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Multicellular Control of T cell Responses 2257084
Abstract Introduction T cells must maintain a precise boundary between self and non-self to effectively clear pathogens while minimizing collateral damage. Traditionally, maintenance of this boundary has been ascribed to mechanisms operating within individual cells. However, self-activated T cells are common even at homeostasis, implying the need for additional controls. We hypothesized that higher order, multicellular mechanisms, enabled by spatial inhomogeneity within lymphoid tissues, provides this regulation, and that the local density of activated T cells (“activation density”) is the key parameter distinguishing self from non-self responses. Methods We immunized mice with protein antigens containing peptides of varying diversity to modulate activation density during the initial priming phase. Using an in vivo dye-labeling approach, we identified endogenous antigen-presenting cells (APCs) engaged in T cell priming. We applied highly multiplexed confocal microscopy to draining lymph nodes to visualize how activation density shapes T cell activation states. We complemented these experiments with a computational model to explore physiological parameters influencing activation density. Results We found that antigen peptide diversity tuned activation density, giving rise to activation dense multicellular domains around antigen-bearing APCs. Elevated activation density was associated with reduced regulatory T cell control, enhanced cytokine sensitivity, and increased proliferative potential. Importantly, spatial coordination among activated T cells was required for achieving high activation density. Conclusion Together, these findings reveal that T cell activation outcomes are not solely determined by cell-intrinsic signaling, but also by collective organization within tissue microenvironments. Such multicellular control mechanisms provide a new framework for understanding how immune responses distinguish self from non-self and may explain how subtle changes in spatial organization give rise to pathological states. Funding Source This work was supported by the Ragon Institute under a Strategic Initiative grant, Massachusetts General Hospital, the Massachusetts Institute of Technology, a Mark and Lisa Schwartz Foundation-Ragon Institute Initiative in Artificial Intelligence, Machin Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Development of a Human Lymphatic Filariasis Vaccine, LFGuard for Clinical Testing 2306046
Abstract Introduction Lymphatic filariasis (LF) is a neglected tropical disease that causes disability and impairment of the lymphatic system. Millions of people in the endemic area still suffer from LF, even after decades of mass drug administration (MDA). The target of MDA is to block the transmission. A preventive vaccine is needed to eliminate the infection in the endemic countries. Our laboratory developed a tetravalent fusion protein vaccine, rBmHAXT, which demonstrated significant protection and immunogenicity in rodent and non-human primate models. The vaccine has now completed the Good Manufacturing Practice (cGMP) guidelines for use in clinical trials. Methods This study aims to confirm whether the final cGMP product (LFGuard) retains the safety, immunogenicity, and efficacy as observed with the parent vaccine in a mouse model. Three doses of the vaccine were given s/c or I/m along with Al-T4 adjuvant, an alum adsorbed TLR-4 agonist to promote a balanced Th1/Th2 response against the filarial infection. To evaluate safety, we measured the body weight, haematological parameters, liver function, and kidney function. Immunogenicity was determined by evaluating the levels of antigen-specific IgG antibody titer. The vaccine efficacy was determined by a challenge study. Results Our results show that vaccine is safe and has no adverse effects on the body weight, haematological parameters, liver or kidney functions in all vaccinated animals compared to controls irrespective of the route of immunization. All immunized mice showed a high titer of antigen-specific IgG antibodies (1:80,000) in their serum as expected. These mice also had a significantly (p > 0.001) higher number of CD4+ TCM in their spleen. The vaccinated mice also showed significantly higher levels of protection (90.7%) compared to the controls. Conclusion This is the most crucial step in bridging the vaccine to phase I clinical testing. These pre-IND-enabling studies will help bring the LF vaccine close to the clinics. Funding Source N/A Topic Categories Vaccines and Immunotherapy (VAC)
Fate mapping with Cd3giCre reveals a subset of ILC2s prevalent in pre- and neonatal stages 2259493
Abstract Introduction Group 2 innate lymphoid cells (ILC2s) are innate counterparts of T cells, and share with T cells transcription factors and effector cytokines. Unlike T cells, they lack antigen-specific receptors, allowing them to respond rapidly to tissue changes during infection, damage, or allergen exposure. Residing in mucosal surfaces, ILC2s also regulate tissue homeostasis. While ILC2s are thought to originate from the bone marrow, our lab has shown that the thymus contributes to ILC2 pools in various tissues. Methods Our previous data suggested that ILC2s of thymic origin express Cd3 genes, therefore we generated Cd3giCre mice and crossed them with Rosa26stop-ZsGreen, to label Cd3g-expressing ILC2s with ZsGreen (ZsG). ZsG signal can be detected by flow cytometry, and RNA sequencing revealed differences between ZsG+ and ZsG- ILC2s. Results We found ZsG expression begins in the thymus at the early stage of T cell development and persists in mature T cells. The absence of ZsG signal in bone marrow progenitors, myeloid and B cell lineages indicates the thymus specificity of Cd3giCre. ZsG+ ILC2s are enriched in neonatal lungs, when lung alveolarization occurs, which suggests a supporting role of ILC2s in lung development and remodeling. RNA sequencing revealed that ZsG+ ILC2s express high levels of Il13, Il4, and Klrg1, indicating their higher activation status. ZsG+ ILC2s also harbor rearranged Tcrb, Tcrg and Tcrd genes, suggesting shared developmental pathways with T cells in the thymus. We used naphthalene lung injury model to explore the role of ILC2s in the lung repair in neonatal mice, which showed that ILC2s are essential for macrophage polarization, highlighting their role in tissue repair. Conclusion This study reveals a previously unrecognized thymic contribution to ILC2s early in life, when bone marrow is not yet fully functional. These findings may help explain differences in pediatric versus adult immunity, and provide hints to therapeutic strategies. Funding Source NIH R01 AI178947, NIH R03 AI175661, AAI’s RESTORE Grants Program Topic Categories Hematopoiesis and Immune System Development (HEM)
Dual-Adjuvanted Meningococcal OMVs C Vaccine: Functionality and Cross-Reactive IgG in BALB/c Mice 2267478
Abstract Introduction Meningococcal disease is a major public health issue in Brazil and worldwide, especially among children and young adults. Broad-coverage vaccines are lacking in Brazil, and the high genetic variability of Neisseria meningitidis strains limits the efficacy of current vaccines. This study evaluated the immunogenicity of outer membrane vesicles (OMVs) from serogroup C combined with Cholera Toxin Subunit B and Alum adjuvants. Methods BALB/c mice received three subcutaneous doses on days 10, 40, and 55. IgG and subclasses (1, 2a, 2b and 3) were analyzed by ELISA; IgG functionality by ELISA-avidity. Immunoblot tested IgG recognition of OMVs from homologous strains, and Dot-blot evaluated cross-reactivity against heterologous strains circulating in Brazil. Results IgG responses increased after the second dose (p < 0.001) and further after the third (p < 0.0001). High-avidity IgG was detected after the second (60.31 ± 16.31) and third doses (76.35 ± 14). Predominant IgG1 indicated a Th2-skewed profile. The third dose enhanced IgG diversity and specificity, with recognition of antigens between 25—58 kDa and 100—190 kDa from the homologous strain. Cross-reactive IgG recognized epidemiologically relevant heterologous strains from serogroups B, C, and W. Conclusion The OMV/C with CTB and Alum formulation induces a robust and functional immune response, supporting its potential as a candidate for broader-spectrum vaccines against diverse Brazilian Neisseria meningitidis strains. Funding Source CAPES — Coordination for the Improvement of Higher Education Personnel Topic Categories Vaccines and Immunotherapy (VAC)
Macrophage Anti-bacterial Activity is Controlled by Adenylate Kinase 4-Mediated Mitochondrial DNA Synthesis 2241601
Abstract Introduction Macrophage anti-bacterial activity requires mtROS production. Macrophage-specific gene upregulation that participates in mtROS-mediated anti-bacteria process remains unclear. Methods We use Listeria monocytogenes (Listeria) and Salmonella typhimurium (Salmonella) as infection models. Results We showed that Listeria and Salmonella infections in human and mouse macrophages increased mtDNA copy number through which to dictate anti-bacterial activity. Interestingly, adenylate kinase 4 (Ak4) expression was upregulated in macrophages after infection. In vivo, Ak4 KO and macrophage-specific Ak4 KO mice became highly susceptible to bacterial infections. We provide evidence that Ak4 is critical for increased mtDNA synthesis and mitochondrial mass in macrophages after bacterial infection. The biochemical function of Ak4 is to transfer a phosphate group from ATP/GTP to (d)AMP for (d)ADP formation, and the K18A and G89S/A166D mutations abolish this function. Unlike Ak4WT, the expression of these mutants in Ak4 KO mice failed to restore the function of Ak4 in mediating the increase of mtDNA synthesis. Conclusion Our results suggest that induction of Ak4 after infection produces more dADP whose conversion to dATP in mitochondria supports mtDNA synthesis and the subsequent increase of mtROS production. Loss of this metabolic coupling in Ak4 KO macrophages diminishes the anti-bacterial activity. Together, our findings highlight the vital role of Ak4 in macrophage defense against pathogenic bacteria. Funding Source Ministry of Education in Taiwan, National Taiwan University (111L892902 and 112L891702) National Science and Technology Council of Taiwan (111-2320-B-002-068-MY3) Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
MOF‐Derived FeNi/C Composites Constructed by Controlled Etching for High‐Performance Electromagnetic Wave Absorption
ABSTRACT The rapid growth of electronic devices has intensified electromagnetic wave pollution. Electromagnetic wave absorption (EWA) materials provide a green solution by converting electromagnetic energy into thermal energy. Achieving high‐performance EWA requires a fine balance between impedance matching and energy dissipation, demanding precise control of microstructure, interfaces, and electronic states of the materials. However, complex multiscale structures involve strongly coupled structural evolution, which reduces controllability and hinders clear structure–performance correlation. Here, a cobalt‐based metal–organic framework (Co‐MOF) is employed as the precursor. By regulating the contents of Ni 2 + and Fe 3 + , the structural evolution and the modulation of localized electronic states during the etching process are systematically elucidated. In addition, the in situ competitive coordination and etching‐competitive coordination systems reveal the mechanistic differences between atomic‐scale induced reconstruction and directional destructive reconstruction. Benefiting from synergistic regulation spanning atomic, nanoscale, and microscale levels, the obtained FeNi/C composite achieves an effective absorption bandwidth of 7.13 GHz at an ultrathin thickness of 1.97 mm. Combining DFT, COMSOL, and CST simulations, the role of etching engineering in enhancing EWA performance is elucidated from electronic, local‐field, and macroscopic perspectives, providing a theoretical basis for its controllable application and the rational design of high‐performance absorbers.
Granzyme A Regulates Uterine Immune-Endocrine Homeostasis in the Female Reproductive Tract 2302876
Abstract Introduction The female reproductive tract (FRT) requires tightly regulated immune responses to maintain tissue homeostasis, ensure hormonal balance, and support fertility. Dysregulation of this balance contributes to infertility, chronic pelvic inflammation, and poor pregnancy outcomes. Although immune cells are known to play a central role in maintaining FRT equilibrium, the underlying mechanisms remain incompletely understood. Here, we identify granzyme A (GzmA), a serine protease predominantly produced by ovarian and uterine natural killer (NK) cells, as a key regulator of the FRT. Methods We used wild-type and GzmA-deficient female mice to assess reproductive inflammation, estrous cycle patterns, uterine and ovarian morphology, and fertility outcomes. Vaginal cytology, gross tissue measurements, immunofluorescence imaging, qPCR, flow cytometry, and breeding records were used to evaluate structural, hormonal, and immune changes associated with GzmA loss. Results Loss of GzmA resulted in marked alterations in uterine morphology, disrupted estrous cycle synchronization, and subfertility. These included increased uterine weight, shortened uterine horns, excessive mucus production, enlarged endometrium, and evidence of hormonal imbalance, features commonly associated with reproductive pathologies such as endometriosis and Polycystic Ovary Syndrome (PCOS). Notably, recent clinical studies report reduced GzmA levels in the peritoneal fluid of patients with endometriosis, aligning with our findings and supporting a role for impaired granzyme signaling in the disease. Conclusion These findings reveal a previously unrecognized role for NK cell-derived GzmA in regulating endometrial growth and preserving immune-hormonal homeostasis in the FRT and suggest that dysregulation of this pathway may contribute to female reproductive disease. Funding Source The Women’s Global Health Institute (WGHI) at Purdue University Topic Categories Mucosal and Regional Immunology (MUC)
The bacterial quorum-sensing molecule 2’-aminoacetophenone reprograms macrophage metabolism and sustains histone lactylation for persistence 2253661
Abstract Introduction Pseudomonas aeruginosa (PA) uses its quorum-sensing molecule 2’-aminoacetophenone (2-AA) to modulate host mitochondrial activity. This study investigates how 2-AA—driven host metabolic reprogramming contributes to macrophage dysfunction and supports bacterial persistence. Methods We used biochemical and molecular assays to show that 2-AA induced host lactate augmentation. Immunoprecipitation identified proteins involved in histone lactylation (Kla), while CUT&RUN and transcriptomics studies deciphered gene regulation and molecular anergy. Results Mechanistically, 2-AA disrupts the ESRRA—PPARGC1α regulatory axis, leading to the downregulation of the mitochondrial pyruvate carrier (MPC1). This impairment affects pyruvate transport into mitochondria, rewiring cellular metabolism to a glycolytic state, leading to increased lactate dehydrogenase A (LDHA) activity, elevated and sustained lactate levels in PA-infected immune cells and host tissues, and Kla. Genome-wide profiling of H3 lysine 18 lactylation (H3K18la) demonstrated distinct chromatin modification at regulatory regions, indicating novel epigenetic regulation by lactylation. The 2-AA-mediated H3K18la involves the GTP-specific succinyl-CoA synthetase (GTPSCS) and its interaction with histone lactyl-transferases CREB-binding protein (CBP) and p300. In agreement with H3k18la signatures, transcriptomic profiling of wild-type PA and its 2-AA-deficient mutant revealed regulatory pathways modulating immune and metabolic responses. Functionally, enhanced H3K18la favors a tolerogenic macrophage phenotype that supports intracellular bacterial survival. Conversely, inhibiting lactate accumulation or blocking 2-AA synthesis diminishes H3K18la and enhances bacterial clearance. Conclusion Collectively, these findings uncover a previously unrecognized QS-regulated metabolic—epigenetic axis through which PA manipulates host immunity, highlighting lactate metabolism as a potential therapeutic target for combating chronic Pseudomonas infections. Funding Source N/A Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
IRF2 deletion in regulatory T cells enhances anti-viral and anti-tumor immune responses 2257347
Abstract Introduction T regulatory cells (Tregs) are vital to limit immunopathology, yet they can also be an immune deterrent during chronic infections and cancer. Interferon Regulatory Factor 2 (IRF2) is a transcription factor that negatively regulates type I interferon (IFN-I) signaling. In response to viral infections, Tregs integrate IFN-I signals to limit their activity and enable antiviral immunity. Methods To understand how IRF2 alters Treg activity during chronic infections, we generated mice with Treg-specific conditional knockout (cKO) of IRF2 and infected them with chronic lymphocytic choriomeningitis virus (LCMV). Mouse melanoma models were used to study role in cancer. Results After infection, Treg numbers were almost entirely abolished in cKO mice, and the few remaining Tregs exhibited high IFN-I signaling and an activated phenotype. Lack of IRF2 expression by Tregs reduced CD8 T precursor exhausted (Tpex) cells and increased the amount and functional activity of effector virus-specific CD8 T cells. cKO mice also had more functional virus-specific CD4 T cells with a skew toward Th1 differentiation during chronic infection. Consistent with the diminished CD8 T cell exhaustion programming in chronic LCMV infection, ∼50% of cKO mice died within the first 2 weeks of infection, while the surviving mice exhibited decreased virus titers and accelerated control of the chronic infection. In response to melanoma, IRF2-deficiency in Tregs enabled better tumor control and substantially extended overall survival. Conclusion Thus, IRF2-serves as a key checkpoint in Tregs to balance IFN-I signaling, with important implications toward immune mediated control of chronic infections and cancer. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Single-Cell Insights into Endothelial Cell Heterogeneity in the Thymus 2267370
Abstract Introduction The thymus contains a complex vascular network that supports progenitor entry, thymocyte egress, and blood-thymus barrier integrity. Endothelial cells (ECs) line the luminal surface of blood and lymphatic vessels and mainly regulate these processes. However, their diversity and distinct functions remain unexplored in the thymus. Methods Thymi will be harvested from 6-week-old C57BL/6 mice, with stromal cells isolated from enzymatic digestion and density gradient centrifugation. ECs will be sorted by flow cytometry for single-cell RNA sequencing. Single-cell barcoding and cDNA synthesis will be performed using 10X Genomics Chromium platform and libraries sequenced on an Illumina platform. Data will be processed with Cell Ranger and analyzed in R using Seurat for dimensional reduction, clustering, and differential gene expression. Results We anticipate that this analysis will reveal transcriptionally distinct EC subsets corresponding to structurally distinct vessels. These subsets are expected to contribute to thymic homeostasis and differentially express genes involved in barrier function/regulation, cytokine signaling, and tissue regeneration. Conclusion This study will be the first to provide a single-cell atlas of thymic ECs, revealing specialized roles of thymic EC subtypes. These findings will establish a framework for further studies on how EC diversity contributes to thymic organization, function, and regeneration. Funding Source National Heart, Lung, and Blood Institute of the National Institute of Health (R01HL171971-01A1), Institute of General Medical Sciences of the National Institutes of Health (T32GM141862) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Smoking Tobacco Increases Extracellular Vesicles and Alters Their Cargo in the Blood of Multiple Sclerosis Patients 2259575
Abstract Introduction Multiple sclerosis (MS) is a chronic disease of the central nervous system that is characterized by a widely variable prognosis. While some patients experience a relatively mild course, others suffer from severe disease progression. Tobacco smoke is an environmental factor known to worsen MS outcomes; however, the mechanism by which it exerts its detrimental effects is unclear. Methods We used the OLINK-Explore assay to profile 1,466 serum proteins in MS patients who were smokers or non-smokers, as well as in healthy controls. Extracellular vesicles (EVs) were isolated, and mass spectrometry was performed to identify differentially abundant proteins. In vitro experiments using cigarette smoke extract (CSE) were conducted to assess EV release from immune cells, including T cells, B cells, and monocytes. Additionally, a 3-D human airway tissue model was utilized to identify the primary cell types producing vesicles in the lung microenvironment. Results Smoking significantly increased levels of proteins associated with the EV cellular compartment, an effect more pronounced in patients compared to healthy individuals. Isolation of EVs confirmed that MS smokers had significantly more plasma exosomes (30—150 nm vesicles) compared to non-smoker patients and healthy non-smokers. Mass spectrometry of exosomes revealed 20 differentially abundant proteins, including elevated SerpinA1 in non-smokers and Trim33 in smokers. In vitro, CSE directly induced EV release from immune cells, with monocytes identified as the major contributors in the 3-D human airway tissue model. Conclusion These findings suggest that tobacco smoke drives the release of extracellular vesicles, potentially exacerbating immune cell dysfunction in MS. Ongoing research aims to further characterize the cargo and functional differences of EVs in smoker and non-smoker patients to better understand their role in disease. Funding Source NIH, Presbyterian Health Foundation Topic Categories Basic Autoimmunity (BA)
Exosome-like nanoparticles derived from Ipomoea aquatica promote dendritic cell—mediated anti-tumor immunity 2267679
Abstract Introduction Plant-derived extracellular-like nanoparticles (ELNs) have recently emerged as natural bio-nanoparticles capable of modulating immune responses and serving as novel platforms for cancer immunotherapy. Ipomoea aquatica (water spinach) is an edible plant known for its antioxidant and immunoregulatory properties, but its nanovesicle fraction has not been studied in the context of anti-tumor immunity. This study aimed to investigate whether I. aquatica-derived ELNs (IA-ELNs) can activate dendritic cells (DCs) and promote anti-tumor immune responses. Methods IA-ELNs were isolated from I. aquatica leaves using differential centrifugation and characterized by transmission electron microscopy and nanoparticle tracking analysis. Bone marrow—derived dendritic cells (BMDCs) from C57BL/6 mice were treated with IA-ELNs, and surface markers (CD80, CD86, MHC-I, MHC-II) were analyzed by flow cytometry. Cytokine production (IL-12p70, TNF-α, IL-10) was quantified by ELISA. Mixed lymphocyte reactions (MLRs) were performed to assess T-cell activation. MAPK and NF-κB signaling were examined by Western blotting. Results IA-ELNs treatment significantly enhanced DC maturation by increasing CD80, CD86, MHC-I and MHC-II expression. Cytokine analysis revealed elevated IL-12p70 and TNF-α secretion, indicating Th1-polarizing activity. In MLR assays, IA-ELNs—treated DCs promoted proliferation of CD4+ and CD8+ T cells. Mechanistically, IA-ELNs activated ERK, JNK, and p38 phosphorylation as well as NF-κB signaling, consistent with an immunostimulatory phenotype. Conclusion IA-ELNs promote dendritic cell activation and enhance T-cell—mediated anti-tumor immunity via MAPK and NF-κB signaling pathways. These findings suggest that IA-ELNs represent a promising plant-based nanoplatform for developing safe and effective immunotherapeutic agents. Funding Source n/a Topic Categories Classical and Non-Classical Antigen Presenting Cells (APC)
GARP+ Erythroid Regulatory Cells (G-ERCs) define spatially organized immunosuppressive niches that restrain anti-tumor immunity 2259805
Abstract Introduction Erythroid precursors have emerged as regulators of tumor immunity, yet their suppressive mediators and spatial context remain unclear. We identified a distinct population of GARP+ Erythroid Regulatory Cells (G-ERCs) that express the TGFβ docking receptor GARP (encoded by LRRC32). Given GARP’s known role in immune regulation, we hypothesized that these erythroid-derived cells create immunosuppressive niches within the tumor microenvironment (TME) that dampen antitumor responses. Methods Erythroid-specific Garp-knockout (KO) mice (EporRCreLrrc32fl/fl) bearing MB49 bladder tumors were analyzed by high dimensional flow cytometry, validated by adoptive co-transfer of Pmel CD8+ T cells with Garp-KO or WT G-ERCs. 10x Visium transcriptomics were used to identify Lrrc32+ regions with positive signature defining G-ERCs. Multiparametric immunofluorescence (mIF) and immune deconvolution validated G-ERC localization and surrounding immune composition. Results Tumor growth was significantly slower in erythroid-specific Garp-KO mice compared to WT controls, and in tumor-bearing mice receiving Pmel cells co-transferred with Garp-KO ERCs versus WT G-ERCs. Spatial transcriptomics revealed organized G-ERC enriched regions, enriched for TGFβ, hypoxia, and oxidative-stress pathways. Integrated mIF and deconvolution confirmed that G-ERC niches were surrounded by areas with reduced dendritic cells and B cells, increased myeloid cells, and lower cytotoxic and interferon-response activity. Garp deletion remodeled the TME, increasing CD8+ infiltration and effector activation, enhanced CD8+ T-cell effector differentiation while reducing exhaustion programs, as also validated by flow cytometry. Circulating G-ERC like cells were also detected in patients with cancer. Conclusion G-ERCs represent a regulatory erythroid subset forming spatially organized niches suppressing T-cell immunity. Targeting erythroid GARP enhances antitumor responses and represents a novel link between erythropoiesis and cancer immunity. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Selective Growth of Bulk‐Like Perovskite in Plasmonic Nanoholes for Enhanced Two‐Photon‐Excited Emission
ABSTRACT Plasmon‐enhanced nonlinear optical phenomena in hybrid perovskite‐plasmonic systems present significant opportunities for photodetection, bioimaging, and quantum light sources. However, achieving uniform growth of luminescent materials within confined plasmonic hotspots remains challenging, as conventional surface deposition or infiltration approaches cannot fill nanocavities effectively. To resolve this, we present a vacuum‐assisted capillary infiltration (VACI) strategy for delivering precursor solutions into gold (Au) nanohole (NH) arrays, enabling in situ crystallization within plasmonic cavities. This work demonstrates selective bulk‐like cesium lead bromide (CsPbBr 3 ) growth inside NHs through precise infiltration and controlled crystallization. By integrating the capillary effect with vacuum pressure, this approach ensures uniform filling beyond conventional methods. The successful integration of CsPbBr 3 was confirmed through peel‐off techniques and structural/chemical analyses. Electromagnetic simulations predict an average field enhancement of | E/E 0 | ≈2.80 at 800 nm, consistent with an experimentally observed ∼40‐fold increase in two‐photon absorption (TPA)‐induced green emission ( λ ≈526 nm). Unlike prior TPA systems employing quantum dots, rare‐earth doped crystals, or organic chromophores, this approach integrates bulk‐like perovskite directly into plasmonic NH cavities. This establishes a general framework for material‐structure integration in plasmonics and nanophotonics, and a foundation for infrared photodetection, high‐resolution nonlinear imaging, and quantum light sources.
Engineered TCR therapy targeting lipid antigens in AML 2306228
Abstract Introduction Acute myeloid leukemia (AML) is the most common form of adult leukemia and has a poor prognosis with a 5-year overall survival of ∼30%. While CAR-T has revolutionized the treatment of other hematological malignancies, it has been difficult to replicate this same success in AML. Healthy myeloid cells often share expression of putative AML targets and have led to significant toxicity in clinical trials. These challenges necessitate examining unexplored target antigens with higher cancer specificity, such as lipids. Methods This project aims to target lipid antigens presented by CD1d, an MHC Class 1-like molecule that presents lipid antigens to unconventional T cells such as iNKT cells. We aim to develop a lipid-targeting cell therapy for AML by engineering conventional T cells with a CD1d-targeting TCR derived from iNKT cells. These engineered T cells (herein called CD1d-targeting T cells) were co-cultured with CD1d+ AML cell lines with a-GalCer, a canonical iNKT agonist, or DMSO as a vehicle control for up to 72 hours and at varying effector: target ratios. Cells were analyzed or cytotoxicity via flow cytometry and supernatant was collected for ELISA. Results CD1d-targeting T cells demonstrated robust cytotoxicity against CD1d+ AML cell lines in the presence of a-GalCer. CD1d KO cells (THP-1) showed no killing in the presence of a-GalCer or DMSO. Cell lines with higher CD1d expression (OCI-AML3) displaying targeted killing without the presence of a-GalCer. IFN-y ELISA results corroborate results from our killing assays and show pronounced IFN-y production in culture conditions that have high CD1d expression and/or presence of a lipid antigen. Conclusion Our engineered T cells showed CD1d-specific killing that was dependent on CD1d expression, and in cases of high CD1d expression, may be dependent on endogenously self-lipids found in AML cells. Further work will be done to identify putative lipid antigen targets and establish in vivo efficacy and safety our CD1d-targeting T cells. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)
Lipin-1 promotes mitochondrial respiration to facilitate macrophage pro-resolving responses 2253765
Abstract Introduction Atherosclerotic cardiovascular disease, a chronic inflammatory disorder, remains the leading cause of death globally. Macrophages (Møs) promote inflammation resolution and atherosclerosis regression by eating up apoptotic cells via efferocytosis. This imposes a metabolic burden on Møs that requires the efficient catabolism of lipids derived from efferocytic cargoes for effective pro-resolving responses and plaque stability. Our data suggest that lipin-1 attenuates atherosclerosis and promotes inflammation resolution by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and efferocytosis. However, the mechanism by which lipin-1 aids OXPHOS and efferocytosis in pro-resolving macrophages is unknown. Therefore, we hypothesize that lipin-1 drives these processes by facilitating cardiolipin synthesis. Methods We used flow cytometry, fluorescence microscopy, mass spectrometry and RNA interference to assess cardiolipin levels and their impact on efferocytosis in lipin-1 knockout (lipin-1 KO) and wildtype (WT) bone marrow-derived Møs. Also, mitochondrial-ER contact sites (MERCs) formation in WT and lipin-1 KO Mø was observed by electron microscopy. Results Lipin-1 KO Møs had a significant reduction in cardiolipin, a mitochondria-exclusive phospholipid critical for mitochondrial structure and OXPHOS. Reducing cardiolipin synthesis in WT Møs significantly impaired their efferocytic capacity. Also, in response to IL-4, lipin-1 KO Møs exhibited significantly fewer MERCs. MERCs regulate the transfer of lipid precursors essential for cardiolipin synthesis in the mitochondria. Conclusion Our findings show that lipin-1 aids MERCs formation and cardiolipin synthesis to support macrophage efferocytosis and pro-resolving responses. This deepens our insight into the cellular and molecular mechanisms driving inflammation resolution and atherosclerosis regression, which may inform future studies on metabolic regulation of immune responses in chronic inflammation. Funding Source 1. NIH under grant number P20GM134974 and R01HL163106, 2. CCDS under grant number CCDS000013, 3. AHA predoctoral fellowship under grant number 25PRE1373728 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Sex-Specific Loss of SMS2-regulated Germinal Center B Cell Tolerance in Systemic Lupus Erythematosus 2257381
Abstract Introduction Systemic Lupus Erythematosus (SLE) is an antibody-mediated autoimmune disease with a striking female predominance. However, the mechanisms underlying this female bias remain poorly understood. Germinal Center (GC) B cell tolerance, a critical self-protective process that eliminates spontaneously generated autoreactive B cells, is essential for preventing SLE. However, whether impairment of GC B cell tolerance is sex-specific remains unclear. We previously identified the sphingomyelin synthetase 2 (SMS2)/protein kinase C δ (PKCδ) pathway as a critical regulator of GC B cell tolerance in mice, and that this pathway is impaired in lupus patients. Here, we investigated whether this impairment is specific to females using a lupus-prone mouse model. Methods Lupus prone NZBWF1 mice were used as the female bias of disease incidence is similar to that seen in humans. C57BL/6 mice were used as non-autoimmune controls. Untouched GC B cells and naïve B cells were isolated from male and female NZBWF1 mice for RT-PCR analysis of Sgms2 mRNA levels. In addition, GC and non-GC B cell SMS2 protein levels in male and female NZBWF1 mice were analyzed by flow cytometry. To understand the significance of reduced GC B cell SMS2 in lupus development, female NZBWF1 mice were treated with 2OHOA, a SMS activator, to test whether activation of the SMS2/PKCδ tolerance pathway could alleviate lupus pathogenesis. Results RT-PCR revealed that Sgms2 mRNA was significantly reduced in female NZBWF1 GC B cells when compared to males, but not in female C57BL/6 mice. Flow cytometry analysis also revealed a significant reduction in GC B cell SMS2 protein levels in female NZBWF1 mice compared to males. Activation of the SMS2/PKCδ tolerance pathway with 2OHOA significantly alleviated lupus pathogenesis in female NZBWF1 mice Conclusion These findings support that loss of GC B cell SMS2 expression is a female-specific deficit in SLE, which likely contributes to female bias in lupus. Funding Source SUNY Downstate Trainee Research Grant Topic Categories Basic Autoimmunity (BA)
TLR9 and Type II IFN signaling in monocyte-derived macrophages orchestrate CD8 T cell activity in DNaseII deficiency-induced autoimmune hepatitis 2309847
Abstract Introduction Aberrant accumulation of nucleic acids underlies a variety of sterile inflammation—induced liver diseases, as evidenced by patients with hypomorphic mutations in DNase2. DNaseII⁻/⁻ IFNαR⁻/⁻ double-knockout (DKO) mice recapitulate key features observed in these patients. However, it remains unclear which nucleic acid sensors are responsible for triggering this autoimmune hepatitis (AIH) phenotype and how they do so. In this study, we aim to examine how nucleic acid—induced signaling alters the heterogeneity of liver macrophages and shapes CD8+ T cell function in autoimmune hepatitis. Methods Deep profiling of liver immune cells was performed by spectral flow cytometry and immunofluorescence staining, scRNAseq and bulk RNAseq analysis. The roles of endosomal TLRs and IFNγ are explored using genetically ablation and use of bone marrow radiation chimeras. Results We observed extensive expansion of myeloid cells and CD8+ tissue resident memory T in the DKO liver that depends on TLR9. The infiltrating Ly6Chi inflammatory monocytes rapidly differentiate into monocyte-derived Kupffer cells and eventually replace the original embryonic Kupffer cells. These infiltrating monocyte-lineage cells exhibit a stronger inflammatory signature marked by elevated expression of genes related to antigen-presentation. Notably, TLR9 is hihgly expressed in monocyte-derived macrophages and is required for the differentiation process. Transcriptome analysis comparing monocyte-lineage cells sorted from DKO and TLR9 TKO livers reveal a robust TLR9-dependent IFN signature that is induced by IFNγ. The initial recruitment of TLR9 expressing monocytes, subsequent activation of lymphocytes, and other aspects of hepatic inflammation in DKO mice are completely dependent on IFNγ signaling Conclusion Taken together, these findings highlight a critical synergy between IFNg and TLR9 signaling in liver infiltrating monocyte-lineage cells that promotes activation of CD8+ T cells in the pathogenesis of AIH. Funding Source NIH grant R37 AI155901 Topic Categories Basic Autoimmunity (BA)
Inside the wavering mind of an NK cell: Mathematical Modeling of NK cell Activation 2256230
Abstract Introduction The paradigm of NK cell activation is the “Missing-Self Hypothesis”; NK cells kill cells that lack MHC. When considering a single interaction between an NK cell and a target cell, the “Missing-Self Hypothesis” offers a plausible explanation for NK cell activation. However, an NK cell’s interaction with a target cell is not an isolated event. Experimental studies have shown that NK cells in an MHC-deficient environment, fail to lyse cells lacking MHC. Curiously, a study by the Yokoyama lab found that when 10% of cells in a well-mixed environment lacked MHC, the NK cells failed to lyse most of the cells. Our hypothesis is that target cell recognition is less about the balance of the presence or absence of markers, and more so about detecting outliers in an environment. Furthermore, we propose that NK cells will tolerate a small population of cells without MHC only if the cells lose MHC slowly. Methods We developed a mathematical model of how NK cells assess and learn from their environment to effectively lyse harmful outliers. To capture NK cell learning, an NK cell estimates the environment’s MHC distribution by interacting with target cells. An NK cell lyses a target cell if its MHC expression is an outlier based on its estimated MHC distribution. Results To model an infection, where the infected cells have lower MHC expression, preliminary results show that slightly varying the mean of the infected cell population results in either complete elimination or tolerance of the infected cells. To experimentally test our model predictions, we have initiated a collaboration with the Weis lab at the Huntsman Cancer Institute. Conclusion Our preliminary results suggest NK cells may be vulnerable to “slow growing danger” which makes detecting outliers challenging. We are currently creating a dynamic model to explore the rate of MHC suppression and viral spreading on NK cell reactivity. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)
Multi-Mechanistic Approaches to Disarming IgE-Mediated Allergic Diseases 2304105
Abstract Introduction Pathogenic IgE production contributes to a wide range of allergic and atopic diseases. Binding of allergen-specific IgE to its high-affinity IgE receptor FcεRI on basophils and mast cells results in allergen-mediated cross-linking of FcεRI-bound IgE triggering the release of inflammatory mediators causing allergic reactions. We briefly highlight here Seismic’s multiple approaches to rapidly disarm the allergic effector cells. These include (i) cleaving and eliminating circulating IgE, (ii) dissociating IgE from effector cells and down-modulating effector function, and (iii) sweeping IgE from circulation for clearance. Here we will focus on the discovery and design of our IgE dissociator that rapidly dissociates IgE from FcεRI on effector cell surfaces and preventing cell activation, as well as preventing binding of free IgE to FcεRI and FcεRII. Methods IgE dissociators, discovered from an immunization campaign, were screened by surface plasmon resonance and in primary human cell-based assays using flow cytometry for their ability to dissociate IgE from FcεRI. Confirmatory basophil activation assays were performed on human whole blood. Pharmacokinetics (PK), pharmacodynamics (PD) and in vivo efficacy were tested using relevant preclinical models. Results The IgE dissociator rapidly dissociates IgE from the high affinity receptor FcεRI on basophil and mast cell surfaces without triggering degranulation, and shows superior IgE dissociation and FcεRI down modulation compared to standard of care in preclinical models. It exhibits multi-pharmacology by preventing free IgE binding to FcεRI and FcεRII. It demonstrates favorable PK, PD and efficacy in murine model of acute anaphylaxis. Conclusion Our IgE dissociator molecules address multiple aspects of IgE pathology by rapidly removing FcεRI-bound IgE and neutralizing free IgE without triggering effector-cell activation, providing a unique and potentially more effective approach to treat IgE-driven allergic and atopic diseases. Funding Source n/a Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)