Browse Articles

Photo‐ and Thermally‐Induced Huge Layer Decoupling in Twisted Bilayer WSe ₂

ABSTRACT Twisted bilayer systems host a wealth of emergent phenomena, such as flat‐band superconductivity, ferromagnetism, and ferroelectricity, arising from moiré superlattices and unconventional interlayer coupling. Despite their central role, direct and quantitative access to the 3D atomic arrangement in these systems has remained elusive due to their nanoscale dimensions. Here, we introduce an automated dark‐field electron tomography technique that enables quantitative 3D structural analysis of atomically thin materials with sub‐ångström precision. By applying this method to twisted bilayer WSe 2 , we precisely visualize the twist‐angle‐dependent structural relaxation appearing as the AB/BA stacking domains separated by 10–20 nm domain walls. In the marginally twisted region (θ ≤ 0.1°), we uncover a significant expansion of the interlayer spacing compared to the bulk configuration, exceeding 0.1 Å, along with a remarkable temperature‐driven interlayer decoupling. Ultrafast measurements further reveal optically induced interlayer separation of ∼0.2 Å on the picosecond timescale, attributed to transient exciton formation. These findings not only establish a powerful approach for visualizing hidden out‐of‐plane structures in atomically thin micro‐flake materials but also uncover the intrinsic fragility and dynamical tunability of interlayer coupling in moiré‐engineered 2D materials.

Conjugated Polymer Semiconductors Enabled Multifunctional Interfacial Engineering for High‐Performance Inverted Perovskite Solar Cells

ABSTRACT The interfacial defect challenge between perovskite and electron transport layer (ETL) in inverted perovskite solar cells have become a critical bottleneck for achieving concurrent high efficiency and stability in the process of industrialization. We developed a novel multifunctional integrated polymer semiconductor material P4N‐Cl as an interface interlayer between perovskite and [6,6]‐phenyl‐C 61 ‐butyric acid methyl ester. Various functional groups including carbonyl group, Cl atom and sp 2 ‐N atom in the polymer backbone effectively passivate defects at the perovskite interface through a synergistic coordination mechanism and significantly suppress non‐radiative recombination losses. Simultaneously, the robust interfacial binding at the heterointerface further optimizes the energy level alignment at the perovskite/ETL interface and enhances charge carrier dynamics. The inverted PSCs based on the P4N‐Cl multifunctional layer achieved a champion efficiency of 26.20% and a high open‐circuit voltage of 1.21 V. The target devices retained 96.2% and 90.2% of their initial power conversion efficiency after 2016 h aging in ambient air (40%–60% relative humidity) and 1500 h maximum power point tracking at 65°C under 1‐sun illumination in nitrogen, respectively. This “one‐stop” design provides exciting research prospects for constructing a new generation of commercially viable perovskite solar cells with high efficiency and long‐term operation stability of devices.

Metagenomic characterization of Acinetobacter in rivers reveals health-related risks, ecological functions, and underrepresented genomic diversity

Multimodal Immune Profiling in Thyroid Eye Disease 2253789

Abstract Introduction Thyroid Eye Disease (TED) is an autoimmune orbital disorder affecting up to half of patients with Graves’ disease. It progresses from inflammation to fibrosis, leading to proptosis, diplopia, and tissue remodeling. While fibroblast diversity is known to influence disease course, the interplay between immune and stromal compartments remains poorly understood. Here, we apply integrated single-cell and TCR sequencing to define cellular programs and immune—stromal interactions across disease stages in TED. Methods Orbital tissue was obtained during decompression surgery from twelve TED patients (active: Clinical Activity Score ≥3, n = 6; inactive: CAS< 3, n = 6) and two non-inflammatory controls. Single-cell RNA and paired V(D)J sequencing characterized cellular heterogeneity and clonality. Differential expression, pathway enrichment, and ligand—receptor interaction analyses were performed to identify mechanisms associated with disease activity and fibrosis. Results Analysis revealed coordinated immune and fibroblast remodeling across disease stages. Active TED was marked by inflammatory—adipogenic PLIN2+/TNFSF8+ fibroblasts, elevated cytokine signaling, and expanded CD8+ T cell clones expressing activation markers (REL, NFKB1, CD69). Inactive TED showed a shift toward fibrotic and contractile fibroblast states (SPARC, GPX3, TAGLN) and reduced T cell clonality. Cross-talk modeling identified a RORA+ fibroblast subset co-expressing TSHR and IGF1R, potentially mediating T cell—fibroblast signaling via ELL2-associated pathways. Conclusion This study provides a multimodal framework linking immune activation with stromal remodeling in TED. By integrating transcriptional and clonotypic data, we uncover immune—stromal circuits that define inflammatory and fibrotic phases, revealing candidate targets for stage-specific intervention. Funding Source Rhodes Scholarship, Canadian Institutes of Health Research (CIHR) Doctoral Foreign Study Award, Kennedy Trust Prize Studentship (KTPS), Wellcome Trust Fellowship Topic Categories Immune Mechanisms of Human Disease (HUM)

Loss of αVβ6 integrin function disrupts intestinal immune homeostasis and increases susceptibility to experimental colitis 2310140

Abstract Introduction Ulcerative colitis (UC) is characterized by inflammation and decreased barrier integrity of the colonic mucosa. Patients with UC experience immune dysregulation, including the production of autoantibodies to the αVß6 integrin expressed by intestinal epithelial cells. αVß6 autoantibodies are not only highly associated with UC, but are also strongly predictive of disease, with seropositivity being detected up to 10 years before diagnosis. Given that αVß6 directly activates TGF-ß, we hypothesized that the disruption of αVß6 function via autoantibodies disturbs intestinal immune homeostasis, predisposing individuals to chronic intestinal inflammation. Methods Colonic tissue from mice lacking αV protein expression in intestinal epithelial cells (αV-Villin) was used for intestinal immunophenotyping, histological analysis, and spatial transcriptomic analysis. αV-Villin mice were also subjected to a DSS colitis model. Results αV-Villin mice presented with transcriptomic and structural changes in the mid-colon, with an expansion of goblet cells in the colonic epithelium. Furthermore, αV-Villin mice experienced a disruption of intestinal immune cell populations, including decreased CD103+ CD8 T cells and dendritic cells in the epithelium and lamina propria, respectively. Finally, αV-Villin mice exhibited increased susceptibility to DSS colitis. Conclusion Disruption of αVß6 function leads to the dysregulation of intestinal immune and epithelial homeostasis at baseline and increased susceptibility to experimental colitis, suggesting that αVß6 neutralization by autoantibodies induces a “pre-colitis” state. Models of αVß6 functional disruption can be used to better understand the factors which interact with the dysregulated intestinal environment to influence UC pathogenesis. Furthermore, αVß6 autoantibodies may serve as a functional target to prevent the development of UC. Funding Source NIH; Kenneth Rainin Foundation Topic Categories Mucosal and Regional Immunology (MUC)

Biodistribution of self-antigens and regulatory cues in lymph nodes induce memory-like Treg to drive antigen-specific tolerance 2299645

Abstract Introduction Multiple sclerosis (MS) occurs when immune cells attack myelin, causing neurodegeneration. Current therapies cannot selectively target myelin-reactive cells. An emerging strategy is to engineer myelin-specific regulatory T cells (TREGs) for selective vaccine-like tolerance. Here, we co-deliver myelin peptide (MOG) with rapamycin (rapa) in polymer microparticles (M/R-MPs) directly to lymph nodes (LN) via intra-LN injection (iLN). We have shown these tissue-retained depots expand MOG-specific TREG and reverse paralysis in a preclinical MS model (EAE). However, how these cues synergize to program long-lasting, highly suppressive TREG remains unknown. Methods Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaged potassiated rapamycin (952 m/z) in LN cryo-sections 0.1 and 48 hours after iLN treatment. MOG TCR specific CD4+ T cells were transferred into iLN-treated mice to test if M/R-MPs promote markers linked to long-lasting T cell responses. Transferred cells from treated LNs were analyzed at 7 days for TREG, early persistence markers including memory cytokine signaling (CD127), and central memory phenotype (CD44, CD62L). Finally, TREG from treated LNs were tested in a suppression assay for their ability to restrain MOG-specific effector T cells (Teff). Results MALDI imaging of rapa revealed that MPs increased the duration and distribution of conditioned LN T cells. M/R co-encapsulation was required for expansion of MOG-specific TREG. These TREG exhibited increased central-memory phenotypes (CD44+CD62L+) and elevated CD127 expression, consistent with enhanced persistence. TREG isolated from M/R-MP treated LNs significantly suppressed MOG-Teff thus demonstrating the importance of synergistic delivery of peptide and rapa. Conclusion M/R-MPs regulate the duration and combination of immunomodulatory cues to condition suppressive antigen-specific TREG. These findings define mechanisms for engineering long-lasting potent TREG in the pursuit of disease-specific therapies for MS. Funding Source 5R01AI169686-03 Topic Categories Therapeutic Approaches to Autoimmunity (THER)

ZL-1503: A Bispecific Antibody Targeting IL-13 and IL-31Rα Sustains Suppression of Pruritus and Atopic Disease in Non-Human Primates 2305220

Abstract Introduction IL-13 and IL-31 are key mediators in atopic diseases. IL-13 promotes type 2 inflammation and tissue changes, while IL-31 drives pruritus and amplifies local immune responses. Their combined effects worsen disease, highlighting the need for dual-targeted treatments. ZL-1503 is a bispecific antibody with extended half-life that blocks both IL-13 and IL-31Rα signaling. Methods In the pruritus model, cynomolgus monkeys (n = 16) received a single IV dose of ZL-1503 (0, 0.3, 3, or 10 mg/kg; n = 4/group). IL-31-mediated scratching and IL-13-induced pSTAT6 were monitored for 112 days. Serum samples were collected for PK analysis. In the atopic disease (asthma/allergic rhinitis) model, cynomolgus monkeys (n = 9; 3/group) were divided into: naive, disease (Ascaris suum challenge), and treatment groups (ZL-1503 10 mg/kg IV + antigen). Results In the pruritus model, a dose dependent effect of ZL-1503 was observed in both the magnitude and duration of the response, including inhibition of IL-31-mediated scratching and IL-13-mediated pSTAT6, during a 112-day observation period following ZL-1503 treatment. PK analysis demonstrated a strong PK/PD correlation. In the atopic disease model, ZL-1503 significantly improved airway resistance (RAW, p < 0.001) and dynamic compliance (Cdyn, p < 0.01), and reduced nasal rubbing (p < 0.001) and sneezing frequency (p < 0.0001) compared with disease-induced, untreated animals. Conclusion ZL-1503 strongly inhibited IL-13/IL-31 pathways in primates and showed significant effects across atopic conditions in NHP models. These findings support its development for human trials targeting moderate-to-severe atopic dermatitis and other IL-13/IL-31 driven diseases. Funding Source n/a Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Epithelial Toll-Like Receptor 9 Upregulation by Influenza Virus Impairs Pulmonary Antibacterial Immunity 2303048

Abstract Introduction Influenza is often complicated by bacterial coinfections. Among the most common is methicillin-resistant S. aureus (MRSA). Viruses and bacteria are recognized by pattern recognition receptors (PRRs) such as Toll-like receptor 9 (TLR9). Most studies have focused on TLR9 function in immune cells. In contrast, its role in structural cells remains less understood. Our laboratory found that TLR9 knockout mice exhibit improved outcomes during influenza and MRSA coinfection, prompting us to explore the role of TLR9 in structural cells during coinfection. Methods To study TLR9 in structural cells, we generated mice that lack TLR9 in lung alveolar type II epithelial cells or in mouse lung fibroblasts. We measured bacterial burden and viral load following coinfection with influenza virus and MRSA. We performed primary isolation of alveolar type II cells and alveolar macrophages for in vitro supernatant studies and phagocytosis of MRSA. Results Influenza infection upregulates TLR9 through a MyD88-dependent pathway in fibroblasts, and we are investigating the signaling cascade that leads to TLR9 upregulation in epithelial cells. Epithelial TLR9 expression is detrimental during coinfection: mice lacking epithelial TLR9 have reduced bacterial burden without changes in immune recruitment or lung injury. Lack of TLR9 in alveolar epithelial type II cells does not affect responses to single infections with influenza virus or MRSA. Additionally, alveolar macrophage phagocytosis of MRSA was significantly enhanced by supernatant from influenza-infected TLR9-knockout epithelial type II cells compared with supernatant from influenza-infected wild type epithelial type II cells. We are further exploring epithelial-derived factors that modulate macrophage function. Conclusion These novel findings reveal that epithelial TLR9 regulates antibacterial immunity and highlight its potential as a target for improving coinfection outcome. Funding Source Beth Moore: NHLBI R35HL144481 Helen Rich: NHLBI T32HL007517, Michigan Postdoctoral Pioneer Program Francina Gonzalez De Los Santos: Immunology Graduate Program AI007413 T32, University of Michigan; Herman & Dorothy Miller Award, Immunology Program, University of Michigan Topic Categories Viral Immunology (VIR)

Sustained bone marrow myeloid-skewing is associated with systemic inflammation in adenine-induced chronic kidney disease mice 2258869

Abstract Introduction Chronic kidney disease (CKD), affecting over 10% of the global population, is frequently accompanied by comorbidities such as cardiovascular disease (CVD), largely driven by chronic inflammation. Emerging evidence implicates trained immunity, a form of innate immune memory triggered by uremic toxins, in sustaining inflammatory responses in CKD. However, its physiological relevance remains unvalidated in appropriate animal models. Methods CKD was induced in C57BL/6 mice using a 0.2% adenine-containing diet administered for 2 or 4 weeks, followed by a resting phase to assess the development of trained immunity. Serum creatinine (Cr) and blood urea nitrogen (BUN) were measured to evaluate renal function, while histopathological changes were examined via Hematoxylin & Eosin (H&E) and Masson’s trichrome (M-T) staining. Flow cytometry of spleen and bone marrow was performed for immunophenotyping, and systemic cytokine responses were analyzed after intraperitoneal lipopolysaccharide (LPS; 5 mg/kg) challenge using ELISA. Results Adenine-fed mice showed significantly elevated serum Cr and BUN levels, with tubular injury and interstitial fibrosis that worsened with prolonged adenine exposure, confirming CKD establishment. Partial recovery of renal parameters and histopathology occurred following the resting period. Nevertheless, flow cytometric analysis revealed a sustained expansion of myeloid-biased multipotent progenitor cells (MPP3) in bone marrow, persisting beyond renal recovery. Upon secondary LPS stimulation, previously adenine-fed mice produced markedly higher serum levels of TNF-α and IL-6, characteristic of a trained immunity phenotype. Conclusion These findings demonstrate that adenine-induced CKD leads to long-term reprogramming of hematopoietic progenitor cells, promoting an enhanced inflammatory response that may contribute to persistent inflammation and the progression of CKD-associated CVD. Funding Source This work was supported in part by grants (RS-2022-NR070612 and RS-2022-NR070765 to W-W. Lee) from the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (MSIT) and by a grant (RS-2023-00238632 to H.Y.K.) of Basic Science Research Program through NRF funded by the Ministry of Education, Republic of Korea. Topic Categories Hematopoiesis and Immune System Development (HEM)

Tissue resident NK cells establish residency via KLF2-independent pathways 2217188

Abstract Introduction Group 1 ILCs, composed of conventional NK cells (cNK), tissue resident NK cells (trNK) and ILC1s, are critical mediators of anti-viral immunity in lymphoid and non-lymphoid tissues. Within this group, it is understood that cNK act as the circulating, cytotoxic population, while trNK and ILC1s provide regulatory protection within tissues. Krüppel-like factor 2 (KLF2) is a transcription factor that controls the migration and activation of various lymphocytes. Recent work has demonstrated that KLF2 is a distinguishing factor for group 1 ILCs with cNK cells expressing KLF2 and trNK and ILC1s largely lacking KLF2 expression. However, its role in shaping the function, development, and residency patterns of group 1 ILCs remain poorly understood. Methods To address this gap in knowledge, we generated conditional knockout (cKO) mice in the group 1 ILC lineage (NCR1-Cre KLF2 fl/fl) and evaluated cell distribution across tissues. Results In lymphoid tissues (i.e. blood, spleen, bone marrow) KLF2 deficiency resulted in a reduction in mature NK cells and a significant increase in immature NK cell subsets. The abundance of trNK and ILC1 in non-lymphoid tissues (i.e. uterus, pancreas, salivary gland) was not affected. Despite their lack of circulating mature NK cells, cKO animals showed no differences in morbidity relative to control animals following MCMV infection. However, Ncr1-Cre-Rosa-DTA mice, which lack all group 1 ILCs, exhibited significantly greater weight loss, suggesting that mature, circulating conventional NK cells are dispensable for preventing excess morbidity during acute infection. Conclusion These data highlight an unappreciated role for KLF2 in controlling Group 1 ILC1 maturation, residency, and function across lymphoid and non-lymphoid tissues. Despite their well known ability to control MCMV infection, our data demonstrate that tissue resident group 1 ILCs are sufficient for preventing excess morbidity during acute infection. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Modular Evaluation of Multi-Platform Human In vitro Systems (MEMPHIS): A Methodological Advancement in Vaccinology 2260451

Abstract Introduction Vaccination is an effective strategy to reduce the burden of infectious diseases. Pre-clinical vaccine development has traditionally relied on animal models, limiting access to human-specific data necessary for rapid clinical translation. With the FDA’s Modernization Act endorsing human in vitro approaches for pre-clinical evaluation of novel vaccine candidates, we developed MEMPHIS — a suite of complementary human immune profiling modules designed to characterize age- and population-specific innate and adaptive immune responses to vaccine adjuvants and formulations. Methods Consisting of six complementary in vitro modules, MEMPHIS is designed to assess the ability of adjuvant formulations to influence innate immune activation, cell migration and fate, as well as antigen-specific B- and T-cell activation. MEMPHIS includes the assessment of type and magnitude of cellular immune memory to pathogens of interest, such that pre-exposure history can be factored into the assessment of new candidate vaccine formulations. Together, these modules enable comprehensive assessment of how adjuvants influence innate immune activation and adaptive immune programming. All MEMPHIS assays have been optimized to use minimal input — requiring only 100 mL of blood from adults or 20 mL from children — allowing integrated testing from a single donor sample. Results In this study, we demonstrate that MEMPHIS accurately modeled the type and magnitude of response to adjuvanted protein-based vaccine, mRNA vaccine, and small-molecule adjuvants, and defined molecular differences in mode of action between these stimuli. In addition, we report on the technical and biological variance of data generated with our modules, and present novel strategies for cross-platform data integration. Conclusion In summary, this system provides a powerful translational platform to inform adjuvant selection and vaccine design by defining potency, reactogenicity, and the breadth, diversity, and durability of immune responses across populations. Funding Source Department Innovation Award Topic Categories Technological Innovations in Immunology (TECH)

CuS‐Cobalt Porphyrin Organic–Inorganic Heterojunction with a Long‐Lived Charge‐Separated State for Efficient CO ₂ ‐to‐CH ₄ Conversion under Infrared Light

ABSTRACT Harnessing infrared (IR) light for the selective reduction of CO 2 remains a significant challenge due to sluggish kinetics and poor carrier dynamics. In this research, a novel organic–inorganic hybrid heterojunction was designed consisting of HKUST‐1‐derived CuS and cobalt porphyrin (CoTPPS), which achieves an exceptional CH 4 generation (178.02 µmol g − 1 h − 1 ) and 96.5% CH 4 selectivity under IR irradiation. Femtosecond transient absorption spectra and synchrotron radiation measurements show that strong interfacial electronic coupling enables a significant charge transfer, creating a robust internal electric field which greatly increases carrier lifetimes (170‐fold). Density functional theory (DFT) calculations further elucidate how the heterojunction lowers the rate‐determining *COOH formation barrier (from 1.43 to 1.07 eV) and stabilizes the critical *CHO intermediate. This effectively steers the eight‐electron pathway towards CH 4 while suppressing CO desorption. The synergy between the porous MOFs‐derived scaffold and the molecular active center provides a versatile paradigm for engineering IR‐responsive photocatalysts with precise intermediate regulation. This work promotes the rational design of hybrid materials for efficient solar‐to‐fuel conversion.

Suppressing Charge Carrier Recombination in Bulk Heterojunction Organic Photocatalyst via Improving Molecular Crystallinity and Reducing Electron‐Phonon Coupling for Efficient Hydrogen Evolution Reaction

ABSTRACT Organic semiconductor bulk‐heterojunction nanoparticles have emerged as promising photocatalysts, due to their strong visible absorption in the Vis–NIR region, excellent optical/electronic adjustability, and spatially abundant interfaces for charge carrier separation. However, organic semiconductors generally suffer from inferior crystallinity and high lattice's susceptibility to molecular vibrations, which leads to the localization of separated charge carriers and severe recombination in nanoparticles, limiting the further improvement of photocatalytic H 2 evolution rate. Herein, a methoxy‐functionalized electron acceptor, ITIC‐OMe, is developed and presents enhanced crystallinity, more compact molecular packing and weaker electron–phonon coupling, compared to the parent ITIC. This enables ZnTPP‐3O:ITIC‐OMe bulk‐heterojunction nanoparticles to afford more ordered molecular stacking, reduced charge transfer resistance, and inhibited charge back transfer for triplet state formation, thereby suppressing charge carrier recombination and facilitating charge transport to the nanoparticle surface for proton reduction. Consequently, the photocatalyst based on ZnTPP‐3O:ITIC‐OMe bulk‐heterojunction nanoparticles achieves an impressive hydrogen evolution rate up to 1017.7 mmol g −1 h −1 under AM 1.5G illumination, which is the record for organic photocatalysts so far. It highlights that suppressing charge carrier recombination via finely molecular design is a powerful route to enhance the photocatalytic H 2 evolution performance.

Lifestyle factors associated with cataract surgery among working age adults in Japan using claims and health screening data

Abstract Cataracts are primarily age-related; however, factors associated with earlier progression to surgery among working age adults remain unclear. Identifying modifiable lifestyle factors may provide insights into delaying surgical intervention. This study used the Japanese Medical Data Center Claims Database (January 2005 to August 2023). Patients who underwent cataract surgery (n = 30,971) were identified as cases. Controls (n = 92,913) without cataract-related diagnoses or surgery were matched 1:3 on age at health screening, sex, and screening year. Conditional logistic regression using linked health screening data was performed to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Subgroup analyses by age group were also conducted. Hypertension (OR, 1.10; 95% CI, 1.06–1.13), diabetes mellitus (OR, 1.97; 95% CI, 1.89–2.06), renal dysfunction (OR, 1.12; 95% CI, 1.06–1.18), hyperuricemia (OR, 1.07; 95% CI, 1.01–1.14), and excessive alcohol consumption (OR, 1.15; 95% CI, 1.10–1.21) were significantly associated with cataract surgery. Age-stratified analyses showed that ORs for hypertension and diabetes mellitus generally decreased with increasing age, and interaction tests were also statistically significant. These findings indicate that modifiable lifestyle-related conditions were associated with earlier progression to cataract surgery in working age adults.

Donor-reactive T cells and innate immune cells promote pig-to-human decedent xenograft rejection 2254507

Abstract Introduction Xenotransplantation of pig organs is a promising strategy to address the organ shortage; however, rejection poses a barrier to its widespread adoption. Pig-to-decedent human transplantation provides an opportunity to study immune barriers to xenotransplantation. We followed donor-reactive T cell dynamics in a long-term (61-day) pig-to-decedent human kidney Methods We developed a method to identify xeno donor-reactive T cell clones (XDRTCCs) by TCRB CDR3 sequencing and tracked them in the circulation and xenograft. scRNA and TCR sequencing of human leukocytes isolated from the kidney graft were used to investigate the functional phenotypes of XDRTCCs infiltrating the kidney as well as additional immune responses against the xenograft. Results We observed an expansion of both CD4 and CD8 XDRTCCs in the circulation. However, CD8 XDRTCCs demonstrated more striking expansions, constituting 10% of the total CD8 repertoire at POD28 and about 30% at POD33 and POD49, both of which were established rejection timepoints. Normalizing the total quantity of circulating XDRTCCs post-transplant to their pre-transplant baseline revealed clear XDRTCC enrichment, with increases up to 11-fold at POD49 for CD8 cells. scRNA sequencing data on graft-infiltrating leukocytes revealed RNA species characteristic of effector T cells and NK cells and APCs only in the POD33 rejecting kidney specimen. Interrogation of scRNA/TCR seq data for XDRTCCs revealed infiltrating XDRTCCs that expressed some effector genes such as GZMA, GZMB and PRF1 as well as the costimulatory molecule ICOS and some coinhibitory molecules, including PDCD1, LAG3, CTLA4, and TIGIT. Conclusion The findings provide insight into the clonal dynamics of xenoreactive T cells and their contribution to rejection in xenotransplantation and demonstrates the importance of tracking xenoreactive T cell repertoires to better understand immune responses in xenotransplantation and suggests that specific T cell clones might serve as biomarkers for rejection monitoring. Funding Source n/a Topic Categories Transplantation Immunology (TRAN)

A novel silicon-based intracellular delivery platform enables multiplexed delivery across cell types and cell states 2310229

Abstract Introduction Immune cell engineering is a critical element of basic immunological research and translational applications. The ability to deliver modifying materials to cells without substantial phenotypic or survival impacts is a limiting factor for many immune cell types. To enable engineering in sensitive cell types or cell states, we have developed a novel intracellular delivery platform that works through mechanoporation. Methods Cells are pushed through pores on a thin silicon membrane which creates temporary openings in the cell membrane that allow the payload of interest to diffuse into the cytoplasm before the membranes repair themselves. This method has been used to introduce CRISPR RNPs, mRNA, circRNA, siRNA and peptides and is compatible across cell types including naive and activated T cells, B cells, NK cells, monocytes, neutrophils, iPSC and HSC. Results Using our device, we simultaneously delivered a B2M targeting CRISPR RNP, GFP mRNA, and fluorescent dextran to primary unstimulated T cells and observed that 85% of the live population was B2M negative, GFP and dextran positive. The temporary membrane disruption is gentle enough to use with sensitive cells like neutrophils which allowed us to deliver dextran and GFP mRNA to neutrophils in whole blood. We measured 83% GFP positivity in the live, dextran positive population the next day. Similarly, we delivered a GFP-mRNA to PBMCs and observed expression in T cells, B cells, NK cells, and monocytes. This technology can also enable the use of viral vectors in challenging cell types. Using our method, we engineered B cells to express a circRNA ‘transduction enhancer’ (TE) and then infected them with a GFP lentivirus. After 3 days, the cells that received the TE had the highest GFP expression. Conclusion Our technology unlocks scalable, gentle, and versatile intracellular delivery for sensitive immune cells, expanding the feasibility of complex genetic and molecular engineering for research and clinical cell therapies. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Acetyl-CoA generation by Acly distinctly regulates CD4 T cell programming during Plasmodium infection 2300397

Abstract Introduction Malaria is a disease triggered by Plasmodium parasite infection, causing over 200 million cases and ∼600,000 deaths each year. CD4 T cells, specifically Th1 and Tfh, are critical for parasite control and clearance. We found that CD4 T cell subsets responding to malaria exhibit distinct energetic demands. These energetic dependencies are supported by metabolic pathways that may differentially represent available metabolites, including Acetyl-CoA (Ac-CoA) that sits at a central node between cytosolic and mitochondrial metabolism. Based on the distinct energetic demands of CD4 T cell subsets, different levels of Ac-CoA may be present in the cytosol regulating acetylation and differentiation. Our scRNA-seq data shows Acly, which converts cytosolic citrate into Ac-CoA, is expressed in malaria-specific CD4 T cells. While the contribution of Acly in CD4 T cells is not well defined, published data suggests a role in Th1 cells in vitro. Therefore, we hypothesize that Acly is required for optimal Th1 cell programming during infection. Methods To address the role of Acly in vivo, we engineered a conditional genetic system to excise Acly from mature, peripheral Plasmodium-specific CD4 T cells. We employed systems of co-adoptive transfer and flow cytometry to assess the phenotype of the transferred cells. We used SCENITH to measure energy profiles and Mitotracker for changes in mitochondrial activity and mass. Results Loss of Acly negatively impacts the Th1 population but sustains the Tfh population. Acly-KO Th1 cells show changes in mitochondrial function and energetic profiles, with no observed changes in Tfh cells. Functionally, Acly-KO T cells produce less IFN-gamma. Conclusion These data establish a role of Acly in CD4 T cell programming during malaria, potentially supporting the Th1 program. Our data provide an opportunity to unravel metabolic programs and regulatory nodes that are critical for Th1 development but dispensable for Tfh differentiation. Funding Source R01 AI125466, AI127481, AI167058, and T32 AI007485 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Menstruation drives large cyclical increases in granulysin in cervicovaginal secretions despite stable production by local immune cells 2300734

Abstract Introduction Granulysin is a potent anti-microbial protein made by NK and T cells. It is abundant in cervicovaginal secretions during the follicular phase of the menstrual cycle but is nearly undetectable during the luteal phase. The mechanism and implications of this change are unknown. Methods Twenty-three participants collected daily vaginal swabs between the mid-follicular and mid-luteal phase and five collected them for a full cycle. Cycle phase was determined by urinary LH and serum progesterone. Endocervical cytobrushes, ectocervical biopsies, and vaginal biopsies were collected at mid-follicular and mid-luteal visits. We measured granulysin levels in the swabs by ELISA and enumerated granulysin-producing cells from the cytobrushes and biopsies by flow cytometry. Results Granulysin concentrations in secretions were highest during menstruation and gradually declined as much as 10,000-fold before becoming mostly undetectable during the luteal phase. In contrast, cellular expression of granulysin in the endocervix, ectocervix, and vagina was stable comparing the mid-follicular to mid-luteal phase. The most abundant granulysin-producing cell type in the mucosa was CD8 T cells, followed by NK cells. NK cells expressed granulysin at a much higher rate, but CD8 T cells were more abundant. In a minority of participants, granulysin was consistently detected in multiple luteal phase swabs; this phenomenon was associated with parity. Conclusion The cyclical changes in granulysin levels in cervicovaginal secretions are driven by menstruation, suggesting that the granulysin derives from uterine NK cells. In contrast, production of granulysin by resident immune cells in the cervix and vagina is independent of the menstrual cycle. These results suggest that cervicovaginal cells remain able to produce granulysin in response to microbial infection throughout the menstrual cycle, despite the large change in granulysin levels in secretions. Funding Source NIH R21 AI164028 Topic Categories Mucosal and Regional Immunology (MUC)

Spatio-temporal mapping of immune cell dynamics during human sequential lymph node metastasis 2309999

Abstract Introduction Regional lymph node (LN) metastasis critically influences distant metastatic progression, anti-tumour immunity, and patient prognosis. While tumour-induced immune modulation in tumour-draining LNs (TDLNs) has been extensively studied using murine models, the systematic reconstruction of the immune system from primary tumours through TDLNs and subsequent lymph nodes in human cancer progression remains understudied. Methods Here, we utilised integrated multi-omics approaches, including imaging mass cytometry, single-cell RNA sequencing, Visium and Xenium spatial transcriptomics, and multi-colour immunofluorescence to systematically characterise immune cell dynamics across 147 paired primary tumours, sentinel TDLNs (S-TDLNs), and secondary axillary LNs (ALNs) obtained from 50 treatment-naïve triple-negative breast cancer patients with different progression statuses. Results We observe depletion of cDC2 and naïve T cells, with expansion of immunosuppressive MARCO+ macrophages associated with survival, as identified by a newly developed single-cell transformer model. Spatial analysis and a unified cell—cell interaction framework reveal disrupted immune communication and impaired dendritic cell—T cell priming circuits within metastatic LNs and matched tumours. In an independent neoadjuvant immunotherapy cohort (36 patients, 52 LNs), preservation of CD1c+ cDC2 predicts pathological complete response and prolonged event-free survival, positioning cDC2 as a biomarker and rational therapeutic target in TNBC. Conclusion Collectively, this systematic mapping of immune landscape alterations during human sequential LN metastasis provides essential insights for understanding cancer metastasis mechanisms and paves the way for innovative immunotherapeutic strategies. Funding Source Royal Society Fund (IEC\NSFC\233577); CRUK Career Development Fellowship (RCCFEL\100095); National Natural Science Foundation of China (Grant No. 81672638, W2421095) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Redox Index and Capacity Analysis (RICA) sheds light on NAD biology in tumor-infiltrating lymphocytes 2251652

Abstract Introduction NAD metabolism is critical for T cell function; however, technical limitations impede interrogation of NAD biology in living immune cells. This is especially true for small, heterogenous cell populations, such as tumor-infiltrating lymphocytes (TILs). We developed the redox index and capacity analysis (RICA) assay, a novel method for measuring mitochondrial NAD content and redox balance. The RICA assay is a flow cytometry-based technique that uses NADH autofluorescence and mitochondrial inhibitors to assess NAD within specific subsets of immune cells. We used this to reveal nuances in NAD biology in T cells from in vitro and ex vivo contexts. Methods We generated CD8 and CD4 T cell subsets in vitro using standard culture conditions. We also assessed antigen-specific CD8 T cell responses ex vivo using an OT-I adoptive transfer system in the context of a vaccinia viral immunization and in a B16 melanoma model. T cell subsets were analyzed using the RICA assay, which measures NADH autofluorescence in specific cell populations at baseline and after rotenone or FCCP treatment to induce maximum and minimum mitochondrial NADH signal. Results We found that effector-like cells had a higher mitochondrial NADH:NAD+ ratio than quiescent cells. We discovered that cells with greater differentiation potential often possessed a larger pool of mitochondrial NAD than terminally differentiated cells in vitro and in our viral model. Tumor localization and differentiation status dramatically affected the mitochondrial NAD pool, but not the NADH:NAD+ ratio, of adoptively transferred CD8 T cells in our melanoma model. Conclusion Our work establishes a tool for evaluating mitochondrial NAD biology in living immune cells at a greater level of detail than previously possible. It also highlights dynamic changes in mitochondrial NAD pool size as a novel element of CD8 T cell biology, which holds significance for improving the efficacy of CD8 T cell-based immunotherapies. Funding Source NIH grants CA257954 and AI115015 to E.J.U., P30CA023108 to Dartmouth Cancer Center Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)