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Dietary antigens gradually inactivate CD4+ T cells to produce oral tolerance 2258629
Abstract Introduction CD4 T cells specific to dietary antigens are inactivated through mechanisms that include deletion, anergy, and acquisition of regulatory capacities. The factors that guide the fate of these cells are unknown. Here, we analyzed the role of antigen-associated factors (e.g., concentration, frequency, affinity) in determining CD4 T cell fate during oral tolerance. Methods We adoptively transferred naïve OTII CD4 T cells and fed mice with different regimes of the model antigen ovalbumin (OVA). A single dose of OVA induced an early but short-lived induction of FoxP3. Gradually, FoxP3+ cells decreased and anergic cells became the dominant population. Fate mapping analyses revealed that regulatory and anergic programs are mostly exclusive. Ultimately, the majority of OTII cells became apoptotic. Results Repetitive administration of OVA was associated with a higher expression of anergy-associated molecules (e.g., Ctla-4, Nrp-1) and transcription factors (e.g., TOX, Helios) in a dose-dependent manner, suggesting that repetitive exposure to cognate antigen gradually induces a more profound CD4 T cell inactivation. Additionally, repetitive exposure to OVA promoted homing of anergic CD4 T cells into the lamina propria. However, tolerance was a short-lived process, as its effect was not detected after OVA was discontinued. Using OVA-specific tetramers, we identified high and low affinity CD4 T cells and explored the transcriptomic changes induced by oral antigen (scRNA-seq). In vitro examination of tolerogenic fates using ligands of different affinities showed that weak TCR activation was associated with the development of anergy. Conclusion Collectively, our results indicate that tolerance to oral antigens represents a gradual and dynamic process, controlled by the intensity of antigenic stimulation, where expression of FoxP3 and anergy-associated markers represent a transient phase that leads to clonal deletion. Funding Source FORDECYT-303046 Topic Categories Mucosal and Regional Immunology (MUC)
SIRPγ limits effector differentiation of human CD8 T-cells in response to subthreshold TCR-signaling 2266789
Abstract Introduction Signal regulatory protein gamma (SIRPγ) is an immunomodulatory protein that is uniquely expressed on the cell surface of human T cells. Variants in the SIRPγ gene have been associated with type 1 diabetes, relapsing-remitting multiple sclerosis, and the maintenance of a long-term vaccine response. However, the mechanistic contribution of SIRPγ to inflammation remains unclear because its function in the immune system is not fully understood. Methods To investigate the functional role of SIRPγ in human CD8 T-cells, we performed siRNA-mediated knockdown in naïve human CD8+ T-cells. T-cell differentiation, effector responses, and cytokine production were studied by flow cytometry. A CD47 neutralization assay was performed to determine whether this known ligand is required for effector-like differentiation of naïve CD8 T-cells. Results We report that SIRPγ expression varies substantially across individuals and stratifies CD8+ T-cell differentiation states. Individuals with low SIRPγ expression exhibit an increased frequency of CD27⁻CD45RO+ effector-like and CD27⁻CD45RO⁻ terminally differentiated CD8+ T-cells, while high expressors retain a predominance of naïve and central memory cells. Under suboptimal TCR stimulation, SIRPG knockdown drove robust effector-like differentiation marked by increased CD45RO expression, T-bet upregulation, and enhanced production of TNF-α, IFN-γ, and Granzyme B. This phenotype was not recapitulated by CD47 blockade, indicating that SIRPγ modulates differentiation through a CD47-independent mechanism. Conclusion These findings identify SIRPγ as a negative regulator of CD8+ T-cell effector programming under limiting stimulatory conditions. Interindividual variability in SIRPγ expression may influence immune homeostasis and susceptibility to immunopathology, highlighting SIRPγ as a potential therapeutic target in settings of dysregulated T cell responses. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
The E3 Ligase RNF11 is a novel regulator of type I Interferon-independent antiviral defenses critical for restricting viral infection in macrophages 2259415
Abstract Introduction Classically, anti-viral defenses are thought to be driven by the production of pro-inflammatory cytokines and type I interferons (IFNs) triggered by the activation of innate immune sensing pathways such as the DNA sensing pathway cGAS-STING. There are also cell autonomous defense mechanisms, such as xenophagy, that are equally important for restricting viral infections, but these are poorly understood. In this study, we examined a potential role of the E3 ubiquitin ligase RNF11 in regulating cGAS-STING signaling and anti-viral host defense in macrophages. Methods We utilized macrophage cell lines including THP-1 and RAW 264.7 to examine the role of RNF11 in cGAS-STING signaling and viral infections in macrophages. Lentiviral vectors expressing control scrambled or RNF11 shRNA were used to knockdown RNF11. We performed western blotting, Incucyte live-cell imaging, qRT-PCR, ELISA, flow cytometry, and confocal microscopy to examine potential roles of RNF11 in cGAS-STING signaling and innate anti-viral responses in macrophages. Results Knockdown (KD) of RNF11 elicited more robust activation of the cGAS-STING pathway in response to cGAS and STING agonists and increased type I IFN production in macrophages. Unexpectedly, despite more cGAS-STING activation, there was increased replication of a DNA virus (herpes simplex virus 1 [HSV-1]). Similar results were obtained with a panel of RNA viruses including Sendai virus (SeV) and vesicular stomatitis virus (VSV) and this phenotype was specific to macrophages. Remarkably, the increased viral loads in RNF11 KD macrophages persisted with a blocking antibody specific for interferon alpha/beta receptor 1 (IFNAR1) interferon, suggesting that the anti-viral function of RNF11 is independent of type I IFN signaling. Conclusion Overall, our results suggest that RNF11 is an essential component of a novel type I interferon-independent form of cell autonomous defense that is critical for restricting viral infection in macrophages. Funding Source NIH-NIAD Topic Categories Viral Immunology (VIR)
A Tetravalent Dengue prM-E mRNA-LNP Vaccine Protects via Antibodies in Susceptible Mice 2255808
Abstract Introduction Dengue virus (DENV) remains a major global health threat, with no optimal vaccine available against its four distinct serotypes: DENV1, DENV2, DENV3, and DENV4. A central challenge in dengue vaccine development is achieving balanced and durable immunity against all serotypes, as partial protection can lead to antibody-dependent enhancement (ADE), increasing the risk of severe disease upon secondary infection. Methods We developed a tetravalent mRNA vaccine encoding the prM-E proteins from each DENV serotype, formulated in lipid nanoparticles (LNPs). The vaccine was administered intramuscularly to C57BL/6J mice. Each dose contained a total of 5 µg mRNA (1.25 µg per serotype) and was given at 4-week intervals for three immunizations. Germinal center (GC) B cell responses and tissue-resident memory T (Trm) cell induction were analyzed in comparison with recombinant Envelope (rE) protein formulated with Alum. Results The tetravalent prM-E mRNA-LNP vaccine elicited strong neutralizing antibody responses against all four serotypes, producing titers of 5481, 4632, 12,179, and 5006 for DENV1-4, respectively. Passive transfer of sera from vaccinated mice conferred complete protection in AG129 mice (IFN-α/β/γR-/-) challenged with lethal DENV2, resulting in 100% survival. By day 14, the prM-E mRNA-LNP group exhibited a two-fold higher frequency of CD38^lowGL7+ GC B cells than the rE + Alum group, indicating enhanced GC B cell activity. Intradermal (ID) vaccination induced significantly higher Trm cell formation in the skin compared to intramuscular delivery, showing approximately four-fold increases following ID prime-boost immunization. Conclusion These findings show that the tetravalent prM-E mRNA-LNP vaccine elicits strong humoral and tissue-resident cellular immunity against all DENV serotypes. The results underscore its robust immunogenicity and protective efficacy, supporting further development of mRNA-based dengue vaccines for broad and durable protection. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)
Single-Cell Transcriptomic Analysis Of Imaging-Sorted Circulating Immune Cell Doublets In Active Tuberculosis 2309416
Abstract Introduction Communication between immune cells through direct contact is a critical feature of immune responses. Our previous work has highlighted that a significant fraction of immune cell doublets detected in non-imaging flow cytometry are not technical artifacts, and instead hold biological relevance. In particular, using non-imaging FACS (fluorescence-activated cell sorting), we have recently shown that circulating T cell-monocyte complexes during infection hold transcriptomic signatures of active immune interactions. However, conventional non-imaging FACS lacks the spatial resolution necessary to characterize doublets accurately. Methods In this study, we employed the recently commercialized FACSDiscoverTM S8 spectral imaging cell sorter to characterize different phenotypes of circulating T cell-monocyte complexes in a cohort of patients with tuberculosis. Using various combinations of imaging parameters, we found that T cell-monocyte doublets can be classified as either synaptic or coincidental. Synaptic doublets were defined by high spatial overlap between the two cells forming complexes, representing true biological conjugates. In contrast, coincidental doublets were non-interacting cells but in close proximity during acquisition, thus representing technical artifacts rather than biological interactions. For each patient, synaptic and coincidental T cell-monocyte doublets were sorted, resulting in physical separation of the two cells forming the doublet, and processed for single-cell droplet sequencing. Results Sorting and single-cell sequencing analysis confirmed that synaptic doublets carried unique biological gene signatures associated with high metabolic activity and immune activation, while coincidental doublets resembled singlet T cells and monocytes. Conclusion Thus, image-based cell sorting provides unprecedented granularity in the study of immune doublets and enrichment for biological doublets over technical artifacts. Funding Source The Tullie and Rickey Families SPARK Awards for Innovations in Immunology, National Institute of Allergy and Infectious Diseases, Human Immunology Project Consortium, The Conrad Prebys Foundation Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Whooping cough disease induces durable protection from asymptomatic re-infection 2310171
Abstract Introduction Bordetella pertussis (BP) is the causative agent of whooping cough (WC), also known as pertussis, a highly contagious respiratory disease. Although BP vaccination remains the most effective preventive measure, the shift from whole-cell pertussis (wP) vaccine to acellular pertussis (aP) vaccines has been associated with the resurgence of pertussis cases in many countries. While differences in immune responses between wP and aP vaccines have been well documented, limited information exists regarding BP-specific memory T cell responses, particularly against the broad range of BP antigens not included in aP vaccine formulations. Methods This study investigates BP-specific responses in donors vaccinated with aP or wP in childhood, as well as donors clinically diagnosed with whooping cough. To identify BP-specific CD4 T cells we stimulated PBMCs with two pools of peptides: one pool encompassing peptides from aP vaccine antigens (including FHA, PRN, PT, and FIM2/3), and a second pool containing peptides from non-aP vaccine antigens. Results Our findings reveal differential BP antigen reactivity based on vaccination and infection history, with notably high reactivity to non-aP vaccine antigens in the aP-vaccinated cohort, suggesting prevalent subclinical colonization. Unexpectedly, adults examined years after recovery from clinical disease showed lower T cell responses. In contrast, robust BP-specific IgG responses were observed in acute and convalescent pediatric donors, suggesting that individuals who experienced symptomatic disease can develop a strong humoral immunity and remain protected from reinfections. Single-cell RNA and T cell receptor sequencing analyses are currently being performed to further elucidate qualitative differences in immune responses to vaccination versus infection. Conclusion This study emphasizes the importance of understanding immune responses to both subclinical and clinical BP infection for identifying specific immune profiles that correlate with protection. Funding Source This project has been funded in part with Federal funds from the National Institute of Allergies and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (Grants No. U19 AI142742) Topic Categories Vaccines and Immunotherapy (VAC)
Fn14 Signaling in Glioblastoma-Associated Myeloid Cells Inhibits Th1-Driven Anti-Tumor Immunity 2255143
Abstract Introduction Immunotherapy approaches against glioblastoma (GBM) such as immune checkpoint inhibitors (ICI), dendritic cell vaccination, and CAR-T have failed to durably cure most patients. Studies analyzing tumors from ICI responders show that abundance of CD4 and CD8 T cells prior to initiation of therapy predicts responsiveness. Thus, targeting the microenvironment to promote CD4 activity may synergize with ICI. The glioblastoma microenvironment contains high proportions of monocyte-derived macrophages which have potent immunosuppressive capabilities. Characterizing and targeting these immunosuppressive programs is a promising strategy for designing effective combination immunotherapies for glioblastoma patients. Methods We analyzed human glioblastoma snRNAseq datasets to find signaling receptors associated with myeloid immunosuppression and employed murine models of glioblastoma for functional validation. Results By analyzing patient-derived -omics datasets, we identified an understudied TNF-superfamily receptor, Fn14, as being upregulated in glioblastoma-associated myeloid cells with immunosuppressive gene signatures. Using in vitro approaches, we found that Fn14 activation in the tumor microenvironment can drive cytokines implicated in GBM immunosuppression such as IL6, IL1B, and IL8. Thus, Fn14 may serve as a surface marker for microenvironment-remodeling myeloid cells recruited to the tumor from peripheral circulation. Furthermore, in murine models of glioblastoma, we discovered Fn14 KO among glioblastoma-associated myeloid cells increases conventional dendritic cell type 1 (cDC1) and Th1 CD4+ T cell abundance in the tumor microenvironment and significantly prolongs survival. Conclusion These results suggest that blocking Fn14 in glioblastoma-associated myeloid cells can enhance Th1 immunity. Thus, Fn14 may represent a promising dual therapy target. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Longitudinal single-cell atlas to identify B cell subpopulations and states associated with human vaccine responses 2309736
Abstract Introduction Vaccine responses vary across individuals, with specific B cell subpopulations driving humoral immune response. Bulk transcriptomic studies have identified pan-vaccine predictive signatures, but have difficulty determining which cell subsets drive these signals. Single-cell approaches offer the cellular resolution to link signatures to specific subpopulations, and recent study have successfully associated B cell subsets with vaccine responsiveness. However, most single-cell vaccine studies use small cohorts (< 10 subjects) with insufficient power to reliably detect rare cell states like functionally distinct plasma cell subsets and subtypes of memory B cells. Moreover, inconsistent cell annotations hinder cross-study comparisons. A large-scale, standardized B cell atlas across multiple vaccines would facilitate detection of rare response-associated populations and establish reference subset definitions. Methods We curated and processed nine single-cell RNA-seq studies (53 subjects) following SARS-CoV-2, seasonal influenza, and malaria vaccinations spanning baseline through various timepoints post-vaccination. All datasets were processed through a standardized pipeline and integrated to preserve biological variation while correcting technical batch effects. Hierarchical celltype annotation was applied to identify B cell subpopulations. Results The integrated atlas comprises 447,493 B cells spanning naïve (50-70%), memory (25-40%), activated, and plasma cell populations in PBMC samples across three different vaccine responses. We observe plasmablast enrichment post-vaccination consistent with peak antibody responses across many vaccines. Conclusion This multi-vaccine B cell atlas enables systematic profiling of rare response-associated subpopulations not detectable in small-cohort studies. Integration of transcriptional states with antibody outcomes may reveal cellular predictors of immune response. Funding Source This work is supported through the NIH U19AI089992 grant. Topic Categories Computational and Systems Immunology (COMP)
Nanoscale Compositional and Strain Gradients Enable High‐Speed and Amplitude‐Resolved Pyroelectric Sensing
ABSTRACT The frequency response of pyroelectric sensors is fundamentally governed by thermal time constant (τ th , determined by thermal mass and thermal conductance) and electrical impedance arising from film capacitance and readout circuit. Conventional bulk LiTaO 3 detectors are optimized for high responsivity at low modulation frequencies (0.1–10 Hz), possessing a large τ th that thermally averages rapid temperature oscillations at elevated modulation frequencies, limiting fidelity in resolving dynamic varying thermal signals. Here, compositional and strain gradients are introduced into 100‐nm‐thick relaxor‐ferroelectric films reducing τ th to ≈2 µs and producing built‐in potentials (≈1.45 V or 145 kV cm −1 ) that enhance the pyroelectric coefficient and suppress the dielectric constant. This enables complementary dual‐mode operation by enhancing current‐mode electrical responsivity and improving the voltage‐mode figure of merit – advantageous for superior temperature resolution (Δ T min ≈ 30 µK). The responsivity peak shifts to near 1 kHz (>2500‐times higher than conventional bulk sensors), with measurable responsivity extending to a carrier frequency of 100 kHz and amplitude‐resolved detection at modulation frequencies up to 15 kHz. These results establish nanoscale internal‐field engineering can reshape electro‐thermal trade‐off in pyroelectric thin films toward zero‐bias, high‐thermal‐sensitivity, and amplitude‐resolved thermal sensing across a wide frequency bandwidth.
Elephant‐Skin‐Inspired Porous Cementitious Tiles with Programmable Crack Networks for Passive Cooling
ABSTRACT Passive evaporation of water reduces a building's cooling energy demand. However, water is often wasted due to rebound, uneven spreading, and rapid drainage. Here, we present an elephant‐skin‐inspired crack network architecture in porous diatomaceous earth (DE)‐cement composites to capture, route, and store water with minimal runoff. DE's micro/nanoporosity enables ultrafast (sub‐50 ms) water imbibition, while crack networks act as capillary conduits that redistribute water across and up inclined surfaces. Substrate‐guided stress concentration converts drying‐induced stochastic fractures into deterministic crack lattices that route and retain water on inclined surfaces, enabling geometry‐tunable, water‐efficient evaporative cooling. Tiles of hexagonal lattices with intermediate crack density maximize lateral redistribution and delay drainage. Infrared imaging reveals edge‐dominated evaporation, sustaining prolonged cooling. In a mockup home model covered with DE‐cement tiles, under repeated water dosing and IR heating, the temperature beneath the DE‐cement tiles is maintained at ∼32°C vs ∼42°C and ∼52°C for cracked and non‐cracked commercial stucco, respectively. The study offers a simple, scalable route for passive cooling.
Structured Exercise Program After Adjuvant Chemotherapy for Colon Cancer: A Cost-Utility Analysis of the CHALLENGE Trial
PURPOSE The phase III CHALLENGE trial (CCTG CO.21) demonstrated that a 3-year structured exercise program (SEP) improved disease-free and overall survival versus health education materials (HEM) in patients with stage III or high-risk stage II colon cancer after surgery and adjuvant chemotherapy. We assessed the cost-effectiveness of the SEP versus HEM. METHODS This prespecified economic evaluation used prospectively collected data from all trial participants (N = 889). The base case adopted the Canadian public payer perspective and included direct health care costs over a 5-year time horizon with a 1.5% discount rate. 36-Item Short Form (SF-36) outcomes were mapped to Short-Form 6-Dimension (SF-6D) utilities using a validated algorithm. Mean costs (2024 in Canadian dollars [CAD]) and effects (life-years [LYs] and quality-adjusted LYs [QALYs]) per participant in each intervention group were used to estimate the incremental costs, incremental LYs/QALYs, incremental cost-effectiveness ratio (ICER, $ CAD/life-year gain [LYG]), and incremental cost-utility ratio ($ CAD/QALY) when applicable. Uncertainty was assessed via bootstrapping (1,000 samples). Notable scenario analyses included a 10-year time horizon and a societal perspective incorporating lost wages because of missed work, measured through the Work Productivity and Activity Impairment questionnaire. RESULTS In the base case, despite an up-front cost of $2,917 (CAD)/participant to provide the SEP, the SEP strategy (total cost: $31,957 (CAD)/participant) was dominant over HEM (total cost: $33,546 (CAD)/participant), being less costly (-$1,589/participant) and more effective (+0.05 LYs/participant; +0.10 QALYs/participant). The SEP was dominant in 53% of bootstrap samples, and 80% fell below a $50,000 (CAD) per QALY willingness-to-pay threshold. Major cost drivers were cancer recurrence or new malignancy and anticancer therapy. Scenario analyses were consistent with the base case. CONCLUSION The SEP was less costly and more effective than HEM. This study can inform payers and health systems with implementation of SEPs in routine care.
4-octyl itaconate as a metabolite derivative to inhibits cytokine-mediated inflammation via alkylation of TYK2 2258772
Abstract Introduction Sepsis is a host’s dysregulated response to infection. Janus kinases and signal transduction factors and transcriptional activators (JAK-STATS) are among the most important signaling components downstream of cytokine receptors. This dysregulated host response includes the exacerbated systemic inflammatory response syndrome (SIRS) that occurs in an attempt to combat the invading pathogens. Itaconate, an anti-inflammatory metabolite synthesized by enzymes encoded by immune response gene 1 (IRG1). Itaconate and its derivatives are potent cysteine modifiers, due to its nature as an α,β-unsaturated dicarboxylic acid. Methods Through transcriptome and metabolite sequencing analysis of intestinal tissues obtained from CLP-induced mouse models. Cell experiments revealed that 4OI inhibited the JAK-STAT pathway.Through protein modification identification, explore the mechanism by which 4O regulates JAK-STAT. To explore the endogenous itaconate effect, IRG1 KO mice were constructed. To explore the role of the JAK-STAT pathway in sepsis, IFNAR1 KO and TYK2 KO mice were constructed. Results Our results suggest that endogenous itaconate is a negative regulator of type I interferon-mediated signaling pathway. 4OI inhibits the binding of TYK2 to IFNAR1 by alkylating the cysteine residue at position 192 of TYK2, and further negatively regulates the JAK-STAT pathway. To provide a theoretical basis for 4OI as a therapy for sepsis. Conclusion We find that itaconate, as a metabolite significantly up-regulated by metabolic reprogramming, can inhibit type I interferon signaling pathway. Exogenous supplementation of itaconate derivative 4OI can inhibit JAK-STAT pathway. Our research has identified the crucial role of itaconate produced by the tricarboxylic acid cycle in restricting JAK-STAT signal transduction, whose function is to connect metabolism and innate immunity and provided a theoretical basis for 4OI treatment of sepsis. Funding Source National Natural Science Foundation of China (82272209) Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Implications of a dysfunctional meningeal lymphatic system in a mouse model of CSF1R-related leukoencephalopathy 2241999
Abstract Introduction Colony stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is an early onset neurodegenerative disease characterized by microglial deficits, abnormal glial responses, white matter pathology, and severe motor and cognitive decline. Methods At 8 months, mice lacking two copies of the Csf1r gene Fms-intronic regulatory element (FIRE) recapitulate some of the brain pathological features of CSF1R-related leukoencephalopathy, namely the deficits in microglia, astrogliosis, and demyelination. Of note, we detected deficits in lymphatic vasculature and altered macrophage activation in the meninges of Csf1rΔFIRE/ΔFIRE mice by flow, prior to the appearance of brain gliosis, neuronal dystrophy, and fine motor impairment. Results Eliminating the remainder CSF1R-expressing innate immune cells, including border-associated macrophages (BAMs), with PLX5622 at 2 months reshaped the immune crosstalk at the meningeal dura and restored lymphatic vascular expansion in Csf1rΔFIRE/ΔFIRE mice Conclusion Ongoing experiments in the lab will clarify the exact mechanisms through which dysfunctional innate immunity at the brain-meningeal interface and lack of meningeal lymphatic vasculature contribute to the development of brain pathology and impaired motor behavior in this model of CSF1R-related leukoencephalopathy. This work has unveiled novel molecular insights involving the meningeal lymphatic system that might be of therapeutic relevance to canonical central nervous system white matter diseases. Funding Source n/a Topic Categories Neuroimmunology (NEUR)
Effector versus memory-biased engineered IL-2 sequencing plus αPDL2 for improved treatment of bladder cancer 2259496
Abstract Introduction Bladder cancer (BC) is immunogenic but remains poorly responsive to immunotherapy. Interleukin-2 (IL-2) stimulates cytotoxic lymphocytes but causes Treg expansion, toxicity, and rapid clearance. A CD122-directed IL-2 complex (IL-2c) can overcome these issues. To further optimize IL-2—based immunotherapy, we engineered two IL-2/anti-IL-2 antibody fusion proteins, F10 IC and F10 H9T, to differentially modulate effector and memory T-cell responses. Methods We fused the CD25-occluding antibody F10 to either native IL-2 (F10 IC) or an engineered IL-2 variant (H9T, Mo et al., Nature 2021). Antitumor efficacy was tested in an orthotopic MB49 bladder cancer model. Tumor growth, survival, and immune cell phenotypes were analyzed by flow cytometry, UMAP clustering, and IHC. Treatments included single agents, sequential combinations, and αPDL2 checkpoint blockade. Results F10 IC activated cytotoxic Teff and NK cells, whereas F10 H9T promoted CD44+CD62L+ central memory and Trm T-cell differentiation. Sequential or combined IL-2 variant therapy enhanced cytolytic activity and prolonged tumor control versus single agents. F10 IC plus αPDL2 significantly suppressed tumor growth and extended survival, while F10 H9T delayed relapse. UMAP analysis of TILs confirmed the predicted effector versus memory bias. Conclusion Distinct IL-2 fusion proteins can be strategically combined to harness effector and memory T-cell immunity. Integrating F10 IC and F10 H9T with checkpoint blockade represents a promising next-generation IL-2—based approach for durable tumor control in bladder cancer. Funding Source N/A Topic Categories Cytokines and Chemokines and their Receptors (CCR)
T-bet high atypical memory B cells exhibit effector memory features in mRNA vaccine recipients 2256789
Abstract Introduction T-bet high B cells, also referred to as atypical or double-negative 2 (DN2) B cells, are defined by high expression of the transcription factor T-bet (TBX21) and typically expand during chronic infections and autoimmune conditions. In severe viral infections such as COVID-19, these cells arise through extrafollicular activation and have been implicated in both protective neutralizing antibody responses and immunopathology. However, their molecular and functional properties in the context of repeated vaccination remain poorly understood. Methods In this study, we examined SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells from individuals who received multiple doses of the BNT162b2 mRNA vaccine. By integrating multi-omics single-cell sequencing, gene regulatory network analyses, immunophenotyping, and functional assays, we characterized the effector memory features of T-bet high memory B cells. Results Our results show that T-bet high memory B cells can be identified by co-expression of CD11c and FcRL5 and are governed by distinct gene regulatory networks linked to effector functions. These cells were affinity-matured and rapidly differentiated into antibody-secreting cells (ASCs) that produced neutralizing antibodies at levels comparable to classical memory B cells, highlighting their contribution to early recall responses. Subclustering further revealed heterogeneous subsets, including one enriched for RBD-specific cells and expressing markers indicative of recent antigen exposure. Conclusion Taken together, our findings provide a comprehensive view of the transcriptional and functional diversity of human B cells and delineate the trajectory of T-bet high B cells from initial activation through repeated antigenic stimulation. The strong effector memory characteristics of these cells underscore their potential as immunological markers and promising targets for optimizing future vaccine strategies. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)
Inherited C5aR2 deficiency leads to uncontrolled inflammatory T cell responses and autoinflammatory disease 2309676
Abstract Introduction Systemic complement protects the vascular compartment, while cell-intrinsic complement components shape tissue immunity by regulating normal cell physiology and metabolism. In human CD4 T helper cells, intracellular C5a generation engages the intracellular receptor C5aR1, promoting Th1 differentiation. Here, we identify a counter-regulatory role for the alternative C5a receptor, C5aR2, in restraining T cell effector responses by studying the first reported family with a heterozygous C5aR2 mutation. The affected individuals present with an autoinflammatory syndrome, and disease penetrance tracks with the mutation: the affected mother and child carry the variant, whereas the unaffected father and siblings do not. Methods To define the impact of the C5aR2 mutation, we performed single-cell RNA sequencing, in vitro stimulation assays, and flow cytometric profiling of patient and control PBMCs. CPM inhibition or deletion, C5aR2 agonism, and transcriptional profiling of Cpm- or C5ar2-deficient mouse CD4 T cells, along with a T cell transfer colitis model, were used to define mechanistic consequences. Results Patient samples exhibited a profound loss of naïve and central memory CD4 and CD8 T cells, accompanied by expansion of IFN-γ—producing effector memory populations. The C5aR2 mutation abolishes C5aR2 β-arrestin signaling. Additionally, we identified carboxypeptidase M (CPM) as a T cell—intrinsic enzyme generating C5a-desArg, a potent ligand for C5aR2. Loss or inhibition of CPM heightened inflammatory T cell responses, which were normalized by C5aR2 agonism. Mouse Cpm- or C5ar2-deficient CD4 T cells displayed overlapping transcriptional perturbations, and Cpm-knockout CD4 T cells induced more severe colitis. Conclusion Collectively, our data uncover a previously unrecognized CPM-dependent mechanism that balances C5aR1 and C5aR2 signaling to limit pathological T cell activation, revealing an intrinsic complement-driven checkpoint that constrains effector T cell immunity. Funding Source NHLBI/NIH intramural program Topic Categories Immune Mechanisms of Human Disease (HUM)
Pro-Inflammatory Response of Human Monocyte-Derived Dendritic Cells to Bacterial Biofilms 2310119
Abstract Introduction Many human bacterial pathogens, including Uropathogenic Escherichia coli (UPEC), which causes urinary tract infections (UTIs), can form biofilms. Bacterial biofilms are multicellular communities reinforced by amyloids that protect the bacteria from environmental stress, including antibiotics, promoting chronic infections. The recognition of biofilms by innate immune cells remains under-investigated. Our labs have previously shown that curli, the amyloid expressed by Enterobacteriaceae like UPEC during biofilm formation, activate murine dendritic cells. Many PAMPs in biofilms can mediate innate immune activation, and it is pivotal to understand their roles in host defense. Methods We investigated the response of human monocyte-derived dendritic cells (hu-moDCs) from healthy donors to UPEC biofilms. To compare the role of two well-known PAMPs in biofilms, LPS and curli, we utilized UPEC lab strain UTI-89 wild type (WT) and its msbB mutant, which produces a pentacylated LPS that is not immunostimulatory for hu-moDCs. Using bulk RNASeq, Flow Cytometry and proteomics, we compared hu-moDC responses to these biofilms and to individual biofilm components LPS and curli. Results We found that exposure to biofilms strongly activated hu-moDCs, inducing upregulation of surface expression of MHC and costimulatory molecules and secretion of pro-inflammatory cytokines and chemokines in a dose-dependent manner, showing similar activation with WT and msbB mutant biofilms, therefore irrespective of the presence of LPS. Curli and LPS also induced similar responses. Transcriptional analysis showed specific molecular pathways for ongoing investigation. Conclusion Our data suggest that not only can biofilms activate hu-moDCs, but they also do that in an LPS-independent manner, highlighting curli as a major biofilm-associated PAMP. Deeper knowledge of the innate immune response to biofilms is an important step in understanding the full immune response to biofilms. Funding Source N/A Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
Defining the Role of the RSV Small Hydrophobic Protein in Viral Pathogenesis 2255472
Abstract Introduction Respiratory Syncytial Virus (RSV) accounts for 33 million infections worldwide each year and is the leading cause of lower respiratory tract infections in infants. Severe RSV-induced disease is associated with the host response to the infection including the increased production of the inflammatory cytokines IL-1β and IL-6. The RSV small hydrophobic (SH) protein has been previously implicated in regulating the secretion of IL-1β following infection. The SH protein is classified as a viroporin, a class of transmembrane proteins that oligomerize to form pores in membranes and can facilitate ion flux to modulate multiple signaling pathways. It is currently unknown what role SH may play in modulating RSV disease severity beyond the modulation of IL-1β production; however, in 2017 there was an outbreak of RSV-B cases containing a mutation in the SH protein that resulted in high hospitalization rates. This outbreak occurred in the summer, an unusual season for RSV cases prior to the COVID-19 pandemic, with the virus expressing a single nucleotide polymorphism within the SH protein that changed an asparagine into an aspartic acid at amino acid 64 (SH N64D). Methods To study the impact of the N64 mutation, we generated a recombinant A2 laboratory strain virus encoded with an aspartic acid at amino acid 63 which is termed A2 SH N63D. We utilized plaque assays, ELISAs, qPCR for cytosolic mtDNA, and human monocyte THP-1 cell lines with a dual reporter for NF-κB and type I IFN response. Results The A2 SH N63D mutant elicited decreased viral titers and early disease compared to the wildtype A2 (WT A2) in mice. We have also observed increased inflammatory cytokines and a type I IFN response following A2 SH N63D infection. We have observed mtDNA release after A2 SH N63D infection within THP-1 cells as well as a STING-dependent type I IFN response Conclusion These results suggest that the aspartic acid within the C-terminus of the SH protein may contribute to RSV pathogenesis and the type I IFN response. Funding Source NIAID R01 AI167249 Topic Categories Viral Immunology (VIR)
An antigen-agnostic approach to understanding antibody responses against blood-stage Plasmodium falciparum 2309989
Abstract Introduction Malaria is a deadly disease that has been managed using approaches that target the mosquito vector or the parasite within the host. However, the emergence of resistance in both the mosquito and parasite has highlighted the need for novel interventions. The discovery of potent monoclonal antibodies (mAbs) against blood-stage Plasmodium falciparum merozoite antigens has elucidated mechanisms of red blood cell invasion and facilitated new clinical developments such as the development of the RH5 vaccine. More, however, remains to be uncovered regarding the merozoite membrane antigenic landscape and its interactions with the human humoral immune system. Methods Here, we use an antigen-agnostic approach to study the human antibody response to merozoite membrane antigens after natural malaria infection. We evaluated circulating antibody reactivity in a Malian cohort to P. falciparum merozoites. To exclude donors that had high reactivity to MSP1, an immunodominant polymorphic antigen on the merozoite surface, we included a pre-blocking step with this antigen. We identified individuals that retained high antibody reactivity to merozoites despite MSP1 blocking. These individuals have been selected for isolation of mAbs targeting merozoites in an antigen-agnostic manner using the Beacon optofluidic system. Results We have started screening B cells for merozoite reactivity from these donors of interest. From a single donor, we have isolated four unique mAbs. These mAbs bound merozoites to varying degrees and will be tested for neutralization using a growth inhibition assay. Conclusion Future work includes screening more donors, identifying target antigens from strong neutralizing mAbs, and performing structural studies to identify key antibody-antigen contact residues. Funding Source This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
A Targeted, Tandem‐Activated Artificial Urinary Biomarker Sensor for Early Detection and Therapeutic Intervention of Thoracic Aortic Aneurysm and Dissection
ABSTRACT Thoracic aortic aneurysm and dissection (TAAD) is a life‐threatening cardiovascular disorder; current clinical imaging approaches primarily detect structural abnormalities at late stages and fail to enable early diagnosis or dynamic therapeutic monitoring. Here, we develop a targeted, tandem‐activated artificial urinary biomarker sensor (TADEI) for real‐time optical imaging and urinalysis of early biomarkers associated with TAAD. After identifying biomarkers from patient tissue samples, TADEI was designed with three key components conjugated to a near‐infrared fluorophore: a VCAM‐1–targeting moiety (overexpressed on inflamed endothelial cells), tandem‐responsive substrates for caspase‐1 (an inflammation mediator) and legumain (a protease relevant to vascular remodeling), and a renal‐clearance–enabling moiety. TADEI specifically targets TAAD lesions and activates its fluorescence upon reaction with these early biomarkers after systemic administration. Meanwhile, TADEI is excreted into urine via renal clearance (≈76%), converting TAAD biomarker levels into urinary signals for optical urinalysis. In a mouse model, a TADEI‐based urine test detected TAAD at least 12 days earlier than ultrasound imaging and 16 days earlier than plasma D‐dimer assays. The correlation between the urinary signal of activated TADEI and disease severity guided intervention that effectively inhibited TAAD progression, achieving 100% survival compared with 73% for ultrasound‐guided therapy and 40% without treatment.