Interleukins are signaling proteins that control immune responses. Defining the range of human interleukins, their associated receptors, signaling pathways, and functions is thus indispensable for understanding and modulating protective and pathological immunity. Interleukin-39 (IL-39), a heterodimer composed of the subunits IL-23α and EBI3, has been described as a proinflammatory cytokine in mice, but its existence in the human interleukin repertoire has remained controversial. Here, we showed that IL-39 did not readily form from IL-23α and EBI3 in human cells. A combined computer modeling and experimental approach identified structural features that destabilize the human IL-23α:EBI3 heterodimer. Using these insights, we rationally engineered human IL-23α and EBI3 to form disulfide-linked heterodimers that were secreted by cells. This engineered cytokine engaged the receptor subunits IL-23R and gp130 on primary human immune cells to stimulate phosphorylation of the transcriptional regulators STAT1 and STAT3, resulting in proinflammatory cytokine secretion. Together, our findings define limitations in the biogenesis of human IL-39 and show that these can be overcome by molecular engineering. This work highlights the potential of rational cytokine engineering for generating active immune signaling proteins and lays the foundation for further defining possible roles of IL-39 in human immunity.

International swine trade can disseminate influenza A viruses across borders, creating opportunities for reassortment with endemic swine viruses and the emergence of novel strains with zoonotic potential. Here, we characterize a novel H1N2 reassortant, A/swine/Korea/1030-3/2023 (1030-3), detected in South Korea and spatiotemporally associated with European swine lineages and international pig movement. 1030-3 harbors seven gene segments (PB2, PB1, PA, HA, NP, M and NS) closely related to a recent Danish zoonotic H1N1 virus of the 2009 pandemic (pdm09) lineage, and an N2 segment derived from an endemic Korean HxN2. In vitro assays demonstrated that 1030-3 preferentially bound human-type α2,6-linked sialic acid receptors and replicated efficiently in polarized human airway epithelial cells, although these properties were also observed in the endemic Korean H1N2 comparator 1027-1. In mice, 1030-3 caused moderate clinical signs and respiratory replication comparable to an early pdm09 reference strain NL09. In guinea pigs, 1030-3 transmitted to a subset of direct-contact animals but showed no detectable aerosol transmission under the conditions tested. In pigs, the natural host, 1030-3 replicated efficiently and transmitted to all direct-contact animals, with high respiratory shedding and a greater cumulative shedding burden in contact pigs than NL09. Contemporary human sera showed reduced neutralizing activity against 1030-3 relative to both human seasonal H1N1 and 1027-1, while antiserum against a recent H1N1 vaccine antigen also neutralized 1030-3 less efficiently. These findings identify a European-associated H1N2 swine influenza reassortant detected in South Korea that combines a distinct pdm09-derived genomic and antigenic background with efficient replication and transmission in pigs, α2,6-linked receptor preference, and reduced neutralization by human sera, underscoring the need for trade-aware surveillance of swIAVs and continued monitoring at the swine-human interface.

Anti-interferon-γ autoantibody (AIGA)-associated adult-onset immunodeficiency is an important underlying condition in patients with severe talaromycosis caused by Talaromyces marneffei, but the mechanisms underlying impaired antifungal immunity remain incompletely understood. This study aimed to define the contributions of CD4⁺ T cells, CD8⁺ T cells, and IFN-γ-dependent immune signaling to host defense against T. marneffei. We used an intranasal BALB/c mouse infection model with antibody-mediated CD4⁺ T-cell depletion, CD8⁺ T-cell depletion, or IFN-γ neutralization, together with immunological assays using sera from AIGA-positive patients and healthy controls. In mice, T. marneffei infection induced stronger and more sustained T-cell activation in the lungs than in the spleen, with pulmonary CD4⁺ T-cell activation representing the predominant response and CD8⁺ T cells showing distinct temporal kinetics. Depletion of CD4⁺ or CD8⁺ T cells, or blockade of IFN-γ, impaired body-weight recovery and reduced survival, aggravated pulmonary and splenic injury, and delayed fungal clearance. In clinical immunological assays involving 28 AIGA-positive patients and 30 healthy controls, depletion of IgG from AIGA-positive sera restored IFN-γ⁺TNF-α⁺ CD4⁺ and CD8⁺ T-cell responses toward healthy-control levels, suggesting that patient IgG suppresses T-cell cytokine responses. AIGA-positive sera contained IgG that bound IFN-γ and attenuated IFN-γ-induced STAT1 phosphorylation and HLA-DR upregulation in THP-1 cells, supporting functional neutralization of IFN-γ signaling and impaired antigen-presenting cell activation. Together, these findings indicate that AIGA-associated talaromycosis involves impaired IFN-γ-dependent coordination of innate effector functions and T-cell responses. Clinically, they support consideration of AIGA-associated adult-onset immunodeficiency in HIV-negative patients with severe or disseminated talaromycosis, particularly in endemic regions, and provide a rationale for future studies aimed at restoring IFN-γ bioactivity.

The bacterium Chlamydia trachomatis causes three distinct conditions - urogenital chlamydia, the eye disease trachoma, and lymphogranuloma venereum. Use of serological testing for exposure to C. trachomatis for evaluating ocular disease risk factors and population-based surveillance has expanded over the past decade. We developed chimeric monoclonal antibodies to the C. trachomatis antigen Pgp3 as standardized reagents for Pgp3 serological assays. Here we use two anti-Pgp3 monoclonals (1G1 and 1C3) to conduct an interlaboratory comparison of Pgp3 Luminex-based multiplex bead serological assays in 4 different laboratories (each with their own protocol) with 5 operators using 3 different instrument types. Antibodies spotted onto filter paper were stable for two weeks at room temperature. Accuracy and precision were evaluated at 3 concentrations for each monoclonal (high, medium, and low). Accuracy within 20% was achieved for 11/21 conditions for 1G1 and 18/21 conditions for 1C3; the high concentration resulted in the poorest accuracy. Precision was within 10% for all low and medium concentrations and was above 10% for several conditions at the high concentration. The fluorescence values differed between instruments but showed no difference when extrapolated to concentrations using a standard curve. These data suggest that 1C3 may be the more reliable standard reagent and that the Pgp3 multiplex assays are highly robust when comparing different instruments and assay protocols.

BackgroundJNJ-73763989 is a 2:1 combination of 2 N-acetylgalactosamine (GalNAc)-conjugated short interfering RNAs (siRNA) targeting the S (daplusiran) and X (tomligisiran) regions of the hepatitis B virus genome. In phase 2b studies, 48 weeks of JNJ-73763989-based combination treatments led to dose-dependent reductions in viral markers, including hepatitis B surface antigen. The objective of the INSIGHT study Panel 3 was to quantify intrahepatic concentrations of the siRNAs in human livers during and after treatment.MethodsIn the open-label, single-arm, single-center study, participants with chronic hepatitis B received 48 weeks of treatment with JNJ-73763989 200 mg every 4 weeks and nucleos(t)ide analog, with optional pegylated interferon α2a 180 µg weekly added after Treatment Week (TW) 40. Core liver biopsies were collected at TW12, TW40, and at Follow-up Week (FUW) 24.ResultsLiver biopsies were available from all 4 participants at TW12 and from 3 at TW40 and FUW24. Between TW12 and TW40, liver concentrations of daplusiran and tomligisiran increased 1.5- to 2.6-fold, consistent with intrahepatic accumulation. Twenty-eight weeks after the last injection of JNJ-73763989, the triggers were still measurable in 2 of 3 participants with 4.5- to 15-fold reduced concentrations from peak levels; the estimated liver half-life ranged from approximately 2.9 to 16.1 weeks.ConclusionsIn this first study to assess liver concentrations of GalNAc-siRNA in humans with sequential biopsies, long persistence of JNJ-73763989 triggers in human livers may partially explain the prolonged suppressive effect on viral parameters observed in phase 2 studies.

Whether astrocytes are fully competent antigen-presenting cells has been debated for over four decades. Fisher et al. show they are, but only conditionally: IFN-γ and microglial cytokines are jointly required, and stimulated astrocytes drive antigen-specific expansion of CD4+ T cells.

High-dose vitamin C (vitC) has shown superior anticancer activity in immunocompetent compared with immunodeficient mice, suggesting an immunodependent mechanism of action. Because high-dose vitC generates substantial reactive oxygen species (ROS), we hypothesized that ROS could induce immunogenic cell death (ICD), a regulated form of cell death characterized by the release of damage-associated molecular patterns (DAMPs) that enhance antigen presentation and promote antitumor immunity. Spatial transcriptomic and immunofluorescence analyses were performed on breast and colon tumors from mice treated with high-dose vitC to quantify oxidative stress, DAMP mobilization, and immune cell infiltration. To dissect the functional role of ROS and ICD, mice were treated with high-dose vitC in combination with the antioxidant N-acetylcysteine or an antibody blocking calreticulin (an early DAMP essential for ICD induction). VitC treatment caused marked oxidative stress in tumors from both immunocompetent and immunocompromised mice; however, tumor growth inhibition occurred exclusively in immunocompetent animals. High-dose vitC triggered ROS-dependent exposure and release of the DAMPs calreticulin and HMGB1, respectively. This was accompanied by substantial remodeling of the tumor microenvironment, including increased infiltration of cytotoxic CD8⁺ T cells and natural killer cells, and a reduction in immunosuppressive regulatory T cells. Blocking calreticulin effectively disrupted the ICD cascade, prevented immune microenvironment remodeling, and completely abrogated the antitumor efficacy of vitC. Collectively, our study indicates that high-dose vitC promotes ICD by mobilizing calreticulin and HMGB1, and that calreticulin is a determinant of its immune-mediated antitumor activity. Implications: vitC induces ICD, supporting its use as an adjuvant to anticancer immunotherapy.

Basal-like double-negative prostate cancer (DNPC) and triple-negative breast cancer (TNBC) are aggressive tumors with few targeted treatment options, raising the question of whether a shared, druggable cell-surface antigen can be leveraged for effective immunotherapy. Here, we integrated analyses of independent bulk and single-cell transcriptomic datasets with mechanistic and preclinical therapeutic studies to evaluate CDH3 (P-cadherin) as a candidate target in basal-like disease. CDH3 expression was consistently enriched in basal-like subtypes across prostate and breast cancer cohorts and was preferentially expressed in basal epithelial tumor compartments. Mechanistically, CDH3 expression was governed by a YAP1-WNT5A/ROR2 signaling axis, linking basal subtype gene expression to non-canonical Wnt signaling. To exploit this target, we evaluated both a CDH3-directed antibody-drug conjugate (ADC) and CDH3-specific chimeric antigen receptor (CAR)-T cells. The ADC exhibited antigen-dependent internalization and cytotoxicity in vitro and reduced metastatic tumor burden in vivo. CDH3 CAR-T cells showed antigen-specific activation and tumor cell killing in vitro and improved tumor control and survival in metastatic prostate and breast cancer xenograft models, with additional benefit when combined with PD-1 blockade (nivolumab). Together, these findings establish CDH3 as a clinically relevant and therapeutically actionable target and support CDH3-directed ADC and CAR-T strategies for aggressive, basal-like DNPC and TNBC.

The efficacy of adoptive T cell therapy against solid tumors is markedly hindered by the immunosuppressive microenvironment. Here, we demonstrate that stimulator of interferon genes (STING) activation in tumor-associated macrophages (TAM) enhances the intratumoral infiltration and cytotoxicity of adoptive T cells. We next design a TAM-targeted nanoagonist and engineer a T cell-agonist chimera (SNTC) via covalent surface anchoring, to achieve synchronized biodistribution of STING agonist with adoptive T cells. The nanoagonist is locally released in tumor tissue in response to the elevated reduction potential on T cell surface, followed by intracellular glutathione-mediated restoration of STING agonist in TAM, thereby enabling AND logic-gated STING activation. SNTC not only reshapes tumor stroma and immunosuppressive tumor microenvironment to augment adoptive T cell function but also enhances the antigen presentation ability of TAM to activate endogenous T cells. SNTC elicits robust antitumor efficacy in melanoma tumor-bearing mice, resulting in over 40% survival beyond 60 d. Moreover, combining tumor-infiltrating T lymphocyte (TIL)-based SNTC with immune checkpoint blockade therapy leads to sustained tumor regression in mouse model of orthotopic pancreatic tumor. Collectively, SNTC holds translational potential to optimize STING activation for adoptive T cell therapy of cancer.

A D-π-A fluorescent probe Phe-SOO integrating intramolecular charge transfer and molecular rotor effects was developed for turn-on fluorescence detection of the food allergen bovine serum albumin (BSA). The probe exhibits high sensitivity with a detection limit of 0.17 µM and a broad linear range of 0.56-50 µM, and notably shows significantly stronger response to BSA than to homologous human serum albumin (HSA), which helps reduce false-positive signals in food matrix detection. Density functional theory calculations and molecular docking studies confirm that binding to BSA restricts intramolecular rotation of the probe, thereby suppressing non-radiative decay and enhancing fluorescence. Benefiting from the dual fluorescence-colorimetric response, a smartphone-based portable platform was established for instrument-free on-site BSA quantification via green channel intensity analysis. Practical assays in milk, yogurt and beef extract achieved satisfactory recoveries of 96.9-106.2% with RSD below 5.6%. This work provides an efficient portable strategy for food allergen monitoring and offers a feasible molecular design paradigm for advanced food biosensors.

Peritoneal metastasis from gastrointestinal cancers has poor outcomes, and whole-abdominal radiotherapy (RT) is limited by toxicity to radiosensitive organs. Spatially fractionated radiotherapy (SFRT) delivers heterogeneous doses, potentially sparing normal tissue while stimulating antitumor immunity. This study aimed to evaluate the efficacy, safety, and immune modulation of SFRT combined with PD-L1 blockade in a syngeneic peritoneal metastasis model. Mouse-specific grid collimators (2-4 mm) were fabricated and tested. CT26-luc cells were injected intraperitoneally, followed by conventional RT (CONV) or 3 mm SFRT at 8-16 Gy, with or without anti-PD-L1. Tumor burden was assessed by bioluminescence, survival and weight monitored, and necropsy performed to evaluate toxicity. Immune profiling of ascites and blood was performed by flow cytometry, and functional outcomes by open-field testing. The 3 mm grid achieved a peak-to-valley dose ratio of ~ 11 and was used for therapy. At 8 Gy, SFRT provided tumor control comparable to CONV RT with less weight loss. At 10 Gy, CONV RT caused early mortality, whereas SFRT maintained 100% survival. SFRT plus anti-PD-L1 induced near-complete tumor regression, reduced ascites, and preserved intestinal length. Immune analysis showed increased CD4⁺/CD8⁺ T cells and M1 macrophages with SFRT; combination therapy further expanded T cells and yielded the lowest neutrophil-to-lymphocyte ratio. Functional testing demonstrated improved locomotion with SFRT plus anti-PD-L1. SFRT combined with PD-L1 blockade achieves potent tumor control, reduces toxicity, and enhances physical performance, supporting its potential as a safer immuno-radiotherapy strategy for peritoneal metastasis.

Bacterial infections account for millions of deaths every year, with many lacking preventative options. Bacterial lipoproteins are strong targets for vaccines, but are often expressed in a truncated form that may not properly recapitulate native epitopes, resulting in a reduced, or even ineffective, immune response. Borrelia burgdorferi, the causative agent of Lyme disease, contains numerous surface expressed lipoproteins, including outer surface protein C (OspC). OspC has been explored as a vaccine target in its recombinant form, but has resulted in a wide variation in immune response. This may be due to structural dissimilarity between the recombinant protein and the native protein expressed on the bacterium, as OspC is hypothesized to present itself in the form of a dimer containing non-linear epitopes. Presentation of OspC in a lipid membrane, such as on an outer membrane vesicle (OMV), may improve quaternary epitope presentation across dimers and better mimic the native protein. Here, we explored expression of two proteins on E. coli-derived OMVs: OspC derived from B. burgdorferi sensu lato, and a fusion protein replacing the OspC leader sequence with Braun's lipoprotein leader sequence (Lpp). Analysis shows that replacing the OspC leader sequence with Lpp improves surface expression and immunoreactivity of the OspC protein on E. coli-derived OMVs exposed to recombinant anti-OspC antibodies derived from B5 and B11, as well as to B. burgdorferi-positive human sera. This strong antibody reactivity indicates that the OMV structure may aid in recapitulating the quaternary epitopes of OspC and provide a strategy for future development of Lyme disease vaccines. KEY POINTS: • E. coli-derived OMVs expressed OspC when the native leader sequence was replaced with Lpp • Lpp-OspC on OMV surfaces has conserved epitopes that react to B5- and B11-derived antibodies • Lyme-positive serum recognizes Lpp-OspC OMVs with minimal cross-reactivity.

Autoantibodies against type I IFNs can severely compromise antiviral defense, yet the mechanisms underlying their development remain elusive. Using high-throughput autoantigen profiling, we observed that a subgroup of severe COVID-19 patients exhibited strong binding to multiple type I IFNs. Integrated single-cell RNA and B cell receptor (BCR) sequencing with functional analysis identified B cell subsets/states enriched for IFN-α2-binding and -neutralizing antibodies in COVID-19. These findings were compared with those from patients with autoimmune polyendocrine syndrome type I, a rare genetic disease characterized by defective immune tolerance and persistent type I IFN autoimmunity. Competition surface plasmon resonance and AlphaFold 3 structural modeling identified three major functional epitope groups and seven predicted structural subclasses among neutralizing antibodies. Our results indicate that IFN-α2 autoreactivity is present in both naive and antigen-experienced B cell compartments, whereas functional IFN-neutralizing antibodies are predominantly associated with memory and atypical B cells, consistent with defective tolerance and affinity maturation-mediated selection of autoreactive B cell clones.