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Lupus Susceptibility Gene Pbx1 Regulates Treg Metabolism 2256201
Abstract Introduction The lupus susceptibility locus Sle1 includes the Pbx1 gene, which is preferentially expressed in Tregs among CD4+ T cells. Its dominant-negative isoform Pbx1-d is overexpressed in Tregs from both human and murine lupus, contributing to Treg instability and loss of suppressive function. Pbx1 regulates the metabolism of cancer and mesangial cells, thus this study aimed to determine whether Pbx1-d regulates Treg function via metabolic reprogramming. Methods Metabolic profiling of Pbx1 WT, KO, and Pbx1-d overexpressing Tregs was performed using Seahorse metabolic assays. A dual-luciferase reporter assay was used to assess human and murine CamkIV promoter activation by Pbx1 isoforms. CRISPR-Cas9—mediated CamkIV knockdown was generated in CD4+ T cells, which will be polarized into induced Tregs (iTregs) and analyzed by Seahorse and in vitro suppression assays. Results Here, we showed that both Pbx1-d overexpressing and Pbx1-deficient murine Treg cells exhibit increased glycolysis and lower respiration to glycolysis ratio compared to WT cells. In addition, Pbx1 deficient Tregs have higher basal and maximum glycolytic capacity than WT independent of respiration. These results indicate that Pbx1 regulates Treg metabolism. We further showed that CamkIV, a glycolysis-driving gene overexpressed in lupus T cells and known to impair Treg function, is a direct Pbx1 transcriptional target with Pbx1-d inducing a higher expression relative to the normal Pbx1-b in both species. CamkIV-knockdown iTregs will be used to assess whether CamkIV deficiency rescues the metabolic and functional alterations driven by Pbx1-d. Conclusion Our findings suggest that Pbx1-d impairs Treg homeostasis by reprogramming cellular metabolism, at least in part through CamkIV upregulation. Understanding this mechanism could guide strategies to enhance Treg efficacy in adoptive therapies and to overcome intrinsic barriers limiting Treg function in autoimmune diseases. Funding Source R01 AI045050-24 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Evaluating AlphaFold 3 immune complex structure prediction and cryo-EM analysis of broadly neutralizing antibodies towards influenza hemagglutinin 2309571
Abstract Introduction Epitope:paratope interactions define the breadth and affinity an antibody has towards an antigen. Computational structure prediction can help to model immune complexes as a method of in silico screening for broadly neutralizing antibodies (bnAbs). However, we do not currently understand how structural predictions and confidence metrics correlate with experimental data. Here, we evaluate the accuracy of AlphaFold 3 (AF3) in predicting immune complexes of a known class of bnAbs towards a conserved epitope on influenza hemagglutinin (HA). Methods We used single-cell RNA sequencing to generate a library of HA reactive, paired antibody sequences. AF3 was used to computationally model antibody-HA interactions. Antibodies were expressed and characterized with BLI, ELISA, and microneutralization assays. Negative stain electron microscopy (nsEM) and single particle cryo-electron microscopy (SPA cryo-EM) were used to structurally characterize immune complexes. Results B cell receptor sequencing of PBMCs from 23 healthy, human donors generated 1161 H5 HA-specific, paired antibody sequences. These sequences comprised new examples within a known class of antibodies (VH1-69, of which previous examples are in the AF3 training set) likely to target the conserved central stem. We modeled these antibody sequences in complex with H1N1 and H5N1 HAs using AF3. Highly confident predictions correlated with antibodies possessing high affinity and breadth. Moreover, in silico epitope footprints strongly matched experimental nsEM data. Finally, we resolved atomistic details with high-resolution SPA cryo-EM that explain challenges with AF3 immune complex prediction. Conclusion AF3 can be used to screen for VH1-69 bnAbs targeting a conserved epitope on influenza HA with high confidence. This method of down selection may be applied to other classes of antibodies and to other epitopes. However, models still need to be tuned to accurately predict immune complexes of novel or less characterized antibody classes. Funding Source Endowed Fellowship in the Skaggs Graduate School of Chemical and Biological Sciences, National Institute of Allergy and Infectious Diseases (NIAID). Topic Categories Computational and Systems Immunology (COMP)
NKG7 is upregulated by IL-2/T-bet pathway during effector differentiation of cytotoxic CD8+ T cells 2309827
Abstract Introduction Cancer immunotherapy has transformed the treatment landscape by leveraging the immune system to eradicate malignant cells. Among immune effectors, CD8+ T cells play a pivotal role in tumor clearance through recognition and destruction of cancer cells. To effectively counter the rapid proliferation and continuous evolution of cancer, robust effector function of cytotoxic CD8+ T cells is essential. The expression of effector genes in CD8+ T cells is tightly regulated, ensuring that cytotoxic molecules are expressed only at appropriate times and in relevant contexts. We previously reported that upregulation of the gene NKG7 following immunotherapy is critical for clinical response and demonstrated that NKG7 expression is both necessary and sufficient for T cell cytotoxicity. However, the mechanisms governing NKG7 expression in CD8+ T cells remain poorly defined. Methods We performed a cytokine screen to identify potential drivers of NKG7 expression at both the transcriptional and protein levels in cell lines and healthy donor peripheral blood mononuclear cells (PBMC). We employed quantitative RT-PCR, flow cytometry, and Western blotting to determine and validate the expression level of NKG7 and relevant signal molecules. We validated the signal pathways identified in single cell RNAseq data analysis in PD-1 therapy-responsive CD8+ T cells. Results In PBMC derived CD8+ T cells, we found that IL-2 increases NKG7 mRNA levels, correlating with a dose dependent increase in NKG7 protein expression. IL-2—induced NKG7 expression is associated with elevated levels of T-bet, a transcription factor essential for effector T cell differentiation. Consistent with this, scRNA-seq analysis revealed enrichment of the IL-2 signaling pathway in CD8+ T cells responsive to anti—PD-1 therapy. Conclusion Our results suggest that NKG7 is upregulated by IL-2 and T-bet signaling. Due to the critical role of this signaling axis during effector CD8+ T cell differentiation, NKG7 expression may be determined during this phase. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Breaking Thermal Conductivity–Electrical Resistivity Trade‐Off in Liquid Metal–Based Thermal Interface Materials via Interface Engineering
ABSTRACT Liquid metal–based thermal interface materials offer superior thermal conductivity and fluidity but are limited in practical applications by their inherently low electrical resistivity. Here, we present an interface engineering strategy that overcomes this fundamental trade‐off, enabling the synthesis of GaIn‐B featuring a bimodal particle size distribution. This structure simultaneously exhibits a non‐contact network feature that effectively prevents electrical percolation while maintaining efficient thermal transport. GaIn‐B exhibits a significant thermal conductivity of approximately 16 W m −1 K −1 and an electrical resistivity exceeding 10 11 ohm cm. We developed a phenomenological model based on effective medium theory to quantitatively describe and predict the critical conditions for breaking the thermal–electrical trade‐off. The simplicity and scalability of the GaIn‐B synthesis process enable kilogram‐scale production, making it highly suitable for industrial applications.
Development of an in vitro Sjögren’s disease tissue chip for mechanistic studies and drug screening 2300053
Abstract Introduction This project aims to develop a Sjögren’s disease (SjD) tissue chip for disease modeling and drug discovery. SjD is a chronic autoimmune disorder affecting up to 1% of the global population, characterized by lymphocytic infiltration and destruction of salivary glands, leading to hyposalivation in over 90% of patients. Current treatments are palliative and do not halt disease progression. Although genetic, epigenetic, hormonal, and environmental factors are implicated in SjD development, its etiology remains unclear. While animal models are commonly used, they are costly, low-throughput, and raise ethical concerns. Methods To address these limitations, we are developing an in vitro SjD tissue chip (SDTC) using microbubble (MB) array technology and diseased salivary gland mimetics (SGm) derived from 12-13-week-old MRL/lpr mice, a validated SjD model. The MB array enables generation of > 20 chips per mouse, each containing ∼40 uniform SGm, thereby reducing animal use and increasing assay throughput. Flow cytometry characterized epithelial and immune cells in glands from MRL/lpr mice and C57BL/6 controls. SDTC validation included viability staining and calcium signaling assay as functional readouts in SGm from young (6-7 weeks) and aged (12-13 weeks) MRL/lpr mice, and aged-matched controls. Results The SDTC maintained >90% viability and promoted SGm spheroid growth by day 7. Aged MRL-SGm showed reduced carbachol-induced calcium flux, consistent with SjD secretory dysfunction. Flow cytometry showed acinar-to-ductal cell ratios in C57BL/6 glands matching previous studies, supporting methodological accuracy. Notably, aged MRL glands exhibited increased CD45+ leukocyte infiltration, predominantly T cells. Ongoing studies will assess SDTC cellular composition, gene expression, and tissue architecture. Conclusion This work establishes the first in vitro SjD tissue chip with increased assay throughput and reduced animal use for mechanistic studies and drug screening. Funding Source University of Rochester Environmental Health Sciences Center (EHSC) and NIH Office of Autoimmune Disease Research Topic Categories Technological Innovations in Immunology (TECH)
IgA Dengue Virus NS1 Immune Complexes Are Associated with In Vitro Neutrophil Activation 2308886
Abstract Introduction Dengue virus (DENV) causes ∼400 million infections annually, with severe disease occurring more frequently during heterologous secondary infections, partly due to antibody-dependent enhancement and heightened inflammation. While IgG responses have been extensively studied, the role of circulating IgA, particularly IgA targeting non-structural protein 1 (NS1), in modulating neutrophil activation remains poorly understood. IgA immune complexes (ICs) can engage Fc receptors on neutrophils and trigger effector functions such as neutrophil extracellular trap (NET) formation (NETosis), but their contribution to dengue immunopathogenesis has not been fully characterized. Methods Serum samples from DENV-seropositive individuals in Puerto Rico (n = 20) were evaluated for IgA and IgG recognition of recombinant DENV-2 NS1 and envelope (E) proteins by ELISA. Neutrophils isolated from healthy donors were stimulated for 6 hours with IgA-NS1 or IgG-NS1 immune complexes. NETosis was quantified using the Incucyte live cell imaging system. Correlation analyses were performed in GraphPad Prism. Results IgG recognized NS1 and E proteins at comparable levels, while IgA favored the recognition of NS1 over E protein. The mean NS1/E ratio was 1.09 for IgG (range 1.02—1.16) and 1.71 for IgA (range 1.18—2.10). NETosis demonstrated a stronger and more statistically significant association with IgA-NS1 ICs (R² = 0.42, p < 0.0001) when compared to IgG-NS1 complexes (R² = 0.15, p = 0.026). Conclusion Collectively, these findings suggest that IgA—NS1 immune complexes may serve as potential modulators of neutrophil-driven inflammation in dengue. This isotype-specific linkage to NETosis suggests a distinct immunopathogenic pathway that may be particularly relevant during heterologous DENV infection and merits further mechanistic and translational investigation. Funding Source R01AI153434 & U54MD007579 Topic Categories Viral Immunology (VIR)
Intratumoral administration of a single dose of the HEPLISAV-B Vaccine and IL-15 complexes induces tumor regression in Triple Negative Brest Cancer 2259444
Abstract Introduction Triple negative breast cancer (TNBC) is a disease with limited treatment options and a poor prognosis, highlighting an urgent need for therapeutic strategies to improve outcomes. Immune checkpoint blockade (ICB) with a PD-1 inhibitor has been an attractive strategy for TNBC treatment; however, the “immune-excluded” tumor microenvironment (TME) often confers resistance. Therefore, a creative solution is needed to turn the immunosuppressive TME into an “immune-inflamed” one with robust CD8+ T cell activity and infiltration. In this study, we determined whether combining IL-15 complexes alone, anti-PD-1 alone, or IL-15 complexes and anti-PD-1 together, with the HEPLISAV-B (HEP B) vaccine, administered intratumorally (i.t.), would enhance tumor immunity in an established 4T1 breast cancer model. Methods For this study female BALB/c mice were injected in the right mammary fat pad with the 4T1-luciferase expressing TNBC cell line, and once tumors developed, either a single dose of HEP-B vaccine or three serial doses of HEP B vaccine was administered i.t., followed by two i.t. injections of IL-15 complexes alone, five doses of anti-PD1 alone (i.p.), or a combination of both IL-15 complexes and anti-PD1 and tumor growth and survival were monitored over 60 days. Tumors were measured using calipers and IVIS imaging. Results We show that, compared with control mice receiving PBS, a single i.t. dose of HEP B induces complete tumor regression in 60% of mice. When HEP B was used with IL-15 complexes and α-PD1, 80% of mice remained tumor-free. Furthermore, when HEP B was combined with IL-15 complexes and +-PD1, tumors regressed and did not recur. CXCL9 expression was downregulated on CD8 T cells in the tumors of mice treated with HEP B alone or with IL-15 and +-PD-1, while CXCR4 was increased. Conclusion Our findings indicate that the HEP B vaccine in combination with IL-15 is an attractive strategy to overcome immune checkpoint inhibitor resistance when added to +PD-1 treatment in TNBC. Funding Source DoD proposal number BC23095 award number HT9425-24-1-0463 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
IL-22 Depletion is a Cornerstone of Gut Immune Dysregulation in Lupus-Prone Mice 2255818
Abstract Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease that features excessive immune activation and inflammation throughout the body. Local gut inflammation causes an impaired mucosal barrier (“leaky gut”), leading to the dissemination of gut bacteria and their immunogenic components throughout the body. SLE patients have elevated biomarkers of leaky gut; however, the mechanism by which this occurs is not clear. Methods To determine whether lupus-prone mice exhibit progressive defects in gut immune regulation, we performed flow cytometry on gut lamina propria cells of lupus-susceptible B6.Sle1.Sle2.Sle3 triple congenic (TC) mice and B6 controls at young, middle, and old ages. We also treated young TC, B6, and single congenic B6.Sle1 mice with two agents linked to lupus exacerbation and leaky gut: resiquimod (R848), a TLR7 agonist that mimics the high type I interferon signature seen in SLE patients, and Ruminococcus gnavus (Rg), a gut pathobiont that cross-reacts with anti-dsDNA autoantibodies. Results With age in TC mice, there were many immune alterations consistent with lupus progression, including increased CD4+ and CD8+ T cell frequencies. Notably, IL-22, a cytokine critical for gut barrier integrity and immune homeostasis, was depleted uniquely in the ileum of TC mice over time. ILC3s, the main immune cell subset responsible for producing IL-22 in the gut, correspondingly decreased with age in TC mice. Interestingly, R848 treatment in young TC mice reduced global IL-22 production and significantly altered the distribution of IL-22-producing cells. Combined Rg and R848 treatment in B6.Sle1 mice led to greater IL-22 depletion in ILC3s than R848 alone, indicating a synergistic mechanism between gut dysbiosis and TLR7 signaling, and increased IFN-y secretion, a strong inflammation marker. Conclusion Our findings demonstrate a gut-localized immune deficit in inducible models of lupus that mimics natural lupus progression and may precede or contribute to gut barrier vulnerability. Funding Source Supported by an NIH grant R01 AI143313-06 to Laurence Morel. Topic Categories Mucosal and Regional Immunology (MUC)
Maternal fructose—induced metabolic reprogramming underlies multi-tissue inflammation and dysbiosis in HF-fed offspring: partial normalization by PCA 2256918
Abstract Introduction Excess maternal fructose intake predisposes offspring to metabolic pathology. p-Coumaric acid (PCA), a phenolic with anti-inflammatory and mitochondria-supporting actions, may counteract such programming across metabolic tissues and the gut microbiome. Methods During gestation and lactation, dams consumed either standard chow, a 60% fructose diet, or the same fructose diet with PCA (1 mg/g in diet). After weaning, all progeny were maintained on a 60%-fat diet for 8 weeks and designated CD-HF, HFr-HF, or HFr+PCA-HF. We profiled metabolic phenotypes; histology of adipose, liver, muscle, and intestine; inflammatory signaling; mitochondrial/glycolytic transcripts; grip strength; and 16S rRNA microbiome composition. Results Maternal fructose exposure impaired glucose control and insulin sensitivity and provoked adipocyte hypertrophy, hepatic steatosis, skeletal-muscle myosteatosis, and reduced grip strength, coincident with amplified inflammatory signaling in adipose, liver, and muscle. HFr-HF exhibited higher Clostridiaceae and Peptostreptococcaceae with lower Bacteroidaceae and Muribaculaceae. Maternal PCA (HFr+PCA-HF) attenuated mitochondrial-stress signatures, improved intestinal morphology, and countered fructose-driven inflammatory signaling, while shifting the microbiome toward a CD-HF-like configuration. Conclusion Maternal fructose perturbs gut—liver—muscle crosstalk, coupling mitochondrial stress, systemic inflammation, and dysbiosis in offspring. Maternal PCA offers partial normalization of inflammatory pathways and microbial composition despite a post-weaning high-fat challenge, indicating a preventive window for intergenerational metabolic health. Funding Source This research was supported by a National Research Foundation of Korea (NRF) 2023-00208776, RS-2023-00221563 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)
GSDME-Mediated Pyroptosis Suppresses Anti-Tumor Immunity via IL-1α—Dependent Recruitment of Immunosuppressive Myeloid Cells 2309550
Abstract Introduction Pyroptosis is widely recognized as an immunogenic form of cell death that promotes anti-tumor immunity. Gasdermin E (GSDME), cleaved by caspase-3 in tumor cells, has therefore been proposed as a tumor suppressor. Here, we report an unexpected immunosuppressive role of GSDME-mediated pyroptosis in cancer development. Methods A genetically engineered spontaneous C-Myc+ Tp53⁻/⁻ murine HCC model was used. Hepatocyte-specific deletion of Gsdme and/or Il1a was achieved using conditional knockout strategies. Tumor growth was assessed in immune-competent and immune-deficient mice. Tumor-infiltrating immune cells were analyzed by flow cytometry. Cytokine release mechanisms were studied in hepatocytes and tumor cells. Human HCC samples were analyzed by immunohistochemistry to assess GSDME expression and its association with tumor stage. Results Hepatocyte-specific deletion of Gsdme significantly delayed tumor growth in immune-competent mice but not in immune-deficient hosts, indicating an immune-dependent mechanism. Although GSDME directly promoted tumor cell death in vitro, its expression in vivo was associated with impaired anti-tumor immunity, characterized by reduced infiltration of cytotoxic T and NK cells and increased accumulation of CD11b+ immunosuppressive myeloid cells. Mechanistically, GSDME pores facilitated interleukin-1α (IL-1α) secretion from tumor cells independently of overt plasma membrane rupture and were associated with calpain activation and nuclear membrane permeabilization. Il1a deletion phenocopied Gsdme deficiency, and combined deletion of Gsdme and Il1a did not confer additional tumor protection. In human HCC, high GSDME expression correlated with advanced tumor stage. Conclusion These findings uncover a previously unrecognized immunosuppressive function of GSDME-mediated pyroptosis in cancer. By promoting IL-1α—driven recruitment of suppressive myeloid cells, GSDME can impair anti-tumor immunity, challenging the paradigm that pyroptosis is uniformly beneficial in cancer. Funding Source n/a. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
PROTAC-Mediated SHP2 and STAT3 Degradation as a Therapeutic Strategy for Rheumatoid Arthritis 2309941
Abstract Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation, fibroblast-like synoviocyte (FLS) hyperplasia, and progressive cartilage and bone destruction. RA FLS acquire an aggressive phenotype that promotes inflammatory cytokine production and drives tissue damage. Despite advances in RA therapies, many patients fail to achieve durable remission, highlighting the need for strategies that target the molecular drivers of joint pathology. The protein tyrosine phosphatase SHP2 (PTPN11) integrates cytokine and growth factor signaling through MAPK, PI3K/Akt, and STAT3 pathways. However, the specific contributions of SHP2—STAT3 signaling to synovial inflammation and RA progression remain poorly defined. Methods Primary FLS isolated from RA patients and healthy donors were treated with selective PROTAC degraders targeting SHP2 (SHP2D26) or STAT3 (SD36). Cell viability, target protein degradation, and downstream signaling were evaluated by MTT and immunoblotting assays. Complementary in vivo studies employed the K/BxN serum transfer arthritis mouse model with conditional deletion of SHP2 in either Prg4+ synovial lining cells or LysM+ myeloid cells. Disease severity was evaluated by ankle measurements, DigiGait analysis, flow cytometry, micro-CT, and IF staining to evaluate synovial inflammation and immune cell infiltration. Results Targeted degradation of SHP2 or STAT3 in RA FLS significantly reduced inflammatory cytokine production and attenuated pro-inflammatory signaling. In vivo, SHP2 deletion in either Prg4+ or LysM+ cells reduced joint swelling, immune cell infiltrates, and synovial pathology, with preservation of joint architecture and articular cartilage. Conclusion These findings identify SHP2—STAT3 signaling as a key regulator of pathogenic FLS behavior, stromal—immune interactions, and RA progression. Targeted degradation of SHP2 or STAT3 represents a promising therapeutic strategy to limit synovial inflammation and joint destruction in RA. Funding Source NIAMS R21 AR081642, NIDDK R01DK138630 Topic Categories Immune Mechanisms of Human Disease (HUM)
Solvation Chemistry Reimagined: LiPF6‐Enabled Suppression of Gas Evolution for Ultra‐Stable 200 Ah Anode‐Free Lithium‐Metal Batteries
ABSTRACT Anode‐free lithium‐metal batteries (AFBs) offer an attractive pathway to achieve cell‐level energy densities >500 Wh kg −1 but suffer from rapid degradation driven by parasitic electrolyte decomposition, gas evolution, and unstable interphases. Here, we report a solvation‐structure engineering strategy using trace LiPF 6 additive in localized high‐concentration electrolytes (LHCEs) to regulate homogeneous Li deposition and stabilize electrode‐electrolyte interfacial chemistry. Molecular dynamics simulations and spectroscopic analyses reveal a synergistic PF 6 − –FSI − coordination that dramatically enriches aggregate (AGG) species in the Li + solvation sheath, increasing AGG populations from 17% to 93% with only 1 wt.% LiPF 6 . Owing to its persistent stability, this LiPF 6 ‐derived AGG‐dominated solvation suppresses ether‐solvent reduction and CH 4 generation, forming an inorganic‐rich solid–electrolyte interphase/cathode‐electrolyte interphase. AFBs with optimized electrolyte achieve substantially improved performance, including a significant reduction in high‐temperature gas evolution alongside increases in cycle life of 64% at 45°C and 28% at 25°C. When scaled to 240 Ah blade cells, this optimized electrolyte delivers stable cycling for nearly 100 cycles at 80% DOD and 45°C with negligible swelling and >1260 Wh L −1 volumetric energy density. This study demonstrates a practical and scalable electrolyte design principle that overcomes the key barriers to commercial deployment of large‐format AFBs.
Regulation of Dendritic Cell Migration by RANK Signalling in Lymphatic Endothelial Cells 2303575
Abstract Introduction Lymph nodes (LNs) are key sites for initiating adaptive immune responses, enabling antigen-specific T cells to interact effectively with dendritic cells (DCs) and B cells. DCs migrate from peripheral tissues to draining LNs via lymphatic vessels lined by lymphatic endothelial cells (LECs), guided by chemokines and adhesion molecules. LEC-derived CCL21 binds CCR7 on activated DCs, while ACKR4 on subcapsular sinus floor LECs modulates the CCL21 gradient to steer DC migration. Recent findings implicate lymphotoxin β receptor (LTβR) signalling in regulating leukocyte trafficking through canonical and non-canonical NF-κB pathways, affecting expression of CCL21 and VCAM-1. However, the role of RANK (Receptor Activator of NF-κB) in LEC-mediated DC migration remains unclear. Methods We generated Prox1-CreERT2 Rank flox/flox mice and induced LEC-specific knockout via tamoxifen and confirmed gene deletion by PCR and flow cytometry. Bone marrow-derived DCs, matured with LPS and pulsed with OVA, were transferred into control or knockout mice. Migration to draining LNs and antigen presentation to CD4+ T cells were assessed. Results RANK-deficient mice showed significantly impaired DC migration, unlike LTβR-deficient mice. To dissect underlying mechanisms, we developed an in vitro LEC—DC interaction model using LECs isolated from wild-type, Rank-deficient, and Rank-overexpressing mice. Gene manipulation was achieved using Cre-expressing adeno-associated virus, and RANKL was administered to stimulate signalling. We observed a RANK-RANKL signalling-dependent MAdCAM-1 and VCAM-1 expression in LECs. We also observed ACKR4 alterations in the loss or overexpression of RANK. Conclusion We have shown that RANK activation affects DC transmigration across the lymphatic barrier and are currently investigating its molecular mechanism. Funding Source The project receives funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement number 101034170 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Tim-4 Effector B Cells Express a Th17-Like Proinflammatory Cytokine Module That Is Restrained by Nur77 2266650
Abstract Introduction B cells expressing various proinflammatory cytokines potentiate immune responses in infection, autoimmunity and transplantation. However, phenotypic markers for such effector B cells (Beffs) are lacking and regulation of their proinflammatory potential is uncharacterized. Tim-4+ B cells express IFNγ and promote islet allograft rejection. We now examine expression and regulation of other proinflammatory cytokines by Tim-4+ B cells. Methods RNAseq/ATACseq: +/- 24h stimulation with anti-IgM+IL-23. Cytokines: intracellular flow cytometry (IFC) of splenocytes stimulated 5h with PMA, ionomycin, BrefeldinA. Transplants: BALB/c islets under the kidney capsule of chemically diabetic B6 mice; heterotopic Bm12 hearts into B6 mice. Results Tim-4+ Beffs uniquely express RORgt, which along with IL-23, drive expression of a pathogenic Th17-like proinflammatory module comprising Il17a, Il17f, Il22, Csf2, Il6, and Il1b. RNAseq/ATACseq identified Nr4a1 (Nur77) as a potential TF regulating Tim-4+ B cells. Nur77floxCd19CRE+/- (Nur77 BKO) mice exhibited a 1.5X increase in Tim-4+ Beffs, and these had increased RORgt (2.5x), IL17(1.7x), IL22(2x), GM-CSF(1.5x), IL6(2x), & IL1b(2x) expression vs. Cre-controls (IFC). Notably, Tim1+ Breg frequency and IL-10 expression were decreased (30% & 60% respectively). Compared to Cre controls, Nur77 BKO mice exhibited shorter islet allograft survival (MST 14d vs 20d; p < 0.05) and accelerated rejection of BM12 hearts (MST 30d vs 65d; p < 0.01). In vitro data reveal tamoxifen-induced KO of B cell Nur77 acutely increases IL-17 expression, now enabling studies to determine how Nur77 regulates the Tim-4+ Beff transcriptome. Conclusion Tim-4 is a broad marker for Beffs that promote allograft rejection via expression of a pro-inflammatory module driven by IL-23 and RORgt. Nur77 constrains Tim-4+ Beffs and maintains a normal balance between Beffs and Bregs. This enhances our understanding of B cell biology and provides insight into immune regulation and tolerance. Funding Source R01 Topic Categories Transplantation Immunology (TRAN)
FCRL1 promotes tonic PI3K/AKT signaling and constrains inducible BCR pathways 2260146
Abstract Introduction Fc receptor-like 1 (FCRL1) is an understudied B cell immunoregulatory protein with tyrosine-based signaling in humans and mice that peaks on naïve and memory subsets. Among malignancies, FCRL1 is upregulated by chronic lymphocytic leukemia (CLL) and germinal center (GC)-derived B cell lymphomas that rely on tonic PI3K/AKT signals. Early studies found FCRL1 could co-activate B cell receptor (BCR) functions, but broader understanding of its regulatory roles in human B cells remains limited. Methods To interrogate its biology, we used a CRISPR/Cas9 strategy to target FCRL1 in a GC-derived B cell lymphoma cell line that highly expresses it. Single-cell sorting was performed to isolate and expand FCRL1-deficient clones. Indel profiling, mRNA quantification, and flow cytometry analyses confirmed FCRL1 knockout at both the transcript and protein levels. To comprehensively define FCRL1-dependent pathways in an unbiased manner, RNA sequencing (RNA-seq) and gene set enrichment analysis (GSEA) were used to compare FCRL1-sufficient and -deficient cells. Transcriptomic findings were validated by Western blot and phospho-flow cytometry under basal conditions and following BCR stimulation. Results Expanded FCRL1-deficient clones showed altered granularity and enhanced BCR-mediated apoptosis. RNA-seq and gene set enrichment analysis (GSEA) revealed that the loss of FCRL1 drove the upregulation of multiple hallmark pathways including mTORC1, NF-kB, apoptosis, INFg, and hypoxia, indicating a repressive role for it in resting B cells. Cells lacking FCRL1 also exhibited reduced global tyrosine phosphorylation (pY) and p-AKT (T308), but had higher Src-family kinase (SFK) and p-PLCg2 activity both at rest and upon BCR ligation. Conclusion These results implicate FCRL1 as a dual-regulator, which promotes tonic PI3K/AKT signals but limits inducible BCR activation, and expose its potential significance in B cell-related malignancies and humoral disorders. Funding Source Leukemia Lymphoma Society and the UAB O’Neal Comprehensive Cancer Center Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)
The effect of type I interferon on infection and the CD8 T cell response to Coxsackievirus B3 2255932
Abstract Introduction Coxsackievirus B3 (CVB3) is the leading cause of viral myocarditis, which disproportionately affects males in both humans and mice. Sex-specific immune responses likely contribute to this bias; however, the mechanisms remain unclear. We previously found that CVB3 induced expansion of protective CD8 T cells in female but not male Ifnar-/- mice. To further define this difference, we evaluated how type I interferon (IFN) influences infection and CD8 T cell responses to CVB3. Furthermore, we bioinformatically predicted CVB3 CD8 T cell epitopes to track these T cell responses. Methods Beta-2-microglobulin knockout (b2M-/-) and bone marrow chimera were intraperitoneally inoculated with 1x104 PFUs of CVB3. Bone marrow chimera mice were prepared by lethal irradiation of male and female Ifnar-/- mice, followed by reconstitution of their immune cells with bone marrow from sex-matched Ifnar-/- and wild-type mice. Splenic T cells were evaluated by flow cytometry, and mice were monitored for survival. Finally, NetMHCcons, PickPocket, and Consensus algorithms were used to predict CD8 T cell epitopes of CVB3. Results We found that CVB3-inoculated female β2M-/- mice, which lack CD8 T cells but exhibit an IFN response, had significantly more CVB3-induced mortality than wild-type mice. Furthermore, to evaluate the contribution of type I IFN on the CD8 T cell response, we found that bone marrow chimera mice that received either Ifnar-/- or wild-type immune cells showed similar responses in CD8 T cells to CVB3 infection. Finally, since CD8 T cell epitopes for CVB3 have not been identified, we employed a bioinformatic approach to identify 33 top candidates that map across the CVB3 genome. Conclusion Overall, these data indicate that CVB3-specific CD8 T cells offer immune protection in female mice, independent of type I IFNs. We are currently confirming the CD8 T cell epitopes so we can track the fate of these CD8 T cells to determine their long-term protection against CVB3 infection. Funding Source K01: DK110216, R03: DK124749, a Biomedical Research Grant from the Indiana Clinical and Translational Sciences Institute, and an American Heart Association (grant 23TPA1141316) to CMR. Topic Categories Viral Immunology (VIR)
The Evolutionary Diversification of CD300s Reveals How Gene Duplication and Structural Innovation Shift the Landscape of Immune Recognition 2257270
Abstract Introduction The CD300 gene cluster encodes activating and inhibitory lipid-binding innate immune receptors that, in humans and mice, play important roles in immune response, including viral entry and autoimmunity. However, their broader evolutionary and functional diversification dynamics across vertebrates remain unresolved. Methods Genomic and transcriptomic analyses were employed to identify CD300 orthologs and paralogs across vertebrates. Numbers and combinations of CD300 genes were quantified and cataloged. AI-based protein folding tools were used to predict changes in protein structures. Recombinant forms of CD300 proteins were generated and subjected to an unbiased lipid-binding assay to identify and quantify ligand binding differences between orthologs and paralogs. Results Our analyses reveal that CD300s are present across jawed vertebrates, with a pronounced pulse of gene duplication coinciding with the origin of placental mammals. Within mammals, the evolutionary trajectory of this cluster is remarkably labile. We observe dramatic lineage-specific expansions and contractions in groups such as chiropterans (bats) and cetaceans (whale and dolphins) that correspond to major events in the evolutionary history of these groups. Structural predictions suggest that these gene duplications are accompanied by corresponding changes in protein shape, including the extracellular domains that mediate lipid binding. Ongoing lipidomics-based ligand screening supports this prediction. Conclusion Our preliminary data show divergent lipid-binding profiles across CD300 paralogs, suggesting functional novelty. Together, these results position CD300s as a tractable model for understanding how the molecular diversification of clustered immune gene families corresponds to their ligand preferences likely reflecting their evolutionary histories. Funding Source NSF IOS 2419126; NSF IOS 2419127; NSF IOS 2419128 Topic Categories Veterinary and Comparative Immunology (VET)
In vitro and In vivo Neuroinflammatory Responses to LPS in Plcg2-Deficient Mice 2259976
Abstract Introduction PLCG2, a critical immune regulator, has emerged as a key factor in Alzheimer’s disease, with genetic variants conferring both risk and protection. In the brain, PLCG2 is predominantly expressed in microglia, influencing cellular functions including calcium signaling and inflammatory responses. Methods We investigated PLCG2’s role in neuroinflammation using constitutive knockout mouse models subjected to lipopolysaccharide (LPS)-induced acute inflammation. Calcium imaging, transcriptomic profiling, and protein analyses were performed, followed by behavioral assessment. Results In primary microglia, PLCG2 loss blunted LPS-induced calcium responses and reduced calcium release even after ionomycin stimulation, indicating impaired intracellular Ca²+ handling or signaling capacity. I vivo, systemic LPS treatment induced over 200 differentially expressed genes in the brain, altering pathways related to innate and adaptive immune responses, inflammatory signaling, and microglial function. Knockout mice exhibited a distinct gene signature characterized by upregulated interleukin, prostaglandin, and interferon pathways. Consistent with the transcriptomic data, protein analysis confirmed elevated brain levels of the chemokine CCL2 (MCP-1), a key mediator of monocyte recruitment during inflammation, in LPS-treated knockout mice. Interferon-responsive genes were markedly upregulated in homozygote knockout mice post-LPS, a phenotype associated with white matter pathology and inflammaging. Principal component analysis of myelin-enriched genes revealed LPS-treated homozygote knockout samples clustering separately from other genotypes. Behaviorally, LPS-treated knockout mice displayed reduced locomotion in open-field tests, correlating with expression of sickness behavior-related genes. Conclusion These findings suggest that PLCG2 deficiency enhances interferon responses, potentially driving behavioral changes and myelin-related pathology. Funding Source This research was supported by the Alzheimer’s Association (AARG-21-846012, SH and JPP); the William & Ella Owens Medical Research Foundation (JPP); and intramural funding (SH and JPP). Topic Categories Neuroimmunology (NEUR)
ImmPort: An AI Eco-System for Reproducible Immunology Research 2310238
Abstract Introduction Immunology research generates diverse molecular and clinical datasets spanning autoimmunity, infectious diseases, allergy, transplantation, vaccine response and beyond. Decades of accumulated data in generalist and domain-specific repositories present an unprecedented opportunity for machine learning and artificial intelligence (AI) - powered analyses of large-scale, multi-modal datasets, accelerating scientific discovery. Methods ImmPort, the NIH-NIAID sponsored open-access immunology data portal, houses over 1,300 studies, 5,000 experiments, and over 7.5 million data points, supporting conventional and emerging technologies for capturing molecular and clinical datasets at subject level. To fully unlock this potential, ImmPort is deploying AI to semi-automate repository operations, reformatting data for AI-readiness, and establishing integrated AI ecosystems that maximize the value of existing immunology data for reproducible research and discoveries. Results AI-driven automation streamlines data curation and enhances metadata quality through intelligent extraction and validation. Standardized provenance frameworks, ontologies, machine-readable metadata, and Application Programming Interface (APIs) transform data into computationally accessible resources. ImmPort has developed methods for integrating Common Data Elements (CDEs) and piloted automated pipelines to facilitate systematic mapping for promoting Findability, Accessibility, Interoperability, and Reuse (FAIR) principles. ImmPort disseminates Fast Healthcare Interoperability Resources (FHIR) formatted data and is piloting Data Mesh architecture using ImmPort as a domain-oriented, interoperable data node to enable AI-assisted biomedical discovery. Conclusion The ImmPort AI ecosystem enables immunology-specific foundation models trained on multimodal data to capture immune system dynamics, transforming repositories into AI-enabled platforms that accelerate biomarker discovery and advance personalized, translational research. Funding Source HHSN316201200036W Topic Categories Computational and Systems Immunology (COMP)
Quinoxaline Terpolymer‐Controlled Miscibility With Oligomeric Acceptors for Over 20% Efficiency, Highly Stable and Stretchable Polymer Solar Cells
ABSTRACT While the emergence of oligomeric acceptors has improved the operational stability of polymer solar cells (PSCs), power conversion efficiencies (PCEs) and mechanical properties remain morphology‐limited, necessitating tailored polymer donors. Using tethered dimeric acceptor (DY2) as a model acceptor, we demonstrate how strategic modification of the classical D18 donor enhances miscibility and thus device performance. Specially, we developed a new quinoxaline (TQx) building block and incorporated it into D18 via random copolymerization, producing D18‐TQxn terpolymers (n = 5, 10, 15) with 5%–15% TQx proportion. The polar moieties in TQx enhance the surface energy of the terpolymers (improving thermodynamic miscibility) while also modulating the backbone conformation of D18, thereby tuning the crystallization kinetics. The optimal D18‐TQx10 exhibits optimized miscibility with DY2, achieving a remarkable PCE of 20.21%, among the highest reported for binary oligomeric acceptor based devices. Furthermore, the D18‐TQx10 blend exhibits significantly enhanced stretchability, with a crack‐onset strain nearly three times greater than that of the pristine D18‐based device. It also demonstrates good compatibility with a range of dimeric acceptors. This work establishes a dual‐control strategy using a quinoxaline terpolymer to regulate thermodynamic miscibility and crystallization kinetics, addressing the key challenges of morphology control for high performance PSCs using oligomeric acceptors.