Browse Articles
Discover research articles across all indexed journals
Stem cell therapy improved nerve regeneration and functional outcomes in vascularized composite allotransplantation 2258692
Abstract Introduction Vascularized composite allotransplantation (VCA) includes limb and face transplants an emerging field to improve the quality of life of limb amputees. We investigated whether adult bone marrow derived mesenchymal stem cell (MSC) therapy can improve functional outcomes in limb transplantation (Tx). Methods We performed orthotopic syngeneic hind-limb Tx in Lewis rats (n = 5/group). MSC (2 million; passage ≤6) or vehicle was administered locally and intravenously. Sensory function (SF) and motor function (MF) of the Tx limb was assessed weekly. At the study endpoint (16 weeks) rats were euthanized and limb tissues were harvested for cell/molecular analysis. Results Total SF recovery (on a scale of Grade 0-3) improved from 1.4 in 8 weeks to 2.6 by week 16. SF in the tibial and peroneal nerve boundaries recovered by ∼5 weeks. MF (on a scale of Grade 0-4) improved (P < 0.05) with MSC compared to control by week 16 (Grade 2.6 Vs 0.6). Gastrocnemius muscle was atrophied (P < 0.05) and rats developed mild flexion-contractures in MSC treated rats (Grade 0.9) compared to untreated control rats (Grade 1.6) on a scale of 0-3. Rat MSCs were CD29+, CD44+, CD90+, MHC Class-I+, CD31-, CD34-, MHC Class II-; and multi-potent. Immuno histochemistry showed increased (P < 0.05) expression of nerve regeneration markers (NF-M, S100, Oct6, Krox20/EGR2, GAP43) in the sciatic nerve of MSC treated rats compared to controls. Conclusion MSC therapy improved limb transplant functional recovery and is attributed to improved nerve regeneration and muscle innervation. Funding Source CDMRP-DoD Topic Categories Transplantation Immunology (TRAN)
35-color CytoFLEX mosaic panel profiling B cells, myeloid cells, and diagnostic markers in chronic lymphocytic leukemia (CLL) 2306495
Abstract Introduction Advances in spectral flow cytometry have enabled high-resolution analysis of immune cell diversity, particularly within the B cell compartment. Building on previous work, we refined and validated a high-dimensional spectral B cell panel using PBMC samples from CLL patients. The panel was optimized for the CytoFLEX mosaic 88 Spectral Detection Module integrated with the CytoFLEX LX platform with UV laser capability. Methods The panel incorporates refined fluorophore selection and marker combinations to resolve major and minor B cell subsets, including transitional, naïve, double-negative, switched and unswitched memory, and antibody-secreting cells, while maintaining robust spectral unmixing and minimizing overlap. It supports simultaneous assessment of surface immunoglobulins (IgA, IgG), light chains, activation markers, and co-receptors, enabling broad phenotypic coverage in a single tube. Additionally, the high multiplexing capacity allows inclusion of markers for progenitors and other immune subsets such as dendritic cells, monocytes, and NK cells. Results Leveraging the multiplexing capacity of spectral technology, the panel design also accommodates markers for progenitor cells and additional immune subsets, including dendritic cells, monocytes, and NK cells. Conclusion Applying this panel to CLL samples can reveal disease-associated immunophenotypic patterns, highlighting its potential for translational research and future clinical applications in B-cell malignancies. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
TCR-Associated Rasal1 rewires T-cell mitochondrial metabolism to regulate checkpoint immunotherapy 2267535
Abstract Introduction Rasal1, a GTPase-activating protein, is a novel component of the T-cell receptor (TCR) complex that associates with ZAP-70 and negatively regulates the p21ras/ERK pathway (Thaker et al., Nat. Commun., PMID: 31641113). Here, we demonstrate that Rasal1 selectively regulates mitochondrial oxidative phosphorylation (OXPHOS) metabolism, while cooperating with anti—PD—1 therapy to limit T-cell exhaustion. Methods Utilizing a knock-in mutant mouse expressing Rasal1 lacking the C2-1 domain (Rasal1c-mut), we analyzed glycolytic- and mitochondrial-driven metabolic programme by measuring the oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR). In vivo studies were conducted in Rasal1c-mut mice using several immune checkpoint blockade (ICB)-resistant melanoma, colorectal, lymphoma, and lung carcinoma models in response to anti—PD—1 treatment. Results Primary T cells from mice expressing impaired Rasal1 exhibited a normal glycolytic shift following antigen stimulation but displayed a pronounced and selective increase in mitochondrial respiration. These findings indicate that Rasal1 fine-tunes TCR-dependent mitochondrial activity to sustain ATP generation and biosynthetic output during activation. Unexpectedly, inactive Rasal1–with elevated ZAP-70 and ERK signaling–synergized with anti—PD-1 blockade to reduce tumor-infiltrating lymphocyte exhaustion and promote the expansion of CD8+PD-1+TCF1+ stem-like progenitor cells, leading to enhanced tumor control. The same mechanism also increased the presence of cytolytic CD4+ TILs. Conclusion Collectively, our data reveal Rasal1 as a unique metabolic brake in T cells whose modulation offers a promising strategy to enhance the durability and breadth of immune checkpoint therapies. Funding Source Canadian Institutes of Health Research (159912) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
All cells are immune cells: the impact of endothelial-T cell crosstalk on protective pulmonary immune responses 2333504
Abstract Introduction Not all natural infections or vaccines elicit long-lived, protective immune responses. The mechanism(s) responsible for waning immunity to pulmonary bacterial infections are poorly understood. We previously found that poor prognosis after virulent Francisella tularensis challenge was associated with rapid IFN-γ production by lung-resident T cells, endothelial IDO1 up-regulation, lung tryptophan depletion, and poor T cell proliferation. We are now studying the crosstalk between T cells and endothelial cells during primary infection with attenuated F. tularensis to understand how cellular communication educates both populations prior to challenge. Methods We utilized single-cell RNA sequencing to identify gene expression changes in pulmonary endothelial cells following F. tularensis infection. Key findings were validated at the protein level using flow cytometry. We are also employing a Cre-lox system to specifically remove IFN-γ signaling from endothelial cells to determine the importance of these pathways during infection. Results Following infection, endothelial cells respond to IFN-γ by up-regulating genes associated with cellular recruitment, T cell activation, and metabolism. Flow cytometry validation confirmed increased protein expression as early as day 5 post-infection suggesting a non-T cell source of IFN-γ, likely NK cells, is responsible for the initial reprogramming of endothelial cells. Indeed, depletion of NK cells attenuated the response in endothelial cells. Finally, elimination of IFN-γ signaling in endothelial cells leads to increased susceptibility to F. tularensis. Conclusion We predict that poor immunity against tularemia results from reprogramming of the endothelial compartment by current live vaccine strategies. Our findings underscore how the pulmonary milieu impacts T cell function and leads to subpar immune responses. These results should inform the development of improved vaccines and vaccination strategies for difficult-to-target pulmonary pathogens like F. tularensis. Funding Source This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Disease, National Institutes of Health Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
H <sub>2</sub> S Self‐Supplied Micelles Reverse Tumor‐Immune Effector Cells Energy Metabolisms to Boost Breast Cancer Immunotherapy With Microenvironment Normalization
ABSTRACT Reversing the immunosuppressive tumor microenvironment by targeting metabolic competition between tumors and immune effector cells could induce tumor starvation and enhance the activity of immune cells, representing a potential approach to boost tumor immunotherapy. However, its actual efficacy is limited by compensatory oxidative phosphorylation (OXPHOS) energy replenishment and low delivery efficiency. Herein, we report a hydrogen sulfide (H 2 S)‐self‐supplying nanoplatform that orchestrates a dual blockade of glycolysis and OXPHOS for improved triple‐negative breast cancer (TNBC) immunotherapy. The micellar system, HA‐ADT@W, achieves tumor‐targeted delivery of a glycolysis inhibitor (WZB117) and H 2 S continually released in GSH‐overexpressed tumor cells. This strategy concurrently suppresses glucose uptake in tumor cells by reversing the acidic tumor microenvironment (TME) and disrupts compensatory OXPHOS via H 2 S‐mediated inhibition of cytochrome c oxidase. Consequently, we demonstrate a significant rewiring of tumor energy metabolism that not only induces immunogenic cell death with remodeling of the immunosuppressive TME but also alleviates nutrient constraints of immune effector cells, leading to enhanced infiltration and function of cytotoxic immune cells. This work exhibits a smart nanoplatform‐based H 2 S self‐supplied micelle for reinforced TNBC immunotherapy via regulated metabolic competition between tumors and immune effector cells with TME normalization.
Targeting glycans for cancer immunotherapy 2308347
Abstract Introduction Despite the curative potential of checkpoint blockade immunotherapy, a majority of patients remain unresponsive to existing treatments. Glyco-immune checkpoints — interactions of cell-surface glycans with lectin, or glycan binding, immunoreceptors — have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer. Methods Here, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a tumor-targeting arm as well as a lectin “decoy receptor” domain that directly binds tumor glycans and blocks their ability to engage lectin receptors on immune cells. Results We demonstrate proof-of-concept for the AbLec platform through the development and characterization of AbLecs combining tumor-targeting antibodies with decoy receptor domains from Siglecs-7 and -9, which have been shown to mediate immune suppression in multiple cancers. We show that tumor-targeting AbLecs enhance antibody-dependent phagocytosis and cytotoxicity of cancer cells in vitro and reduced tumor burden in vivo compared to the parent monoclonal antibody. We demonstrate that the AbLec platform can be applied to target diverse tumor-associated antigens, and cancer cells with varying levels of antigen expression. Further, we designed and validated dual blockade AbLecs that simultaneously block protein-based immune checkpoints (e.g., PD-1/PD-L1, CD47/SIRPα) as well as glyco-immune checkpoints that restrain productive anticancer immune responses (e.g., Siglec-7, galectin-9). We found that glyco-immune checkpoint blockade was synergistic with blockade of these established checkpoints. Conclusion Our results indicate that AbLecs target a non-redundant axis of immune suppression in cancer and could expand the subset of patients who respond to immunotherapy. In sum, AbLecs represent a generalizable approach for glyco-immune checkpoint blockade and a new modality for cancer immunotherapy. Funding Source Burroughs Wellcome Fund Career Award at the Scientific Interface 1017790, Society for Immunotherapy of Cancer Steven A. Rosenberg Scholar Award, V Foundation V Scholar Grant V2024-005, NIH/NCI Cancer Center Support Grant 5P30CA014051, NIH/NCI F32 Postdoct Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Programmable regulatory T cell platform for control of neuroinflammatory diseases 2310203
Abstract Introduction Inflammation is a fundamental component of immune defense but also a major driver of pathology across autoimmune, neurodegenerative, and trauma-associated diseases. Dysregulated inflammation propagates chronic tissue damage that current therapies fail to adequately control. Existing treatments rely on systemic immunosuppression or single-target biologics, which are limited by toxicity and insufficient tissue penetration. There is a critical need for tissue-targeted, dynamically controllable anti-inflammatory therapies. Methods We developed a multifunctional, programmable cell therapy platform that engineers regulatory T cells (Tregs) into living cellular pharmacies. These SMART Tregs are designed to home to disease-specific tissues, deliver anti-inflammatory disease-modifying molecules, and express biologics such as neuroprotective factors or functional enzyme replacements. We have established proof of concept for the platform in models of ALS and Alzheimer’s disease. Results Preliminary studies demonstrate our engineered Tregs can persist within the central nervous system and maintain functional activity, supporting feasibility for neuroinflammatory and neurodegenerative indications. The platform introduces three key innovations: integration of immune regulation with localized delivery, disease-context activation, and the ability to secrete persistence factors to extend therapeutic activity. Ongoing studies will evaluate disease-modifying molecule activity, in vivo efficacy, and biodistribution. Conclusion This Treg platform represents a paradigm shift in cell therapy, enabling precision immune modulation and localized therapeutic delivery across multiple disease contexts. The approach offers significant translational potential to several indications, including mitigation of neuroinflammation, immune-mediated injury, and protein replacement. This technology provides a validated, scalable cellular therapy platform ready for preclinical transition and partnership with translational stakeholders. Funding Source This work was supported by grants from the National Institutes of Health (NS102556, NS117895, and NS132666), Department of Defense (AL230089), and funds from the Center for Synthetic Immunity, Geisel School of Medicine at Dartmouth. Topic Categories Neuroimmunology (NEUR)
Multifactorial Immune Response Between Male and Female BALB/cJ and C57BL/6J Mice Exposed to Aspergillus fumigatus 2310270
Abstract Introduction Aspergillus fumigatus is an opportunistic airborne fungus that poses significant health risks, particularly to immunocompromised individuals and patients with preexisting conditions such as asthma. Despite its clinical relevance, the interplay between host sex, genetic background, and immune response to A. fumigatus exposure remains underexplored. This study investigated how immune cell counts, mucus production, collagen, and antibody production vary with sex and strain in BALB/cJ and C57BL/6J mice exposed to A. fumigatus. Methods Mice were challenged with airborne A. fumigatus spores weekly for three consecutive weeks. Bronchoalveolar lavage (BAL) was performed to collect immune cells, and histological analysis of lung tissue was conducted to assess mucus and collagen production, and an ELISA was conducted to analyze IgE antibody levels. Immune cells were identified using morphometric differences; mucus and collagen production were scored using Periodic Acid-Schiff (PAS) staining and Pico-Sirius Red (PSR) with Fast Green (FCF), respectively. Results Results showed that granulocyte counts peaked at day 3 post-challenge and declined by day 28, with male C57BL/6J mice exhibiting significantly higher granulocyte numbers than male BALB/cJ mice. While immune cell counts were not significantly different between sexes, mucus production varied. Male C57BL/6J mice had higher mucus production on day 3 post-challenge, whereas female BALB/cJ mice showed elevated mucus levels at day 28. Across all time-points and genetic background, females had significantly higher IgE production compared to their male counterpart. Conclusion This study highlights that genetic background (strain), timepoint and sex are important variables affecting , mucus, collagen, and antibody production dynamics in A. fumigatus mediated immune responses. These findings have implications for improving fungal disease models and developing sex-specific therapeutic strategies. Funding Source USR, Undergrad Students Research, SAFAC, CSHE and Judith Strong grant Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)
C5 Inhibitor Nomacopan Preserves Mitochondrial Integrity and Function in Combined Traumatic Brain Injury and Hemorrhagic Shock 2259501
Abstract Introduction Combined traumatic brain injury (TBI) and hemorrhagic shock (HS) induce robust complement activation and systemic inflammation that impair mitochondrial homeostasis. Mitochondrial dysfunction contributes to multi-organ failure following trauma. Nomacopan, a dual inhibitor of complement component C5 and leukotriene B4 (LTB4), has shown anti-inflammatory effects, but its impact on trauma-induced mitochondrial injury remains unclear. Methods In Sprague—Dawley rats subjected to TBI + HS, mitochondrial ultrastructure and oxidative phosphorylation (OXPHOS) were assessed in different tissues using transmission electron microscopy and high-resolution respirometry to characterize trauma-induced mitochondrial injury. To evaluate therapeutic potential, a porcine TBI + HS model was treated with Nomacopan, and mitochondrial morphology, bioenergetic function, and structural proteins were analyzed by electron microscopy and Western blot. Results In rats, TBI + HS caused severe mitochondrial swelling, disrupted cristae, and a significant decrease in OXPHOS capacity across cortex and myocardium. Respiratory chain complex activities were markedly reduced, indicating impaired bioenergetic function. In pigs, Nomacopan treatment markedly improved mitochondrial ultrastructure, including restoration of cristae integrity and reduction of swelling. Functional analysis revealed significant recovery of OXPHOS and respiratory chain activity compared with untreated TBI + HS controls. Furthermore, Nomacopan preserved mitochondrial structural proteins, including Mitofilin and OPA1, suggesting stabilization of the inner membrane architecture. Conclusion These findings demonstrate that TBI + HS induces profound mitochondrial injury in rodents and that complement C5 inhibition with Nomacopan effectively restores mitochondrial structure and function in a translational large animal model. Complement blockade represents a promising therapeutic strategy for mitigating systemic mitochondrial dysfunction following severe trauma. Funding Source DOW Topic Categories Immune Mechanisms of Human Disease (HUM)
Myoglobin expression boosts T-cell metabolism and antitumor effector function 2306919
Abstract Introduction The tumor microenvironment is often highly hypoxic and typically suffers from a marked lack of essential nutrients, including limited glucose availability. Because effector T cells have substantial energy requirements, the altered metabolic conditions created by the tumor can significantly contribute to impaired T-cell function and the development of T-cell exhaustion. Methods In this study, we assessed hypoxia in spleen and tumor tissue of tumor-bearing C57BL/6J mice using RT-PCR, histology, and flow cytometry. CD8+ T cells from C57BL/6J or P14+ mice were transduced with Thy1.1 (control) or Thy1.1—myoglobin (Mb) retrovirus, and Mb expression was verified by RT-PCR and western blot. Cellular metabolism was analyzed using flow cytometry, electron microscopy, Seahorse assays, metabolomics and luminescence-based methods. Mb-expressing or control P14+ or OT-I+ T cells were transferred into B16F10-gp33 or MC38-ova tumor-bearing mice and examined by flow cytometry and histology. Additionally, B16F10-gp33 tumor-bearing mice received anti—PD-1 treatment. Results We show that expression of the oxygen-binding protein myoglobin (Mb) in T cells enhances their mitochondrial and glycolytic metabolism, increasing metabolites, tricarboxylic acid cycle intermediates and ATP. Mb-expressing T cells showed reduced HIF-1α after activation and during tumor infiltration. Consequently, Mb boosted effector T-cell function against tumor cells in vitro while lowering superoxide. After adoptive transfer into tumor-bearing mice, Mb promoted greater T-cell infiltration into the tumor microenvironment. Although Mb-expressing T cells produced more effector cytokines, they still expressed PD-1 and responded to anti—PD-1 treatment, which combined with Mb-T-cell transfer most effectively delayed tumor growth. Conclusion Taken together, our findings show that Mb expression in T cells enhances their metabolic activity, promotes their infiltration into tumor tissue, and improves their effector function against cancer cells. Funding Source This study was supported by the German Research Foundation (DFG, GRK1949, LA2558/8- 1, 417677437GRK2578 & XU 160/3- 1), the Jurgen Manchot Foundation (Molecules of Infection, MOI4/MOI5), the Volkswagen Foundation (9B797), the Medical Faculty of the Heinrich Heine University (Forschungskommission), and the NIH Tetramer Facility, the Deutsche José Carreras Leukämie- Stiftung (DJCLS 18R/2021), the German Childhood Cancer Foundation (A2023/31), the German Federal Office for Radiation Protection (BfS), the DZIF (TTU 07- 711), the German Ministry for Education and Research (Bundesministerium für Bildung und Forschung BMBF, grant no. 01KD2410A (EDI- 4- ALL)), the Luxembourg National Research Fund (FNR) CORE grant (C21/BM/15796788) and the Fondation Luxembourg (Treg HTS). Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
The role of interferon-stimulated gene IFIT2 in a sex-dependent Treg modulation in CNS autoimmunity 2267558
Abstract Introduction Foxp3+ regulatory T (Treg) cells are essential for maintaining immune regulation and homeostasis by preventing excessive immune activation in autoimmunity, such as multiple sclerosis (MS). However, the mechanisms that control Treg function remain largely unknown. Type I IFN signaling serves as an important immune modulator in MS pathogenesis. We previously reported that IFIT2 (interferon-induced proteins with tetratricopeptide repeats), one of the IFN-stimulated genes, limits autoimmune inflammation by promoting myelin debris clearance and remyelination. Here, we investigated whether IFIT2 plays a role in Treg functions to suppress autoimmune inflammation. Methods We used germline and a novel Treg-specific IFIT2-/- mouse models and RNAseq analysis to examine the role of IFIT2 in Treg function in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. Results We found that IFIT2 controls Treg suppressive functions and metabolic profiles selectively in males. Ifit2-/- Tregs exhibited higher glycolysis and lower oxidative phosphorylation, hallmarks of functionally defective Tregs. Bulk RNA-seq analysis further confirmed that Myc and mTORC1 signaling pathways are highly enriched in KO cells. IFNβ stimulation enhances Treg functions and regulates Treg metabolism. However, IFNβ-induced metabolic changes and suppressive activity were completely lost in Ifit2-/- Tregs. At the molecular level, IFIT2 expression was required for Treg expression of PPARα, a ligand-activated transcription factor involved in cellular metabolism. Interestingly, IFNβ-induced, IFIT2-dependent PPARα expression was observed only in male Tregs, or in testosterone-treated female Tregs. Conclusion Together, this study reports a novel function of IFIT2 by which IFNβ signaling controls Treg metabolic activity and suppressive functions through the IFIT2-PPARa axis-dependent and sex-dimorphic manner in autoimmune inflammation. Funding Source NIAID Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Evolution and characterization of the Fc-mediated response in bats 2331633
Abstract Introduction Bats (order Chiroptera) serve as important reservoirs of known and potentially zoonotic pathogens, driven partially by novel immune adaptations. However, their adaptive immune systems remain largely uncharacterized, and the extent of immune diversity across bat species is unknown; this is a fascinating non-model system to understand the evolution of antiviral immunity. Some bat species fail to generate neutralizing antibodies during viral infection, suggesting the importance of non-neutralizing antibodies and Fc-mediated signaling in the antiviral immune response. Here, I characterize components of the Fc-mediated response, specifically the immunoglobulin heavy chain constant (IGHC) locus, Fc receptors (FcR), and Fc receptor-like proteins (FCRLs) in high-quality bat genomes. Methods High quality Bat1K and publicly available genomes were annotated using the Tool to infer Orthologs from Genome Alignments 2 (TOGA2). Due to the complexity of the IGHC locus, Fc genes were annotated manually in Geneious. Fc, FcR, and FCRL sequences were extracted and screened for N-linked glycosylation sites, immunoreceptor tyrosine-based activation (ITAM) and inhibition (ITIM) motifs. Results Bats contain canonical IGHC genes (IGHM, IGHG, IGHE, IGHA, variably IGHD), and most human FcR. However, IGHC genes vary in copy number, and contain bat-specific glycosylation sites not found in human Fc. There are also taxon-specific losses of FcεR2, the low-affinity IgE receptor. Further, there may be a tradeoff between FcαR and FcαμR; species that have retained FcαR have lost FcαμR, and vice versa. Other analyses are in progress. Conclusion Bats and their immune systems are extremely diverse. Further biochemical, structural, and functional analysis of Fc-FcR interactions and how the Fc-mediated immune response varies across species will contribute to our growing knowledge of bat antiviral immunity, which could inform the development of vaccines and therapeutics to improve human health. Funding Source NSF DBI 2515340 Topic Categories Veterinary and Comparative Immunology (VET)
Sequence‐Defined Short Peptide‐Derived Coacervate Vesicles for Targeted Therapeutics
ABSTRACT Compartmentalization underpins biological organization and the design of programmable therapeutic carriers. Here, we report a sequence‐defined short peptide, Biotin‐Ser(‐OPO 3 2 − )‐Phe‐Phe‐Arg ( Biotin‐SR ), that undergoes physiological liquid–liquid phase separation to form lipid‐free coacervate vesicles (CVs) with enhanced structural persistence. The designer peptide integrates oppositely charged functionalities that drive pH‐responsive coacervation, incorporating an alkaline phosphatase (ALP)‐cleavable anionic phosphoester, a diphenylalanine motif that promotes hydrophobic packing, and a cationic guanidinium group that stabilizes the coacervate architecture while serving as a precursor for nitric oxide (NO) generation. These CVs selectively target cancer cells via biotin‐mediated recognition. CVs of Biotin‐SR with pronounced morphological persistence, as confirmed by restricted molecular exchange in FRAP analysis, and encapsulate glucose oxidase (GOx) within this stabilized vesicular architecture, undergo ALP‐induced enzymatic dephosphorylation in ALP‐rich tumor environments to trigger an irreversible loss of charge complementarity, GOx release and a synergistic therapeutic cascade integrating enzymatic starvation, oxidative stress amplification, and NO‐mediated mitochondrial dysfunction. The resulting glucose depletion couples with NO generation to elevate reactive oxygen species (ROS), inducing mitochondrial dysfunction and suppressing tumor growth in both spheroids and xenograft models. This work demonstrates how minimal peptide sequences can translate liquid‐liquid phase separation into stable coacervate vesicles with enzyme‐responsive supramolecular function for targeted therapeutics.
T-bet-deficient CD4+ T cells are sufficient for acute host resistance against Toxoplasma gondii 2308791
Abstract Introduction The transcription factor T-bet is classically considered as the master-regulator of CD4+ T cell-derived IFN-γ, which is essential for host immunity against the intracellular parasite Toxoplasma gondii. Our group and others have previously demonstrated that whole-body T-bet-deficient (Tbx21⁻/⁻) mice rapidly succumb to infection despite retaining CD4+ T cell-derived IFN-γ, indicating T-bet has additional roles in mediating host resistance. Recently, our group demonstrated that T-bet-dependent IFN-γ production by group 1 innate lymphoid cells (ILC1s) is critical to maintain type I conventional DCs (cDC1s) that are essential for parasite control and host survival. To further define the role of T-bet in Th1-mediated immunity, we investigated whether T-bet expression in CD4+ T cells is required for acute resistance to infection. Methods Using a Cre-Lox system to specifically delete Tbx21 in CD4+ T cells (CD4-Tbx21⁻/⁻) and adoptive transfers into Rag2⁻/⁻mice, we assessed CD4+ IFN-γ production, parasite burden, cytokine responses, and host survival following systemic or mucosal infection. Results We observed CD4-Tbx21⁻/⁻ CD4+ T cells produced less IFN-γ and exhibited partial migration defects. In striking contrast to Tbx21⁻/⁻ mice, CD4-Tbx21⁻/⁻ mice controlled infection and did not succumb rapidly. Moreover, adoptively transferred T-bet-deficient CD4+ T cells migrated to lymphoid and intestinal sites and had no defect in IFN-γ production. Furthermore, Tbx21⁻/⁻ dendritic cells preserved functional antigen presentation. Conclusion These findings demonstrate that T-bet-independent CD4+ T cell effector functions are sufficient for parasite control and host survival during acute T. gondii infection. Understanding how T-bet-independent CD4+ T cells mediate acute immunity against T. gondii can provide translational implications for targeting T-bet-independent cell-mediated immunity for not only T. gondii, but other intracellular pathogens such as Listeria, Mycobacteria, and Salmonella. Funding Source NIAID Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency contributes to Treg Dysfunction and Susceptibility to Systemic Lupus Erythematosus 2328440
Abstract Introduction G6PD deficiency, the most common enzymopathy worldwide, is associated with an increased risk of systemic lupus erythematosus (SLE). The G6PD gene lies near SLE susceptibility genes such as IRAK1, and G6PD is the rate-limiting enzyme of the pentose phosphate pathway which is essential for the regulatory T cell (Treg) metabolism. Because impaired Treg cell function drives autoimmunity, we investigated how G6PD deficiency disrupts Treg cell function and promotes lupus pathogenesis. Methods Naïve CD4+ T cells from healthy donor PBMCs were treated with G6PD siRNA or a pharmacologic inhibitor and differentiated into iTreg cells. Treg cell function and metabolism were assessed using flow cytometry, suppression assays, Seahorse analysis and mass spectrometry-based metabolomics. Human studies included a single-center longitudinal cohort of adults with SLE (2006—2025) genomically tested for G6PD mutation status. Clinical outcomes were compared between mutation and non-mutation groups. Results G6PD inhibition or silencing reduced iTreg cell differentiation, impaired suppressive function, altered Treg metabolic profile and induced mitochondrial damage with increased levels of depolarized mitochondria. G6PD silencing in Treg cells lead to reduced NADPH, perturbation of the redox control, one-carbon metabolism, nucleotide balance, and amino-acid metabolism, all consistent with impaired pentose phosphate pathway activity and compensatory metabolic rewiring. In the SLE cohort, G6PD was reduced in SLE Tregs in comparison to healthy controls. G6PD mutations were associated with higher baseline disease activity (SLEDAI), and clinical visits. Conclusion G6PD deficiency disrupts Treg cell development and function through metabolic and non-metabolic mechanisms, contributing to SLE pathogenesis. These findings highlight a potential mechanistic link between a common enzymopathy and autoimmunity. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)
Investigating the role of the innate immune system in releasing neutrophil extracellular traps (NETs) in response to acute Toxoplasma gondii infection 2310314
Abstract Introduction Approximately one-third of the global human population is infected with the parasite Toxoplasma gondii. The immune system depends on neutrophils and one of their key mechanisms, neutrophil extracellular traps (NETs), to trap and neutralize T. gondii. Additionally, NETs help amplify both innate and adaptive responses to this parasite. NETs consist of DNA, myeloperoxidase, citrullinated histone 3, and elastase. Traditionally, it was believed that NET release caused neutrophil cell death. However, vital NETosis, which enables NETs while keeping the cell alive. While T. gondii induces NETs, the question remains whether they are released via vital NETosis. Furthermore, the cellular interactions and signals that trigger vital NETosis are unknown. Methods In this study, the promyelocytic cell line HL-60 was differentiated into neutrophil-like cells (NLCs) using retinoic acid and DMSO. NLCs were then infected with type I (RH) and type II (PA7) T. gondii at a multiplicity of infection of 2, in the presence of SYTOX, a dye that binds to extracellular DNA. Fluorescence levels were measured and compared with PMA-stimulated NLCs, which induce suicidal NETosis. Results Results showed an increase in extracellular DNA in T. gondii-infected NLCs. Immunofluorescence staining for DNA and citrullinated histone 3 at 4, 6, and 16 hours confirmed NET formation via confocal microscopy. The viability of T. gondii-infected NLCs at late time points (16 hours post-infection) was verified by live/dead viability staining and by measuring extracellular ATP. We observed a higher proportion of NET-releasing neutrophils that maintained membrane integrity, consistent with vital NETosis. Conclusion The next phase of this research will involve determining whether neutrophil complement receptors mediate T. gondii-induced vital NETosis. Understanding how neutrophils remain viable after entrapping T. gondii is essential to determine whether they can also deploy other effector mechanisms to prevent uncontrolled parasite replication. Funding Source N/A Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Cytokine-Mediated Preconditioning of Naïve CD4 T Cells Biases Subsequent Effector Differentiation 2329947
Abstract Introduction Naïve CD4 T cells are activated in secondary lymphoid organs, where integration of T cell receptor, co-stimulatory, and cytokine signals directs effector differentiation. Previous studies have shown that exposure to individual cytokines, such as IL-27 or IL-6, or helminth infection—associated environments can alter transcriptional programs and functional potential of naïve CD4 T cells. However, it remains poorly defined which cytokine receptors are constitutively expressed on naïve CD4 T cells and whether cytokine sensing at this stage broadly biases subsequent effector fate decisions. Methods Single-cell RNA sequencing was performed on murine splenic naïve CD4 T cells to profile cytokine receptor expression. Selected receptors were validated by quantitative RT-PCR and flow cytometry. Cytokine responsiveness was assessed by short-term cytokine stimulation followed by phospho-flow analysis of STAT signaling. Cytokine-conditioned naïve CD4 T cells were then activated via T cell receptor stimulation to evaluate downstream functional outcomes. In parallel, systemic inflammation was induced in vivo using poly I:C, and naïve CD4 T cells were isolated to determine whether inflammatory conditioning biased their functional potential. Results Naïve CD4 T cells constitutively expressed IL-4R, IL-6R, IL-7R, IL-21R, IL-27R, and IFNAR. Exposure to these cytokines induced distinct STAT phosphorylation patterns, indicating functional signaling without altering CD62L or CD44 expression. Cytokine conditioning by IL-6, IL-7, and IL-27 favored Th1-like responses, IL-4 enhanced Th2-associated programs, and Th17 differentiation was broadly suppressed. Consistently, in vivo Poly I:C exposure induced Ly6C expression in naïve CD4 T cells and promoted enhanced Th1 commitment. Conclusion Together, our findings demonstrate that cytokine-mediated conditioning at the naïve stage induces biased effector potential without overt phenotypic activation, thereby preprogramming subsequent T cell receptor—driven differentiation. Funding Source RS-2025-25422376, RS-2024-00334289, RS-2025-02232977 Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Olfr2 Regulates oxLDL Signaling and Processing, Driving a Pro-inflammatory Foamy Macrophage Phenotype 2259845
Abstract Introduction Atherosclerosis is a lipid-driven, chronic inflammation of arterial wall, characterized by plaque formation within the intima. A central pathogenic event is the accumulation of oxidized LDL (oxLDL), which drives macrophages toward a lipid-laden “foamy” state. Under sustained lipid stress, foam cells transition into a proinflammatory phenotype that fuels lesion progression, a process whose molecular mechanisms remain incompletely understood. Methods Here, we identify Olfr2, a G-protein-coupled receptor previously shown to confer protection when deleted, as a critical regulator of inflammatory switch in macrophages, by in vitro and in vivo assays. Results Loss of Olfr2 in bone marrow-derived macrophages (BMDMs) attenuated oxLDL uptake and foam-cell formation, as evidenced by a markedly reduced BODIPY and DIL-oxLDL lipid signal compared to wild-type (WT) cells. In vivo, pharmacologic blockade of Olfr2 using its antagonist citral for four weeks significantly decreased BODIPY staining in aortic macrophages of high-fat-diet-fed mice. Moreover, inhibition of Olfr2-mediated Ca²+ influx with diltiazem suppressed oxLDL internalization, suggesting that oxLDL engages the Olfr2 receptor to trigger downstream lipid uptake pathways. Mechanistically, Olfr2 deficiency dampened oxidative stress: oxLDL-induced cellular and mitochondrial ROS generation was reduced in Olfr2-/- macrophages compared with WT macrophages. Transcriptomic profiling revealed activation of the LXR pathway and upregulation of cholesterol efflux genes (Abca1, Abcg1), alongside increased expression of anti-inflammatory genes such as Il10 and Il1r2. Similarly, Olfr2-/- macrophages exhibited diminished Nos2 expression and nitric oxide production, indicating reduced proinflammatory signaling. Conclusion These findings reveal Olfr2 as a key regulator linking lipid metabolism, calcium signaling, and macrophage inflammatory response. By modulating this pathway, it may be possible to rebalance macrophage function to restore vascular homeostasis. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)
Development of strategies to visualize spatially resolved, stimulated cytokine secretion in live tissue slices 2267589
Abstract Introduction The lymph node (LN) is a spatially organized tissue where lymphocytes communicate by secreting cytokines in response to stimuli. Since most cytokines act locally, detection at the secretion site is needed to understand signaling within tissue. However, no current technologies detect cytokine spatial distribution in ex vivo stimulated tissue samples except for genetically modified mice; an expensive option with limited cytokines. Methods We show two methods to visualize cytokine distribution in vibratome-sectioned, 300µm thick, murine LN slices. In the first method, we developed a cell-surface based sandwich immunoassay using a bispecific antibody reagent to detect IFN-γ at its secretion site in slices using microscopy. In the second, we developed a procedure to block cytokine secretion in live slices using Brefeldin A (BFA). Through adding BFA to slices during stimulation, cytokines accumulated in cells for intracellular staining in the fixed tissue. Results With both methods, we successfully visualized IFN-γ distribution in LN slices after anti-CD3ε stimulation. We show that IFN-γ is located primarily in the interfollicular region. Using intracellular staining, we have multiplexed to include both IFN-γ and IL-17. Conclusion Next steps for both assays include applying this method to LN slices after in vivo vaccination and expanding it to human tonsil tissue. We envision that these assays will allow us to better understand cytokine signaling in complex disease states within live tissue samples. Funding Source Moore Inventor Fellowship, National Institute of Allergy and Infectious Diseases (Award # R21AI160547) Topic Categories Technological Innovations in Immunology (TECH)
Differential Protein Synthesis Rates in Tregs, conventional T cells, and ex-Tregs following LPS Stimulation 2332109
Abstract Introduction Protein synthesis is a metabolically demanding process that differs among T cell subsets based on their functional states and suppressive capacity. While regulatory T cells (Tregs) are known to exhibit distinct metabolic profiles compared to conventional T cells (Tconv), the translational dynamics of ex-Tregs cells that have lost the classical Treg phenotype remain poorly characterized. Methods We employed a 10-color flow cytometry panel with puromycin incorporation to measure protein synthesis in human CD4+ T cell subsets during LPS-induced inflammation. Whole (human) blood was stimulated with LPS (TLR4 agonist, 24h) ± cycloheximide (CHX, translation inhibitor) and pulsed with puromycin to label nascent proteins. Results Tregs (CD3+CD4+CD25highCD127low), Tconv (CD3+CD4+CD25-CD127+), and ex-Tregs (CD3+CD4+CD25-CD127high). Tconv and ex-Tregs exhibited significantly higher puromycin incorporation (MFI: 1173±71 and 1209±68) compared to Tregs (557±52, p &lt; 0.01). LPS modestly increased Tconv synthesis (1318±96 MFI) while Tregs remained quiescent. CHX reduced synthesis ∼60% across all subsets (p &lt; 0.01), confirming translation-dependent incorporation. Memory phenotyping revealed Tconv and ex-Tregs were predominantly central memory (&gt;85%), while Tregs showed heterogeneous distribution (25-45% naive). Treg frequencies remained stable (∼4-5% of CD4+) while ex-Tregs constituted ∼15%. Ex-Tregs exhibit translational profiles similar to Tconv compared to Tregs, which suggests functional reprogramming after CD25 loss. Conclusion Differential protein synthesis rates may reflect distinct metabolic requirements for suppressive versus effector functions during TLR4-mediated inflammation. These subset-specific translational differences may contribute to immune dysfunction in ribosomopathies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Single-cell RNA sequencing with ribosome profiling will be critical to define translational landscapes driving distinct T cell fates. Funding Source Supported by a grant from NIH-NIGMS (P20 GM103446) and the State of Delaware. Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)