Browse Articles

Bioinspired Artificial Bioenergetic Organelles: Design Principles, Nanofabrication and Therapeutic Translation

ABSTRACT Disruptions in cellular energy metabolism have emerged as central contributors to a broad spectrum of human diseases. While conventional therapeutic strategies can alleviate symptoms, they typically target downstream disease manifestations and often fail to address the underlying energetic dysregulation fueling disease progression. Bioenergetic organelles‐engineered from mitochondria and thylakoids‐represent a transformative approach by restoring cellular energy homeostasis. Functioning as autonomous metabolic modules, they generate ATP, reductive equivalents, and oxygen in situ, while concurrently modulating redox balance, oxygen tension, and immune‐metabolic signaling to restore cellular homeostasis. Recent preclinical evidence highlights their therapeutic versatility, including alleviating tumor hypoxia, restoring bioenergetic function in myocardial and neuronal tissues, reducing inflammatory damage, and normalizing immune cell metabolism. Unlike nanocarriers that primarily serve as delivery vehicles, these bioenergetic organelles actively remodel pathological microenvironments by integrating metabolic restoration with multi‐targeted therapeutic actions. This review outlines the design principles, mechanistic basis, and disease‐specific applications of artificial bioenergetic organelles, while also addressing key translational challenges such as targeted delivery, immunocompatibility, functional longevity, critical considerations regarding biological safety, and the long‐term metabolic fate of these constructs. Positioned at the convergence of bioenergetics, nanotherapeutics, and synthetic biology, these biomimetic systems offer a flexible platform for redefining disease intervention through precise metabolic regulation.

Ratiometric Mycotoxin Detection in Living Plants With Dual‐Emissive Nanosensors

ABSTRACT Early diagnosis of fungal infections in crops is essential for mitigating yield losses, yet most detection strategies rely on destructive assays that are incompatible with in situ monitoring. Among major fungal pathogens, Fusarium species pose a significant threat to global agriculture and produce the phytotoxin fusaric acid (FA), a key biomarker of early infection. Here, we develop a non‐destructive, ratiometric optical nanosensor that integrates aggregation‐induced emissive carbon dots within a zeolitic imidazolate framework (CD@ZIF‐8) for selective FA detection in living plants. By rationally tuning the balance between dispersed and aggregated CDs within the ZIF‐8 framework, we generated a dual‐emissive nanosensor with distinct blue and red fluorescent signals that enable self‐referencing readouts. Upon exposure to FA, interactions with CD@ZIF‐8 quench the blue emission while leaving the red aggregation‐induced emission stable, thereby affording highly selective ratiometric sensing. Integrating CD@ZIF‐8 into plant‐compatible microneedle patches enables minimally invasive sampling of plant interstitial fluid, allowing FA detection in Fusarium ‐infected crops before visible symptoms and discrimination from abiotic and bacterial stresses. This work establishes an integrated, self‐referenced nanosensing platform for non‐destructive plant diagnostics, laying the foundation for early disease surveillance in precision and climate‐resilient agriculture.

Flexible Dielectric Acoustic Resonator Patch for Tissue Regeneration

ABSTRACT Ultrasound (US) therapy offers a non‐invasive means of stimulating cellular activity, promoting tissue regeneration, and wound healing. However, current US transducers based on piezoelectric ceramics are rigid, mechanically incompatible with soft tissues, and thus unsuitable for wearable applications. To overcome these limitations, we developed dielectric patches that generate high‐frequency acoustic waves using electrostatic resonance between interdigitated electrodes and a flexible polymer substrate. We identified the resonance frequency by analyzing the streaming motion of a droplet placed on the device surface while sweeping the frequency of the applied voltage. We fabricated a dielectric acoustic resonator patch (DARP) exhibiting acoustic resonance at 20 MHz by regulating IDE spacing and substrate thickness. The developed DARP maintained >95% of its acoustic output in the bending tests with 300 cycles regardless of the bending direction. The polyimide‐based device exhibited ∼50% optical transmittance, which is beneficial for in situ imaging. Wound healing assays revealed that the migration speed of fibroblasts increased by 1.5‐fold after 5 min of DARP stimulation. We also demonstrated that DARP was able to deliver acoustic energy into tissue‐mimicking hydrogels with curved and planar surfaces. DARP can be applied to develop a platform for ultrasound‐mediated wearable medical devices and regenerative therapies.

Experimental investigation and Monte Carlo validation of PMMA-based WO3- Bi2O3 radiation shield

A novel double-stranded RNA adjuvant markedly amplifies the immunogenicity of nucleoside-modified mRNA-LNP vaccines 2256634

Abstract Introduction mRNA vaccines have transformed vaccinology in recent years, shortening timelines and enabling precise response to infectious diseases. One key modification that made this platform so successful is the replacement of uridine (U) with N1-methylpseudouridine (m¹ψ), which improved antigen expression and tolerability. The use of adjuvants for mRNA platforms represents a promising approach to increase immunogenicity of these vaccines, lowering the doses and offering more potent CD8+ T cell priming. However, successful adjuvant strategies remain largely undefined. Methods Here, we developed a novel and structurally defined double-stranded RNA (dsRNA) and evaluated its immunogenic potential to induce immune responses against infectious diseases using an mRNA encoding influenza hemagglutinin (HA) as a model. C57BL/6 mice were immunized with 3 doses of HA m¹ψ mRNA-LNP (HA-LNP) alone or in co-administered with dsRNA-LNP using intramuscular-prime/ intravenous-boost administration. Peripheral blood mononuclear cells (PBMCs) were collected after each dose and stimulated ex vivo with HA peptides for intracellular cytokine staining (ICS) or ELISpot. Sera was collected 21 days after immunization for humoral analyses. Results Co-administration of HA-LNP and dsRNA-LNP induces more robust humoral responses with ∼3-fold higher specific IgG-antibody titers and increased neutralizing ability compared to HA-LNP alone. Evaluation of cellular arm showed that dsRNA-LNP potentializes the cellular immune responses with enhanced frequency of IFNγ production by CD4+ and CD8+ subsets, T cell activation (CD69+), CD8+ T cell polyfunctionality (IFNγ/TNFα), and higher cytotoxic profile (Granzyme B+/ Perforin+). Conclusion Overall, the dsRNA-LNP acts as a potent adjuvant for nucleoside-modified mRNA vaccination, inducing a strong immune activation, representing a potent platform for eliciting balanced, durable antiviral immunity. Funding Source This work was supported by the National Institute of Allergy and Infectious Disease (NIAID) and National Cancer Institute (NCI) of the US National Institutes of Health (NIH) under award numbers R01AI153064 (NP) and R01CA283736 (NP and CGR), respectively. Topic Categories Vaccines and Immunotherapy (VAC)

Microvascular Alterations in Infection-Associated Chronic Illnesses: Insights from Nailfold Capillaroscopy and NASA Lean Testing 2260753

Abstract Introduction Infection-associated chronic illnesses (IACI) such as Long COVID and Chronic Lyme Disease are characterized by persistent symptoms including fatigue, pain, and brain fog. The MIT MAESTRO study aims to advance knowledge of these conditions by investigating immune pathways and physiologic changes using novel methods, such as capillaroscopy. Capillaroscopy is a non-invasive imaging technique that visualizes the microcirculation at the nailfold to detect vascular abnormalities. Methods Within the MAESTRO study, capillaroscopy was performed before and after the NASA Lean Test (NLT) to investigate microvascular changes under autonomic stress. The NLT, a test to evaluate dysautonomia, requires patients to lie down for ten minutes and then remain upright against a wall for ten minutes while heart rate, blood pressure, and symptoms are monitored. Results In several participants with IACI, striking microvascular alterations after the NLT compared to baseline were observed, including enlarged capillaries, increased tortuosity, and microhemorrhages. These findings suggest dynamic microcirculatory dysfunction that becomes more evident when the autonomic system is challenged. AI-based software was employed to quantify capillary abnormalities, providing objective measures of enlargement, tortuosity, hemorrhages, and other features. In parallel, data was integrated from the Lumia STAT earpiece, a non-invasive wearable device that captures carotid blood flow variables in real time. Reductions in carotid flow time of up to 30% were observed during the NLT, aligning with patient-reported symptoms of dizziness, brain fog, and worsening fatigue during upright posture. Conclusion By combining high-resolution visualization with quantitative metrics and real-time physiologic data, the MAESTRO study establishes a framework for advancing understanding of pathophysiology while also identifying potential biomarkers for diagnosis, disease monitoring, and therapeutic responses for people living with IACI. Funding Source Fairbairn Foundation, NIH NIAID RO1 AI 178713-01, Massachusetts Life Sciences Center, Manton Foundation Topic Categories Immune Mechanisms of Human Disease (HUM)

AhR—MMP—NK Cell Crosstalk Regulates Colonic Inflammation and Colitis-Associated Colorectal Cancer Progression 2256044

Abstract Introduction Inflammatory bowel diseases (IBD), a group of chronic inflammatory disorders, constitute a major risk factor for colorectal cancer (CRC), accounting for 10—15% of IBD-related deaths. Aryl hydrocarbon receptor (AhR) regulates mucosal immunity, while nature killer (NK) cells detect stress ligands via NKG2D. Matrix metalloproteinases (MMPs)-mediated shedding of these ligands promotes tumor immune escape. In this study, we identified a novel AhR—MMP—NK axis in colonic epithelial cells (CECs) that regulates immune escape during colitis. Methods We employed AhR-deficient mice and indole-3-carbinol (I3C) treatment in a dextran sulfate sodium (DSS)-induced colitis model. To assess the severity of colitis, we analyzed body weight loss, colon length, and histological scores. Molecular assays included RNA sequencing, Western blotting, quantitative polymerase chain reaction (qPCR), and immunocytochemistry/histochemistry. Additionally, flow cytometry was performed to identify and quantify natural killer (NK) cells. Results Using proteomics, scRNA-seq, patient samples, and murine colitis models, we observed reduced AhR activity with decreased expression but increased shedding of murine NKG2D ligands such as RAE1, MULT1, and H60 in CECs, impairing NK cell activation. Colitis-inducing agents (Dextran sulfate sodium, B. acidifaciens, C. rodentium) triggered ligands shedding in CECs. We also found that MMPs mediate the shedding of NKG2D ligands. Importantly, AhR activation in mice with colitis, using dietary metabolite, indole-3-carbinol (I3C), restored ligand expression, reduced NKG2D ligands shedding, and enhanced NK responses in WT mice with colitis but not in intestinal epithelial cells-specific AhR knockout (AhRΔIEC) mice. Conclusion Our findings identify an AhR—MMP—NK cell axis as central to mucosal immunity, with AhR agonists offering a promising approach to limit colonic inflammation and block colitis-associated CRC. Funding Source P20GM103641; P30GM154631; R01ES030144; R01AI160896 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Cooperative molecular mimicry drives prolonged autoinflammation in multisystem inflammatory syndrome in children 2308508

Abstract Introduction Multisystem inflammatory syndrome in children (MIS-C) is a pediatric hyperinflammatory disease manifesting 4-6 weeks after SARS-CoV-2 infection. While the immunological hallmarks of MIS-C have been defined, few details regarding the underlying disease pathology have been resolved. Methods To address this, we used a multiomics approach to profile the plasma and peripheral immune cells of 13 acute MIS-C patients, 18 recovered MIS-C follow-ups resampled over multiple time points (1-18 months), and 15 healthy pediatric controls. Results Despite rapid clinical disease resolution, circulating pro-inflammatory (IL-8, IL-6, IL-1α, IL-1β, TNF-β) and TH2-type cytokines (IL-4, IL-5, IL-13) remained elevated up to three months post-MIS-C onset, revealing a subclinical inflammatory state that endures in recovered children. Surprisingly, the majority of patient-expanded TCRs recognizing SARS-CoV-2 epitopes (84.2%, 16/19 expanded TCRs) were cross-reactive for autoantigen epitopes targeting prostaglandin biology (PTGS2, SLCO2A1) and insulin metabolism (SLC30A8), suggesting the breakdown of self-tolerance via SARS-CoV-2 molecular mimicry. Indeed, autoantibody screening confirmed that 13 candidates exhibited elevated autoantibodies in MIS-C patients. Further, autoreactive TCR expansions lasted over time and correlated with cytokines involved in allergic inflammation. Conclusion Together, our findings point to a mechanism of sustained autoimmunity wherein promiscuous TCRs recognize both viral and self-antigens that are activated during primary SARS-CoV-2 infection in children who will develop MIS-C. Upon onset, these circulating cross-reactive T cells drive clinically apparent sterile autoinflammation that persists subclinically into convalescence. Funding Source Helen Hay Whitney Postdoctoral Research Fellowship Topic Categories Immune Mechanisms of Human Disease (HUM)

γγ17 coordinate innervation of white adipose tissue 2335094

Abstract Introduction Recent work from our lab shows that adipose tissue-resident γδ T cells produce homeostatic levels of interleukin (IL)-17 in a circadian rhythm, regulating of de novo lipogenesis. Innervation of adipose tissue promotes energy storage during caloric intake, utilizes energy stores during demand, and prevents activity when energy stores are low. IL-17 producing γδ T cell (γγ17) promote innervation of brown adipose tissue indirectly via adipocytes; however, it remains unknown whether IL-17 acts directly on neurons. This led us to ask whether IL-17 receptor signaling coordinates adipose tissue innervation to regulate whole-body metabolism. Methods White adipose tissue depots were isolated from wildtype (WT) or IL-17A/F KO mice. Flow cytometric analysis was used to characterize changes in the γδ T cell population. Whole adipose tissue depots were cleared using iDISCO and imaged via light sheet microscopy to map the nerve networks. Results We measured reduced volume of adipose tissue innervation in IL17 KO tissue as compared to control (p < 0.01). Interestingly, the adipose tissue of IL17 KO mice has 18% increase in the frequency of the Vγ6 subset of γγ17 (p < 0.001). In contrast, the inguinal lymph node has 15% reduction in the frequency of Vγ6 T cells (p < 0.0001). RNAseq analysis indicates that γγ17 are enriched for neuropeptide receptors. Conclusion Our results indicate that disrupting IL-17 signaling in adipose tissue results in reduced tissue innervation. Our changes in γγ17 frequency suggest that Vγ6 are recruited to the adipose in response to reduced innervation. The presence of neuropeptide receptors on γγ17 may provide a novel mechanism of neuroimmune crosstalk between neurons and γδ T cells. Funding Source n/a Topic Categories Neuroimmunology (NEUR)

Glutamine-Dependent Biosynthetic Pathways Fuel Autoreactive T and B Cells in Foxp3 Deficiency-mediated Disease 2259241

Abstract Introduction Foxp3 deficiency causes a profound loss of immune tolerance, unleashing autoreactive T and B cells, lymphoproliferation, cytokine-driven inflammation, and autoantibody production. This autoimmune pathology is fueled by increased glutamine usage, but it remains unresolved whether glutamine is necessary to produce energy, or for biosynthetic pathways leading to production of inflammatory modulators. Methods By using a Foxp3 deficiency model. Here, we demonstrate that glutamine utilization supports Foxp3-deficiency mediated disease independently of pathogenic Foxp3-deficient Treg cell energetic reprogramming Results Mechanistically, glutamine biosynthetic pathways sustain conventional T cell activation and proinflammatory cytokine production preventing inosine accumulation and signaling, thus implicating adenosine pathway modulation in autoreactive T cell dysregulation. Conversely, autoreactive B cell activation and autoantibody production depend on glutamine-dependent asparagine synthesis, which we reveal as a targetable vulnerability for autoantibody formation. Conclusion These findings highlight glutamine-driven biosynthetic processes as critical drivers of autoimmunity and reveal distinct metabolic vulnerabilities in autoreactive T and B cells that can be targeted for therapeutic intervention. Funding Source NIH NIAID Topic Categories Translational and Interventional Immunology (TI)

Unravelling gene regulatory networks and driver TFs in CD4+ regulatory T cell development in the human thymus 2308489

Abstract Introduction CD4+ regulatory T cells (Tregs) are essential for maintaining immune homeostasis and preventing autoimmunity. Tregs primarily develop in the thymus, but can also arise from naïve CD4+ T cells in the periphery (pTregs) or be generated in vitro (iTregs). However, a major limitation of Treg-based therapies is the instability and plasticity of pTregs and iTregs. In contrast, tTregs have been shown to exhibit stable suppressive capacity, largely due to thymic-derived signals that epigenetically reinforce the Treg program. Elucidating the mechanisms governing Treg differentiation, stability, and function is therefore critical for improving Treg-based therapies. Methods We generated single-cell multiome data from human fetal and pediatric thymuses. We developed various analytical frameworks for unravelling gene regulatory networks (GRNs) involved in Treg lineage commitment. We identified candidate transcription factors (TFs) involved in thymic Treg differentiation and validated these TFs using a CRISPR-Cas9 KO system in primary human thymocytes. Results GRN analysis revealed key driver TFs, such as FOXP3, REL and IKZF2 within CD4+ Tregs. Comparison of GRNs between mature CD4+ Tregs and conventional CD4+ T cells further reveals TFs related to TCR signaling and other novel TFs. Finally, candidate TFs including IRF4, REL, FOXO1, BATF and others were validated utilizing a CRISPR-Cas9 KO system. Conclusion We generated a single cell multiome atlas of fetal and pediatric thymuses and unravel GRNs involved in Treg lineage-specific differentiation. We develop novel analytical frameworks to identify lineage-specific driver TFs in Tregs and validated these by KO of primary human thymocytes. This framework provides an important mapping of GRNs involved in thymic T cell differentiation, particularly focusing on Tregs and will serve as an important basis for understanding Treg biology and improving Treg-based therapies. Funding Source Creative-Pioneering Researchers Program (800-20230490) Seoul National University Topic Categories Hematopoiesis and Immune System Development (HEM)

Aantibody response to Burkholderia pseudomallei in a 13-year chronic infection: Relationship of antibody response to bacterial genomics and proteomics 2308991

Abstract Introduction Burkholderia pseudomallei (Bp), a Gram-negative soil bacterium, causes the disease melioidosis. The majority of melioidosis cases are classified as acute disease. However, some patients present with a chronic infection (symptoms ≥2 months) or activation of infection from latency. In this study, we tracked the antibody response of a patient with a ∼ ≥ 13-year chronic infection to monitor how humoral response pressures may alter the bacterial proteome or genome. Methods We compared Bp isolates from the patient with a closely related environmental isolate from the patient’s home of the same sequence type. Isolates and patient immune responses were compared using genomic, proteomic (mass spectrometry, Western analysis), and immunological assays (Western Blot, ELISA, and nucleic acid-programmable protein array (NAPPA). Results We found that cultured strains persisting in the patient had a significant reduction in bacterial proteome. The reduction was partially correlated with a decrease in bacterial genome size. However, not all reductions were associated with gene deletion. Early patient serum samples showed that many antibodies reacted to the environmental bacterium. Both capsular polysaccharide (CPS) and lipopolysaccharide A (LPSA)-specific IgG antibodies were detected early during the patient’s infection. The reactivity peaked during early phase and then waned over the remaining monitoring period. The carbohydrate reactivity correlated with expression in patient isolates . We screened 1660 reactive Bp antigens by the NAPPA protein array and determined that 160 proteins showed reactivity and outer membrane protein reactivity increased over time after the loss of CPS and LPS antigens. Conclusion In combination, these data suggest that reduced or eliminated CPS and LPS virulence factor expression could be selected for in response to antibody selection. This antibody selection could increase immune cell availability of outer membrane proteins but were insufficient to clear Bp colonization. Funding Source Defence Threat Reduction Agency HDTRA1-22-C0013 Defence Threat Reduction Agency HDTRA1-12-C0022 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Bubble Formation Control: Fabrication of Centimeter‐Sized Tissue‐Like Constructs by Catalase‐Coated Oxygen‐Releasing Hydrogel (Adv. Mater. 47/2026)

Leveraging Mitochondria‐Endoplasmic Reticulum Functional Interplay With an On‐Demand Nanoparticle to Boost mtDNA‐Based STING Immunotherapy

ABSTRACT Endogenous STING activation by mitochondrial DNA (mtDNA) offers a tumor‐context‐dependent strategy with potentially reduced off‐target toxicity, but is often limited by weak and transient mtDNA‐driven STING signaling that fails to sustain robust STING clustering. Here, we developed an on‐demand nanoparticle system that harnesses mitochondrial–ER functional interplay by concurrently inducing mtDNA release and ER stress, thereby relieving the STIM1‐mediated brake on STING and enabling robust STING–TBK1 assembly and downstream signaling. This strategy markedly increased the phosphorylation levels of STING, TBK1, and IRF3 by 11.50‐, 9.70‐, and 8.95‐fold, respectively, compared with PBS, outperforming the commercial STING agonist MSA‐2 by more than 2‐fold. In addition, the nanoparticles enabled spatially controlled co‐delivery, allowing extracellular release of a PD‐1/PD‐L1 inhibitor and intracellular release of mtDNA‐releasing and ER stress‐inducing agents. Consequently, this on‐demand nanoparticle system potently enhanced both innate and adaptive antitumor immunity in vivo, significantly increasing CD8 + and CD4 + T cell infiltration while reducing Tregs, ultimately suppressing tumor progression, metastasis, and recurrence in mouse models of breast and colon cancer. This strategy advances STING‐based immunotherapy by integrating spatially staged drug release with organelle‐level immune modulation.

A new family of cascaded high-order DC–DC converters for renewable energy applications

A universal CD4 epitope enhances cytotoxic T-cell priming and memory in glioblastoma peptide vaccination 2257077

Abstract Introduction Immunotherapy has yet to show efficacy in glioblastoma (GBM). To enhance immune priming, the ETAPA phase 1 trial (NCT05283109) evaluated P30-EPS, a multicomponent peptide vaccine linking class I tumor-associated antigens (CMV pp65, Survivin, EphA2) to the universal class II tetanus toxoid epitope P30 (TVSFWLRVPKVSASHLE), designed to promote coordinated CD4+ and CD8+ activation. Methods Adults with newly diagnosed, MGMT-unmethylated GBM (n = 18) received 7 intramuscular vaccinations with the TLR3 agonist Hiltonol (20 µg/kg) at doses of 300 µg or 400 µg P30-EPS. Peripheral blood mononuclear cells were assessed longitudinally by IFN-γ ELISPOT, spectral flow cytometry, and TCR sequencing to evaluate cytokine secretion, T-cell phenotype, and repertoire diversity. In parallel, baseline and selected on-treatment tumor samples are being analyzed by Xenium spatial transcriptomics and CODEX multiplex proteomics to map local immune remodeling and spatial coordination of CD4+ and CD8+ responses. Results The vaccine was well tolerated (all adverse events ≤ grade 2). P30-EPS induced robust antigen-specific IFN-γ responses in 11 of 12 evaluable patients, with stronger recall responses at 400 µg. Patients with longer progression-free survival exhibited higher baseline frequencies of naïve CD4+ T cells and reduced terminally differentiated effector-memory (CD45RA+CCR7⁻) subsets, indicating a less exhausted, more adaptable helper T-cell pool conducive to effective priming. Single cell 5’ TCR sequencing revealed expansion of unique clonotypes following booster vaccination, consistent with durable memory formation. Conclusion ETAPA achieved its objectives and the universal CD4 epitope enhanced magnitude and quality of CD8+ cytotoxic and memory responses. Ongoing spatial profiling will define intratumoral correlates of systemic immune activation. These results are informing an epitope-linked and retroviral antigen—targeted vaccine that we are developing next to broaden anti-tumor immunity in CNS malignancies. Funding Source NCI P50 CA190991-08 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Neutrophil diversity in the host defense niche 2261060

Abstract Introduction Neutrophils are powerful antimicrobial innate immune cells, without which a host is highly susceptible to morbidity and death caused by infections. However, in recent years, their role in noninfectious contexts has been highlighted, including in cardiac stress, autoimmunity, and cancer. Across settings, the concept of neutrophil heterogeneity has emerged, yet the basis and functional implications of this diversity remain largely unknown. Many devoted studies have examined contexts of cancer or other lifestyle diseases–arenas largely departed from the selective pressures that shaped neutrophil biology. Therefore, we sought to study neutrophil diversity in an evolutionarily powerful setting: infection and acute injury to the skin. Methods Using spectral flow cytometry, along with intravital microscopy and transgenic mice that enable tracking neutrophils over time, we assessed neutrophil dynamics and plasticity, as well as relationships between environment, phenotype, function, and behavior in vivo. Results We found that neutrophils rapidly specify their phenotype upon extravasating to a site of challenge. During response to a biofilm infection, neutrophil phenotypes shifted over time, reflecting both plasticity and turnover. Several phenotypic changes seen during the infection response were absent in a noninfectious wound, highlighting the challenge-specific nature of neutrophil phenotypic adaptation. Notably, we identified an unexpected population of CD101-low mature neutrophils, expressing elevated levels of PD-L1 and ICAM-1, associated with the infection response. This population was characteristic of a highly structured neutrophil swarm. Conclusion This work provides fundamental insights into the evolved capacity of neutrophils to diversify in vivo, revealing that neutrophil phenotypes are dynamic even in acute settings of inflammation, and tightly linked to a cell’s environment. It further aligns observations of phenotypic adaptation with specific host defensive behaviors. Funding Source Damon Runyon Postdoctoral Fellowship Award Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Assessing Antibody-Mediated Protection Against Acinetobacter baumannii Infection Induced by Multi-Epitope Vaccines AMEV2 & AMEV7 2335246

Abstract Introduction Acinetobacter baumannii (Ab) is an increasingly multi-drug resistant (MDR) nosocomial pathogen. Due to limited antibiotic options, there is an urgent need to develop alternative or adjunct treatments such as immune-based therapeutics against Ab. Methods Two novel subunit vaccines, Acinetobacter Multi-Epitope Vaccine (AMEV) 2 & 7, were developed against Ab using immunoinformatics. Passive immunization and opsonophagocytic killing assays (OPKAs) were used to assess the antibody-mediated immune protection of AMEV2 and AMEV7 against Ab. Results We identified 5 antigenic peptides containing both putative B- and T-cell epitopes from proteins associated with Ab pathogenesis to construct two multi-epitope vaccines. AMEV2 was constructed with all 5 peptides in tandem while AMEV7 contained 3 copies of the characterized protective peptide, pTonB. All 5 peptides reacted strongly to the antisera from Ab-infected mice validating their antigenicity. Subcutaneous vaccination with AMEV2 and AMEV7 protected mice against pulmonary and systemic challenge, respectively, with a hypervirulent Ab clinical isolate. AMEV vaccines improved survival rates by 40-60% and reduced organ Ab burden by 1-2 logs. Passive immunization with AMEV2 immune sera protected 66.7% of mice against pulmonary Ab infection, while only 16.7% of mice received nonimmune sera were protected. While AMEV2 sera did not demonstrate complement-mediated direct Ab killing, the immune sera did enhance killing of Ab in the presence of primary bone marrow-derived macrophages. This opsonophagocytic killing was mediated by Fc receptor and complement fixation. Conclusion We have shown that protection afforded by AMEV2 is antibody-based, acting through opsonophagocytic killing. While we hypothesize AMEV7 has a similar mechanism, further analysis will be performed to confirm. Together, our studies highlight the importance of antibody-mediated protection and broaden the possibilities of immune therapies against this MDR pathogen. Funding Source NIH R21 Topic Categories Vaccines and Immunotherapy (VAC)

Potentiating Natural Killer Cell-mediated Cytotoxicity in Medulloblastoma 2308814

Abstract Introduction Medulloblastoma (MB) is the most common malignant pediatric brain tumor and comprises four molecular subgroups (WNT, SHH, Group 3, Group 4). Although generally presenting an immunologically “cold” tumor microenvironment, flow cytometric (FC) profiling of primary tumor samples from our laboratory demonstrates the presence of natural killer (NK) infiltrates in the tumor microenvironment, prompting investigation into their functional relevance and therapeutic potential. Methods Healthy human NK cells were expanded under standardized IL-2/IL-15 conditions and co-cultured with MB cell lines (DAOY, D341) and patient-derived cell lines (n = 5, WNT=, SHH=1, Group 3=2, Group 4=1) at graded effector:target ratios and time points. Tumor cell death was quantified by ToxiLight assay and Annexin V/PI by FC analysis. NK activation was assessed by CD45, CD56. And CD107a expression. IFN-γ production was measured by intracellular FC and ELISA. CD226-CD96-TIGIT axis (CD155/PVR,CD112) and PD-1/PD-L1 inhibitory pathways were analyzed by FC and IHC. Multiplex spatial proteomics was performed on FFPE patient samples (n = 26 total; WNT=4, SHH=6, Group 3/4=16) to map NK infiltration and inhibitory signaling across subgroups. Results NK cells induced time- and effector-dependent apoptosis in MB cell lines and primary cultures. Co-culture triggered robust NK activation with IFN-γ production, accompanied by PD-L1 upregulation on tumor cells. NK cytotoxicity varied by molecular subgroup, with SHH tumors showing the greatest sensitivity and Group 3 tumors exhibiting reduced susceptibility. These differences aligned with subgroup-specific inhibitory receptor expression and signaling patterns. Conclusion NK-cell cytotoxicity in medulloblastoma is subtype dependent, with Group 3/4 tumors showing reduced susceptibility. Spatial proteomics revealed distinct subgroup-specific inhibitory architectures that align with differential NK sensitivity, supporting the need for subtype-informed NK-based therapeutic strategies. Funding Source Project to Cure, Olivia Hope Foundation and Hyundai Hope on Wheels Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Distinct effects of different programmed cell death modalities on immune microenvironment of major molecular subtypes of breast cancer 2258818

Abstract Introduction Programmed cell death (PCD) is now classified as immunogenic or non-immunogenic based on its ability to trigger immune responses. The three major PCD types are apoptosis, autophagic cell death (ACD), and necroptosis. Immune cells like macrophages and lymphocytes play a vital role in tumour microenvironment. Induction of cell death is a desirable end point for anti-cancer therapies. However, they may sometimes result in immunosuppressive microenvironment with altered immune cell properties that favour the tumour progression or relapse. Methods MCF-7 (hormone receptors+) and MDA-MB-231 (triple negative) BC cells were treated with paclitaxel, shikonin and metformin to induce apoptosis, necroptosis, and ACD respectively. The conditioned medium (CM) was collected and JURKAT (T lymphocyte) and THP-1 (macrophage) cells were incubated with it. Post-incubation, RT-qPCR was used to assess lymphocyte (Th1, Th2, Th17, Th22, and Treg) and macrophage polarization (M1, M2) markers in the above cell types. Functional assays including cell cycle analysis and differentiation and phagocytosis assessment (flow cytometry and confocal microscopy) were carried out to gauze phenotypic effects on the immune cells. Further, PD-1/PD-L1 expression was quantified at transcriptional and translation level on lymphocytes and cancer cells. Results Paclitaxel (1 µM), metformin (2.5, 5mM), and shikonin (2.5 µM) induced cell death in MCF-7 and MDA-MB-231 cells. CM significantly (P-value< 0.005) changed the expression of IL-1β, STAT-5, and iNOS (M1 phenotype) and IL-4, GATA-3, and IFN-á¿« (Th1, Th2 subtype). RT-qPCR revealed drug-specific shifts in T lymphocyte and macrophage molecular and phenotypic properties, indicating distinct immuno-modulatory effects. Conclusion PCD-induced breast cancer cell supernatants modulate immune cell behavior, showing drug-specific effects that can guide therapies to enhance anti-tumor immunity and prevent immune suppression. Funding Source ICMR Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)