -- or blood markers of body fat -- leptin and adiponectin --are associated with binding and/or neutralizing antibody responses to DENV in 85 children in the Nicaraguan Pediatric Dengue Cohort Study who experienced a primary DENV infection in 2019. Associations were estimated using linear models adjusting for age, sex, and DENV infection outcome. Results. Compared to children with normal weight, those with obesity had higher quantities of DENV binding antibodies (fold-change [FC] 1.89, 95% confidence interval [CI] 1.02, 3.48) but no difference in neutralizing antibodies. Likewise, leptin concentration was associated with higher quantities of binding antibodies (FC 1.22, 95%CI 1.09, 1.37), while adiponectin was associated with lower quantities (FC 0.79, 95%CI 0.67, 0.94), and neither was associated with neutralizing antibodies. Lower neutralizing efficiency (neutralizing/binding antibodies) was observed in children with obesity (FC 0.67, 95%CI 0.48, 0.93). Conclusions. Our results indicate that obesity is associated with higher antibody quantity (binding) but not higher quality (neutralization) post-primary DENV infection -- implying that antibodies generated by children with obesity have lower neutralization efficiency, requiring greater quantities to reach similar levels of neutralization than children with normal weight. Further, the agreement among the three models using distinct proxies of body fat -- BMIz, leptin, and adiponectin -- demonstrates that adipokines are useful in supplementing BMIz analysis or as independent predictors of immune responses.

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

Staphylococcus aureus persistently colonises the nasal tissue (NT) of a significant proportion of the population. The long-lasting impact that asymptomatic S. aureus exposure has on immune memory at colonised barrier sites is incompletely understood, potentially impacting vaccine responsiveness in a pre-exposed population. Tissue resident memory (TRM) cells are long-lived T-cells which remain poised at barrier sites for localised reactivation following antigen exposure. This study demonstrates an increase in NT CD4+ and {gamma}{delta}+ TRM cells in response to S. aureus colonisation, which undergo expansion and IL-17 production upon secondary S. aureus exposure. Interestingly, these cells were also capable of non-specific reactivation, with IL-17+ TRM cells in S. aureus colonised mice enhancing protection against K. pneumoniae infection. Ex-vivo data suggest that non-specific CD4+ TRM cell re-activation is pro-inflammatory cytokine dependent, but antigen independent. Overall, these findings demonstrate that S. aureus nasal colonisation shapes long-lasting TRM cell responses in the NT, which have the potential for non-specific bystander reactivation during subsequent heterologous infection.