Browse Articles

Low-affinity CD8+ T cells drive cerebral malaria and redefine the afifnity-effector paradigm 2262761

Abstract Introduction Cerebral malaria (CM) remains the deadliest form of Plasmodium infection. Its pathology stems from CD8+ T cell-mediated disruption of the blood-brain barrier (BBB). High affinity T cells are classically viewed as dominant effectors in polyclonal immune responses. Methods Our data reveal a striking inversion of this paradigm: low-affinity CD8+ T cells are the main drivers of disease in experimental cerebral malaria (ECM). Using Plasmodium bergei ANKA infections of C57BL/6 mice we show that brain-infiltrating CD8+ T cells during ECM had predominantly low-affinity T cell receptors. The pathogenic capacity of those cells was assessed through adoptive transfers into TCRα-/- recipient mice using 3 different sources of high and low affinity T cells: flow sorted, viral-derived and retrogenic. Results In all cases low-affinity CD8 T cells were pathogenic leading to lethal disruption of the blood brain barrier, whereas high-affinity cells failed to cause disease. Both high and low affinity T cells make interferon-y but high affinity cells upregulate more markers that are typical of exhaustion. Conclusion We hypothesize that the high affinity cells may undergo exhaustion or apoptosis explaining their reduced pathogenic potential. Interestingly, high affinity TCRs formed longer-lived bonds under force than low-affinity ones, suggesting that distinct mechanodynamic properties may underlie the transcriptional control leading to divergent profiles. Our data challenges a core principle of T cell biology and reveals unexpected complexity in TCR affinity-disease relationships. Funding Source NIH 5R01AI167422-04 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Gluconolactone restores immune regulation and alleviates skin inflammation in lupus-prone mice and in patients with cutaneous lupus 2328666

Abstract Introduction Systemic lupus erythematosus (SLE) is characterized by impaired regulatory T cell (Treg) function and dysregulated immune metabolism. We previously identified protein phosphatase 2A (PP2A) as a critical regulator of Treg suppressive activity. Emerging evidence suggests that the pentose phosphate pathway (PPP) plays an essential role in immune cell fate decisions. Methods Phosphoproteomic and metabolomic analyses were performed on Treg cells from PP2A—wild-type and PP2A-deficient mice. The PPP metabolite gluconolactone (GDL) was evaluated for its effects on CD4+ T cell differentiation and Treg suppressive function in murine and human cells in vitro. The therapeutic efficacy of GDL was assessed in vivo using imiquimod-induced dermatitis and spontaneous lupus-prone MRL.lpr mouse models. Finally, the clinical and histologic effects of topical GDL were evaluated in a pilot study of patients with cutaneous lupus erythematosus. Results PP2A regulates Treg function through modulation of the PPP. We identified GDL as a key PPP metabolite that enhances induced Treg differentiation and suppressive function while inhibiting T helper 17 (TH17) cell differentiation in murine T cells. In vivo, GDL treatment increased Treg function and significantly ameliorated skin inflammation in both imiquimod-induced and spontaneous lupus mouse models. Ex vivo studies using T cells from patients with SLE demonstrated that GDL similarly promoted Treg differentiation and function. Notably, topical application of a GDL-containing cream in patients with cutaneous lupus erythematosus led to marked clinical and histologic improvement of skin lesions within two weeks Conclusion Our findings identify gluconolactone as a metabolite of the pentose phosphate pathway that restores immune regulation by enhancing Treg function and suppressing TH17 responses. GDL represents a promising metabolic-based therapeutic strategy for the treatment of inflammatory and autoimmune diseases. Funding Source NIH Topic Categories Immune Mechanisms of Human Disease (HUM)

IL-17C—activated MAIT cells drive fibrosis in immune-mediated liver disease 2329709

Abstract Introduction Primary sclerosing cholangitis (PSC) is an immune-mediated chronic fibrotic liver disease with no effective medical therapies. We showed that bile duct epithelial cells, called cholangiocytes, express IL-17C, a cytokine that modulates immune responses. Mucosal-associated invariant T cells (MAITs) have been implicated in fibrosis, but their mechanistic role in PSC remains unknown. SPARC (secreted protein acidic and rich in cysteine) is an extracellular matrix protein whose role in hepatic fibrosis is unexplored. Methods Flow cytometry, RNAscope, ELISA, RNA sequencing, immunofluorescence, immunocytochemistry, spheroid culture. Results Flow cytometry of bile duct brushings from PSC patients (n = 8) revealed that 41.32% of T cells were MAITs, indicating their proximity to cholangiocytes within the epithelial-immune niche. MAITs co-cultured with PSC patients-derived cholangiocytes showed increased activation than with normal human cholangiocytes (p = 0.018, n = 4). RNAscope on human livers identified increased IL17C expression in PSC cholangiocytes compared to controls (p = 0.064, n = 3), and IL-17C levels were elevated in PSC bile (p = 0.035, n = 25). Surprisingly, the receptor of IL-17C is enriched in MAITs in PSC (re-analysis of single cell-RNA seq, PMID: 33774059). Bulk RNA sequencing of IL-17C-treated peripheral blood MAITs revealed upregulation of T cell activation markers and pathways associated with hepatic fibrosis. Moreover, IL-17C-treated MAITs increased SPARC secretion (p = 0.025, n = 7), an effect attenuated by IL-17C blockade. Consistently, conditioned media from IL-17C—treated MAITs activated hepatic fibroblasts (HF), evidenced by increased PDGFRβ expression (p = 0.048, n = 6). SPARC directly promoted HF activation, marked by increased α-SMA (p = 0.039, n = 5). Co-culture of primary human HF, MAITs and cholangiocytes in the presence of IL-17C enhanced HF activation. Conclusion IL-17C drives MAITs toward a profibrogenic phenotype in PSC, promoting HF activation through SPARC. Funding Source C-SIG P30DK084567 Topic Categories Immune Mechanisms of Human Disease (HUM)

Sub‐3 nm Ruthenium Nanopatterns Enabled by Pattern Transfer From Supramolecular Dendrimer Templates

ABSTRACT The fabrication of inorganic‐based nanopatterns with feature sizes below 3 nm remains a fundamental challenge in nanoscience and advanced manufacturing. Existing approaches, including extreme ultraviolet lithography, block copolymer self‐assembly, and colloidal nanocrystal patterning, struggle to reliably achieve such dimensions while maintaining long‐range order and structural fidelity. Here, we present a pattern transfer technique from a supramolecular dendrimer templating strategy that enables highly ordered sub‐3 nm ruthenium‐based nanostructures over large areas (1 µm × 1 µm). The pristine dendrimer template forms a hexagonally packed cylindrical morphology with sub‐5 nm periodicity, exhibiting structural stability and long‐range order. Thermal annealing induces cooperative self‐assembly into a single‐domain array, which is selectively stained with RuO 4 to generate an inorganic RuO 2 core while preserving structural registry. This Ru‐incorporated dendrimer architecture shows markedly enhanced thermal and structural stability. Electron microscopy and elemental mapping confirm vertically aligned cylindrical domains with high pattern fidelity. Subsequent calcination at 250°C removes unstained organic components without structural collapse, yielding periodic Ru‐based nanostructures with a characteristic feature size of ∼2.5 nm. This work establishes a versatile bottom‐up platform for ultrahigh‐resolution patterning beyond current lithographic limits and provides a scalable route toward next‐generation nanoelectronic, catalytic, and quantum device architectures.

Introduction to the thematic review series on intracellular protein degradation. The ubiquitous biology of intracellular protein degradation: A tribute to Alfred L. (“Fred”) Goldberg

Optimized sowing rate and fertilization regulate photosynthetic characteristics, grain yield and quality of winter wheat

ES302, A Novel TL1A/IL-23p19 Bi-specific Antibody Demonstrates Robust Efficacy and Developability for Inflammatory Bowel Disease 2305836

Abstract Introduction The TL1A/DR3 and IL-23 pathways exhibit well-documented synergy in driving chronic intestinal inflammation, with TL1A enhancing IFN-γ and IL-17 production in a T cell-intrinsic manner and IL-23 stabilizing the Th17 lineage. We hypothesize that simultaneously co-targeting these two non-redundant axes with a single bispecific agent will deliver superior efficacy by fundamentally reshaping the dysregulated immune landscape in conditions like inflammatory bowel disease (IBD). Methods We developed a fully human, symmetric 1 + 1 IgG-formatted BsAb. And the Fc portion was engineered to extend serum half-life. Binding affinity for both TL1A and IL-23p19 were determined by surface plasmon resonance (SPR). The dual functionality was assessed using cell-based reporter assays: Inhibition of TL1A-induced NF-κB activation and IL-23-induced STAT3 phosphorylation as well as IL17 secretion from PBMCs. The immune complex formation was assessed by SEC-MALS. In vivo efficacy was determined in human TL1A/IL-23 KI mice using TNBS-induced colitis model. Developability was assessed and PK profile was evaluated in FcRn transgenic mice. Results The ES302 demonstrated high-affinity binding to both targets. It potently neutralized both TL1A and IL-23 functionality from in vitro assays. In animal model, the ES302 showed significantly superior efficacy over monospecific therapies, markedly reducing disease activity, histopathological scoring, and pro-inflammatory cytokines. The molecule exhibited low immunogenicity risk, and excellent developability properties, including high Tm value, low viscosity, and superior stability under stress conditions, compatible with high-concentration formulation for subcutaneous administration. Furthermore, ES302 exhibited excellent PK profile (e.g. very long in vivo half-life) in humanized FcRn mice and NHP. Conclusion ES302 is a highly differentiated antibody with strong potential for the treatment of inflammatory bowel diseases. Funding Source n/a Topic Categories Therapeutic Approaches to Autoimmunity (THER)

Persistent IL-17 Cytokine Patterns and Th17 Expansion Characterize Chronic Critical Illness after Severe Trauma 2310055

Abstract Introduction Severe trauma triggers a dynamic disturbance of immune function that can progress from early hyperinflammation to prolonged dysregulation, increasing patient vulnerability to infection and chronic critical illness (CCI). While underlying mechanisms that drive vulnerability in CCI remains unclear, we hypothesize that intrinsic T-cell functionality is impaired. Methods To identify immune features linked to different post-injury clinical trajectories, we prospectively evaluated severely injured trauma patients and categorized them as rapid recovery (RR), intermediate (IM), or CCI by day 14 of ICU admission. Results CCI patients developed infections earlier and more frequently than RR or IM patients, with nearly one-third experiencing their first infection (primarily pneumonia) within 4 days of ICU admission and >80% did so within 8 days. Immune profiling showed trauma-associated alterations across all groups, including neutrophilia and early T-cell lymphopenia. However, CCI patients exhibited a distinct immunophenotype characterized by elevated neutrophil counts, preserved inducible IFNγ production, and a selective expansion of CD4+ Th17 cells. Functional assays revealed sustained or amplified T-cell cytokine responses in CCI patients, including persistent IFNγ and IL-17A production. Plasma cytokine analysis further demonstrated prolonged elevation of IL-17A, IL-17C, IFNγ, and IL-10, indicating a mixed but enduring pro- and anti-inflammatory state. Conclusion These findings suggest CCI after trauma is not driven by intrinsic T-cell impairment but by an IL-17-skewed immune program coupled with ineffective pathogen control. The persistent Th17/neutrophil axis may contribute to heightened infection risk and progression to CCI, highlighting dysregulated IL-17-mediated inflammatory circuits as a potential mechanism of ongoing immune dysfunction following severe trauma. Funding Source National Institutes of Health: GM142481 Topic Categories Translational and Interventional Immunology (TI)

Differential signaling through the PD-1 receptor allows for a distinct inhibitory signature and unique signal dichotomy between PD-L1 and PD-L2 2261044

Abstract Introduction Checkpoint blockade therapies, especially those targeting the PD-1/PD-L1 axis, have revolutionized cancer therapy. Despite these advances, numerous patients fail to respond or relapse. Poor detection reagents and lack of equivalence between mouse and human function fostered a lack of appreciation for the role of PD-L2 in tumor immune suppression. Given its distinct expression pattern and greater binding affinity relative to PD-L1, we hypothesize that PD-L2 engages PD-1 in a manner which propagates a unique downstream inhibitory signature in T cells. Methods Jurkat T cells with a NFAT luciferase reporter were cocultured with CHO cells transduced to overexpress an anti-CD3 scFv and huPD-L1 or huPD-L2, resulting in depression of the bioluminescent signal if T cell activation is suppressed. Western blots were used to assess the dampening of the TCR phosphoproteomic signatures, and microarrays along with GSEA helped elucidate the ensuing transcriptomic effects. Recovery assays, in the form of anti-CD3/CD28 activation post PD-L1/PD-L2 inhibition, allowed for the assessment of marked differences in functional cytokine production. Results PD-L1 and PD-L2 differ in the temporal dephosphorylation of membrane proximal proteins in the TCR signaling cascade. RPPA and GSEA corroborate PD-L1 having a greater effect on the Ras/Raf/MEK/ERK/MAPK pathway while PD-L2 negatively affects the PI3K/Akt/mTOR pathway. Despite the consistent use of the PD-1 ITSM canonical motif, differential recruitment of downstream phosphatases appears to drive the differential signaling. Moreover, unlike PD-L1, PD-L2 inhibited T cells respond to anti-CD3/CD28 restimulation by recovering high-level IL-2 production. Conclusion We speculate that during primary T cell responses, induction of PD-L2 may transiently inactivate T cells which can reactivate if needed when pro-inflammatory conditions persist, and actively screening for PD-L2 in tumor biopsies would help improve patient screening for immune checkpoint blockade. Funding Source This work was supported by CPRIT Grant RP170067 and a generous donation from the Big Easy Ranch Foundation. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Microplastics Trigger Antigen Presentation and CXCL1-Mediated Inflammation in Glioma Tumor-Associated Myeloid Cells 2260962

Abstract Introduction Glioblastoma, a WHO grade IV glioma, is a lethal tumor with a 5-year survival rate below 7 percent, largely due to its immunosuppressive tumor microenvironment (TME). Tumor-associated myeloid cells contribute significantly to this immunosuppressive environment by downregulating key antigen-presenting markers, including MHC II, CD80, and CD86. Developing literature on macrophage activation indicates that microplastics, once phagocytosed, play a key role. We hypothesize that microplastic particles will activate macrophages within the tumor microenvironment after intertumoral injection. Methods To test this, we used a murine intracranial glioma model, implanted on day 0 and intratumorally injected with fluorescent microplastics 10 days after implantation. Brain and lymphoid organs were harvested 24 hours after and analyzed using flow cytometry and bulk RNA sequencing. Results Plastics directly induced upregulation of activation markers MHC II and costimulatory markers CD80 and CD86 in microplastic-treated mice compared with sham-treated mice. The largest subpopulation of myeloid cells was neutrophils, which showed upregulation of activation markers indicative of antigen presentation, a phenomenon traditionally not observed in neutrophils. Bulk RNA sequencing also reveals a strong emphasis on neutrophil activation, with upregulation of the activation and recruitment marker CXCL1. Conclusion Our finding suggests that myeloid cells in the TME retain the ability to respond to acute stimuli by upregulating antigen presentation markers. Through CXCL1 upregulation, microplastics activate inflammation responses in myeloid cells, particularly neutrophils. We conclude that microplastics could induce myeloid activation and provide novel therapeutic insight into brain tumor biology. Funding Source N/A Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Teachers’ dilemma: Are we knowledge dispensers, coaches, mentors, or career advisors? 2264660

Abstract Introduction In the rapidly changing landscape of education, the role of educators is going through a significant transformation. Traditionally seen as the main sources of knowledge sharing, teachers are now taking on more diverse roles as coaches, mentors, and career advisors. This change is mainly driven by using advanced technologies–such as artificial intelligence, adaptive learning platforms, and digital content systems–that increasingly handle routine parts of knowledge transmission. As a result, educators are freed from the constraints of rote instruction, enabling them to focus on encouraging critical thinking, creativity, emotional intelligence, and personalized support. Methods We at Chicago State Univ used appropriate surveillance methods, such as direct observation, digital monitoring, biometric sensors, and self-reporting tools. Ensured ethical practices by obtaining informed consent, protecting privacy, and maintaining transparency. Results Teachers overwhelmingly responded, and the responses have been summarized. This shift in thinking requires redefining teaching styles, shifting the focus from simply delivering content to creating learning experiences that are student-focused and inquiry-based. Teachers are becoming guides in students’ learning paths, helping them set goals, overcome challenges, and make informed choices about their academic and career futures. Additionally, the teacher’s role as a mentor is growing in importance, with more emphasis on students’ social and emotional growth and well-being. Conclusion The transition requires strong professional development and systemic support to give educators the skills needed for coaching and advising. Institutions must invest in training programs that improve interpersonal communication, career counseling, and technological skills. In the end, this change reflects a larger societal movement toward lifelong learning and adaptability, positioning educators as key agents in preparing learners for an unpredictable and ever-changing future. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)

Chronic Inflammation Induces a Molecular and Functional State Divergent from Physiological Aging in Hematopoietic Stem Cells 2328049

Abstract Introduction Chronic inflammation is postulated to drive hematopoietic stem cell (HSC) aging, largely based on functional decline observed in transplantation assays. However, transplantation imposes severe regenerative stress that may mask native HSC behaviors. It remains unclear whether chronic inflammatory stress truly recapitulates the physiological aging program in situ. We revisited this paradigm using lineage tracing model to distinguish inflammatory adaptation from genuine senescence. Methods We utilized the DARLIN mouse model for high-resolution in situ lineage tracing. Mice received multiple doses of LPS or Poly(I:C) over 2 months to mimic the chronic inflammation and followed by another 2-month break. We integrated clonal tracking with bulk RNA-seq, ATAC-seq, and scRNA-seq to profile the functional and epigenetic landscapes of inflammation-exposed HSCs compared to naturally aged HSCs. Aged mice were also treated to assess if chronic inflammation exacerbates HSC aging signatures. Results While inflammation-treated HSCs showed reduced self-renewal in transplantation, in situ barcoding revealed sustained multilineage output without the myeloid skewing characteristic of aging. Multi-omics demonstrated that chronic inflammation induces an activated state–marked by increased chromatin accessibility at lymphocyte loci and expansion of primed HSC subpopulations–diametrically opposing the repressive program of physiological aging. Furthermore, chronic inflammatory challenge in aged mice re-activated quiescent HSCs to enter the cell cycle rather than exacerbating the aging signature. Conclusion These findings uncouple chronic inflammation from physiological aging in HSC, identifying it as a distinct, active host defense response that preserves differentiation flux despite compromising regenerative potential under transplantation stress. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Cannabidiol Attenuates UVB-Induced Skin Inflammation via STAT3/c-Jun Suppression and Silicone Polymer—Based Topical Delivery 2330062

Abstract Introduction UVB radiation induces epidermal damage and inflammatory responses through immune-mediated pathways in the skin. Cannabidiol (CBD), a non-psychoactive phytocannabinoid derived from Cannabis sativa, has demonstrated anti-inflammatory effects in various disease models; however, its therapeutic application in UVB-induced skin inflammation remains limited by poor stability and low bioavailability, largely due to oxidative and photodegradative instability. Methods This study was designed with two objectives. First, we aimed to elucidate the intrinsic anti-inflammatory mechanism of CBD in UVB-induced keratinocyte inflammation, with a particular focus on STAT3/c-Jun signaling axis. Second, we sought to evaluate a silicone polymer—based delivery strategy to improve the therapeutic efficacy of CBD in an in vivo UVB-induced skin inflammation model. Results To address the first objective, we investigated the effects of CBD on UVB-induced inflammatory responses in the human keratinocyte cell line. UVB irradiation markedly increased IL-6 expression, whereas CBD treatment significantly reduced IL-6 production at both the transcriptional and protein levels. CBD suppressed UVB-induced STAT3 and c-Jun phosphorylation, implicating a previously poorly understood STAT3—c-Jun signaling axis in its anti-inflammatory effects. To address the second one, a silicone polymer—formulated CBD was topically applied to the dorsal skin of UVB-irradiated hairless mice. This formulation was used to assess changes in epidermal thickness and inflammatory skin pathology, thereby evaluating the capacity of polymer-based delivery to enhance local therapeutic efficacy in vivo. Conclusion These findings demonstrate that CBD attenuates UVB-induced skin inflammation through modulation of STAT3/c-Jun—dependent signaling in keratinocytes and that a silicone polymer—based delivery represents an effective strategy to overcome stability limitations and improve the in vivo efficacy of topical CBD for UVB-mediated inflammatory skin conditions. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)

Breaking Interfacial Charge Transport Limitations Enabled by Heterojunction Dielectric Equilibrium in Perovskite Solar Cells

ABSTRACT Heterointerface properties of perovskite solar cells (PVSCs) are critical in determining efficient carrier transport. Conventional interface engineering is insufficient to completely address the dilemma of low charge transport efficiency at the heterointerface with side effects, especially at the perovskite buried interface with complex chemical environments. Here, we have improved effective dielectric matching and constructed an efficient charge transport channel at the heterointerface of perovskite/charge transport layer by narrowing the difference in dielectric constants. The charge transfer resistance and electron trapping radius at the perovskite heterointerface are effectively reduced by the heterojunction dielectric equilibrium strategy for the first time, thus accelerating the interfacial charge transfer. Meanwhile, we further elucidate the effect of dielectric equilibrium on the charge carrier transport across the heterointerface. The resulting device acquires a record efficiency of 26.77% (certified as 26.01%) for n‐i‐p PVSCs prepared by a unique heterojunction dielectric equilibrium strategy and excellent operational stability.

An N-terminal amphipathic helix governs activity and conformational dynamics of Nramp metal transporters

Integrated solar PV energy management and adaptive control of IPMSM drives for lightweight electric trikes

Identification of Integrin αvβ3 as a Receptor for the Novel Macrophage-Secreted Microprotein U9-ORF Encoded by U90926 2307000

Abstract Introduction Gene expression in macrophages is a highly dynamic and tightly regulated process to prevent inflammation-driven pathology, such as septic shock. Previous studies in our lab identified the putative long non-coding RNA (lncRNA) gene U90926 as highly induced in macrophages following pattern recognition receptor activation by lipopolysaccharide (LPS) and other microbial ligands. Using an LPS-induced sepsis model, we found that U90926 knockout mice exhibited greater disease severity and mortality, indicating a protective role of the gene. Surprisingly, analysis of U90929 sequence identified an 87 amino acid open reading frame (ORF), encoding a novel protein, U9-ORF, containing a functional N-terminal secretion signal. We therefore hypothesized that protective effects of U90926 in sepsis are mediated by the secretion of U9-ORF and binding to receptors on other cells. Methods To screen for potential receptors for U9-ORF, we developed recombinant U9-ORF-Fc fusion proteins to measure binding via flow cytometry, and for use in vitro functional assays. Transfection of HEK293 cells was used to express potential receptors for U9-ORF, and cellular adhesion assays were used as a functional readout. Results Multiple sequence alignments of predicted U9-ORF homologs revealed a conserved C-terminal RGD motif, suggesting that U9-ORF could bind specific integrins. Flow cytometry assays demonstrated that U9-ORF preferentially bound cells expressing integrin αvβ3, but not αIIbβ3, in an RGD- and divalent cation-dependent manner. Plate-bound U9-ORF-Fc supported cellular adhesion and spreading of cells expressing αvβ3 integrin, in an RGD-dependent manner. Conclusion U9-ORF is a novel ligand for integrin αvβ3, which is upregulated on endothelial cells during inflammation, and thus could mediate protective effects of U90926 in sepsis. Ongoing experiments aim to determine the effects of U9-ORF on endothelial barrier integrity in the presence of inflammatory stimuli. Funding Source NIAID/NIH R03 AI190196-01; NIAID/NIH R21 AI151116-01; Department of Education CMB-GAANN Fellowship Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Decidual Natural Killer cells utilize the antimicrobial protein granulysin to control bacterial infection 2226506

Abstract Introduction During pregnancy maternal decidual Natural Killer cells (dNK) balance the competing needs of immunity and maternal-fetal tolerance. While dNK were initially shown to play essential roles in placentation, we recently discovered their unique ability to provide immunity for placental viral and bacterial infections. A particularly intriguing mechanism is the utilization of the anti-microbial protein granulysin (GNLY) by dNK to kill intracellular bacteria without cytotoxicity of the infected trophoblasts. Methods Using primary human dNK isolated from healthy term placental tissues we utilized high dimensional flowcytometry, functional bacterial killing assays, RNA sequencing and computational analysis of several dNK populations to define the molecular and cellular drivers of GNLY expression and the anti-bacterial functions of dNK. Results First we confirmed that similar to dNK in early pregnancy, term pregnancy dNK i) produce GNLY; ii) process GNLY into the 9kDA active GNLY form; iii) secrete GNLY constitutively; iv); directly transfer GNLY into trophoblasts; and v) most importantly have the capacity to kill cell free and intracellular bacteria in a GNLY dependent manner. Utilizing the vast heterogeneity of dNK types at term pregnancy, we performed RNA-sequencing on FACS purified GNLY-neg, GNLY-pos and 9kda GNLY-HI dNK. Differential gene expression and gene set enrichment analysis identified immune activation pathways, dNK receptors, metabolic pathways, transcription factors and proteolytic enzymes uniquely upregulated in 9kda GNLY-HI dNK. Further computational analysis using Nichenet identified several receptor — ligand interactions between dNK and EVT as potential drivers of GNLY expression and anti-microbial activity of dNK. Conclusion These findings support a key role for dNK in providing placental immunity throughout pregnancy. Discovering the drivers of GNLY expression, activation and transfer by dNK contributes to designing therapeutic strategies to prevent placental and congenital infection. Funding Source Cincinnati Children’s Research Foundation RIP award Topic Categories Mucosal and Regional Immunology (MUC)

Vitamin D Supplementation Improves Human Natural Killer Cell Responses to JC Polyomavirus Infection 2261205

Abstract Introduction JC polyomavirus (JCPyV) is a DNA virus that causes the rare but often fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) upon reactivation. Reactivation is due to severe immunosuppression. JCPyV infects the kidney tubule epithelium and replicates in renal tissues in most healthy individuals. Vitamin D deficiency is highly prevalent in clinical settings. Studies in renal transplant recipients showed that vitamin D deficiency is associated with increased BK polyomavirus burden; but the role of vitamin D in JCPyV infection is undefined. Vitamin D could exert immunomodulatory effects on immune cells, including human natural killer (NK) cells. NK cell cytotoxicity is key to controlling JCPyV. Methods We sought to determine whether vitamin D could suppress JCPyV replication in organ-specific primary cells and modulate NK cell responses to JCPyV, by co-culturing NK cells with JCPyV-infected primary renal and brain cells. Viral quantification and NK cell functions were assessed by immunochemical staining, intracellular staining, and flow cytometry. Additionally, immunofluorescence was performed on infected target cells, SVGAs, to assess the presence of JCPyV viral proteins after vitamin D treatment in vitro. Results Preliminary data demonstrates that vitamin D may decrease JCPyV viral replication and augment NK cell cytotoxicity. Additionally, vitamin D treatment decreased the amount of JCPyV viral proteins detected in SVGA cells. Conclusion Understanding the role of vitamin D in the context of JCPyV infection and its ability to modulate NK cell responses may offer novel insights into therapeutic approaches for controlling JCPyV infection and reactivation. Funding Source R01NS116278 Topic Categories Viral Immunology (VIR)

Investigating the Spatial Immune Tumor Microenvironment of Dermatofibrosarcoma Protuberans 2266312

Abstract Introduction Dermatofibrosarcoma Protuberans (DFSP) is a rare, locally aggressive cutaneous sarcoma driven by the COL1A1-PDGFB fusion. While its molecular drivers are well-defined, the DFSP tumor immune microenvironment remains poorly characterized. Understanding immune-stromal interactions in DFSP may reveal opportunities to complement surgical resection with immunotherapy or targeted approaches. Methods We performed spatial transcriptomics on 16 DFSP patient samples using 10X Genomics Visium v2 (n = 12) and Visium HD (n = 4), integrated with single-cell RNA sequencing. Results Our analysis revealed distinct tumor, stromal, and immune populations with high PDGFB-high tumor cells spatially segregated from infiltrating immune compartments. We identified transcriptionally distinct cancer-associated fibroblast (CAF) subsets, including populations enriched for extracellular matrix remodeling and inflammatory signatures that may modulate local immunity. Immune profiling uncovered spatially heterogeneous T cell and macrophage infiltration, with distinct immune-rich and immune-desert zones across patients. Notably, we observed tertiary lymphoid structure (TLS) formation in multiple samples, characterized by elevated TLS signature scores and spatial co-localization of B and T cell markers. CXCL13 and CCL19 emerged as key chemokines defining TLS-associated immune-stromal communication networks. Using cyclic immunofluorescence, we validated the spatial organization of TLS-associated immune niches, confirming co-localization of CD3+ T cells, CD20+ B cells, and CD68+ macrophages within TLS-enriched regions at protein level. Conclusion This work provides the first spatial immune atlas of DFSP, revealing organized lymphoid structures and CAF-mediated immune regulation that could inform combination strategies targeting PDGFR signaling alongside immune checkpoint or stromal modulation. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)