Browse Articles

Comparative Analysis of Work Function Measurement Techniques Based on Scanning Probe Microscopy

ABSTRACT In this study, we compare three scanning probe techniques— spectroscopy, field‐emission resonances (FER), and Kelvin probe force spectroscopy (KPFS)—for quantitative local work function measurements. Using Ag(111) as a reference and applying the methods to Ir(111) and Au(111), we find a strong method dependence. spectroscopy shows substantial uncertainty due to broad data dispersion and is reliable only on flat surfaces. We show that the triangular‐barrier FER extrapolation method lacks robustness, whereas numerical FER analysis based on an image potential‐corrected Schrödinger model yields consistent values. KPFS provides the most direct and reliable work function determination once the tip is calibrated. Applying these methods to superconducting Nb, we extract the work functions of (111) and (110) surfaces and its distinct oxygen‐reconstructed phases.

Histotripsy and the IDEAL Framework: A Clarion Call for the Responsible and Evidence-Based Application of New Technology

The evaluation of novel health technologies is increasingly complicated by a landscape of pervasive social media influence and intense product marketing, factors that may distort evidence appraisal and contribute to suboptimal medical decision-making and patient outcomes. Within this context, interest in histotripsy, a novel technology approved for the treatment of liver tumors, has expanded rapidly. This accelerated adoption has outpaced the available evidence, challenging established principles of safety, efficacy, and appropriate patient selection and has generated substantial debate within the oncologic community. In this editorial, we provide an overview of the technology and available data, examine the external forces contributing to its accelerated application, and propose a well established, structured, evidence-based framework to guide its rigorous evaluation, responsible adoption, and optimal clinical implementation.

Tissue-Resident Cytotoxic γδT Cells in Anti-tumor Immunity 2258876

Abstract Introduction Tissue-resident αβT cells are well-documented to play a critical role in controlling solid tumors. In contrast, the characteristics and function of tissue-resident γδT cells remain poorly understood. γδ;T cells are a heterogeneous T cell population characterized by phenotypic diversity and functional variability in response to infection and cancer. The IFN-γ-IL-17 dichotomy among γδT cells is particularly related to their effector functions in cancer. Methods To investigate the heterogeneity of tumor-infiltrating γδT cells, we performed single-cell RNA sequencing (scRNA-seq) on liver CD45+TCRγδ+CD3+ cells isolated from hepatocellular carcinoma (HCC)-bearing mice. A distinct cytotoxic γδT cell population was identified. Using a parabiosis model, we confirmed that this cytotoxic γδT cell subset represents liver-resident cells. Both in vitro cytotoxicity assays and in vivo adoptive cell transfer experiments demonstrated that these liver-resident cytotoxic γδT cells effectively controlled tumor growth. Results In this study, we report a subset of tissue-resident γδT cell population which possess potent cytotoxic activity. These cytotoxic γδT cells are present across various organs but particularly enriched in the liver. These cells resist the immunosuppressive tumor microenvironment (TME), which distinguish them from conventional γδT cells and exhibit robust anti-tumor activity both in vitro and in vivo. Notably, the cytotoxicity of this tissue-resident γδT cells is independent of IFN-γ. Conclusion These findings offer valuable insights into the subset of tissue-resident cytotoxic γδT cells and their therapeutic potential in cancer immunotherapy. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Clostridioides difficile-Driven Expansion of IL1B+ Monocytes Amplifies Neutrophil Recruitment in Ulcerative Colitis 2258811

Abstract Introduction Clostridioides difficile colonization, defined by PCR detection of C. difficile and Toxin B genes in fecal samples, modulates the course of ulcerative colitis (UC). Colonized patients harbor the pathogen at the mucosal surface without overt infection, yet colonization may exacerbate early disease. Methods We applied single-cell RNA sequencing (scRNA-seq) to intestinal biopsies from 24 UC patients and integrated 5 healthy control datasets from the Human Cell Atlas, defining colonization status by PCR/ELISA detection. Results We hypothesized that C. difficile—driven inflammation sustains chronic colonic injury via expansion of a distinct IL1B+ monocyte subset. scRNA-seq revealed this subset uniquely enriched in colonized UC tissue, exhibiting gene programs related to neutrophil recruitment and leukocyte chemotaxis. We further identified a marked expansion of infiltrating inflammatory monocytes along the chemokine axis, accompanied by upregulation of CCL2, CCL3, CXCL3, and CXCL8. Trajectory analysis demonstrated that these monocytes differentiate into macrophages within the tissue, enhancing recruitment in inflamed lesions. Moreover, interactions between CCL2-expressing glial cells and monocytes were implicated in amplifying local inflammation. Conclusion These findings identify C. difficile—induced IL1B+ infiltrating monocytes as key amplifiers of innate immune recruitment and drivers of persistent inflammation in UC, providing mechanistic insights into pathogen-associated disease exacerbation. Funding Source This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2022-NR067309). Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Green-synthesized gold nanoparticles derived from Spirulina and Leptolyngbya, modulate innate immune response via M1 polarization of THP-1 macrophages 2259710

Abstract Introduction Macrophage polarization plays a pivotal role in regulating immune responses against infections and cancer. Cyanobacteria such as Spirulina and Leptolyngbya are rich in immunomodulatory metabolites; however, their mechanistic roles in macrophage polarization remain unexplored. This study investigates whether cyanobacterial extracts and their green-synthesized gold nanoparticles (AuNPs) modulate human macrophage polarization. Methods Methanolic extracts of Spirulina and Leptolyngbya and their AuNPs were characterized by UV—Vis, DLS, TEM, and FTIR. THP-1 macrophages were treated with extracts or AuNPs. Expression of M1/M2 gene markers (TNF, IL1β, IL8, CXCL10, CD163, TGFβ, and STAB1) was analyzed by qRT-PCR, while surface markers (CD80 and CD86) were assessed using both qRT-PCR and flow cytometry. Agonist—antagonist assays were conducted to trace the potential pathway. Statistical significant difference was observed using Student t-test and ANOVA (n = 3). Results THP-1 macrophages treated with extracts or AuNPs exhibited significant upregulation of M1 markers TNF-α (p < 0.01), IL-8 (p < 0.01), and IL-1β (p < 0.01), along with a moderate increase in CXCL10 (p < 0.05). Conversely, there was significant downregulation of M2 markers CD163 (p < 0.01), TGFβ (p < 0.05), and STAB1 (p < 0.01). Algal extract-induced polarization occurred via the TLR2 pathway, as it was significantly reversed by TLR2 blockade via a specific antagonist. In contrast, algal-derived AuNPs retained their M1-polarizing capability even after TLR2 inhibition, suggesting activation of TLR2-independent pathways. Conclusion The immunostimulatory response induced by cyanobacterial extracts is partly dependent on TLR-2 signaling. However, extracts AuNps appear to evade TLR2-mediated signaling by internalizing into the macrophages, suggesting activation of alternative pathways leading to M1 polarization. The M1 polarizing ability of extracts and AuNPs highlights their potential as promising therapeutic candidates for cancer immunotherapy. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

Staining and Characterizing Extracellular Vesicles from Platelet-Poor Plasma Using the CytoFLEX nano Flow Cytometer 2306502

Abstract Introduction Extracellular vesicles (EVs) are recognized as important mediators of intercellular communication and potential diagnostic biomarkers. However, their precise characterization, particularly from complex biological samples like platelet-poor plasma (PPP), demands highly standardized methods. Our work establishes a complete process for the enrichment, fluorescent staining, and multicolor flow cytometric analysis of EVs from PPP using the CytoFLEX nano Flow Cytometer, leveraging its improved violet side scatter (VSSC-1) for sensitive detection. Methods Izon qEV SEC fractionation was used to enrich EVs from PPP, followed by serial dilutions of EV-enriched fractions to determine the optimal dilution at which the swarming effect is avoided. Thorough antibody titration was performed to minimize antibody aggregation and maintain background noise at the level set at the beginning of acquisition. Results During our investigation, we determined three crucial factors that could affect EV staining and thus their characterization: 1. Variation between different donors within the same fraction. 2. Differences among CD9 antibody clones in both the detected amount of PPP EVs and their Median Fluorescence Intensity (MFI), highlighting the importance of selecting the optimal clone for staining. 3. Confirmation of the importance of proper clone selection in multicolor analysis, along with demonstration of fluorochrome combinations using different fluorochromes conjugated to the same CD9 clone. In our multicolor flow cytometry analysis, we further showed that besides CD9, PPP EVs also express CD61, whereas CD63 and CD81 exhibited very low–if any–expression levels. Conclusion In conclusion, our standardized process demonstrates the importance of optimizing EV and sample preparation, along with careful attention to the EV fraction, donor variability, and antibody choices (clone, fluorochrome, and dilutions). These factors influence the accuracy and consistency of EV staining and characterization. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)

Regulation of gamma delta (γδ) T cell effector programming by the SLAM/SAP signaling pathway 2310142

Abstract Introduction Gamma delta (γδ) T cells are innate-like T cells that shape the developing adaptive immune response through their ability to rapidly produce large amounts of cytokines such as IL-17 (γδT17) and IFN-γ (γδT1). Unlike conventional αβ T cells, γδ T cells acquire their effector function during thymic development through mechanisms that remain unclear. Previously, we reported that the SLAM/SAP signaling pathway regulates both thymic γδT17 and γδT1 development. Here, we investigated whether the SLAM/SAP signaling pathway regulates thymic γδ T cell developmental programming through its ability to modulate TCR signal strength. Methods Using a novel B6.Nur77GFP.SAP-/- reporter strain, we used multi-parameter flow cytometric analysis to compare the expression of canonical markers of γδ TCR signaling, Nur77 transcription and CD5 protein expression, between B6 and B6.SAP-/- mice during embryonic and adult thymic developmental stages. Results Ex vivo analysis of embryonic day 17 thymic γδ T cells revealed both decreased CD5 expression and increased Nur77-GFP expression in B6.Nur77GFP.SAP-/- γδ T cells. Re-analysis of our previously published E.17 scRNAseq dataset revealed enrichment of Nr4a1 in the γδT1 subsets and enrichment of Cd5 in the γδT17 subsets. In vitro co-stimulation of thymic B6.Nur77GFP γδ T cells with anti-TCRδ and anti-SLAMF antibodies revealed increased Nur77-GFP compared to anti-TCRδ alone. Further in vitro stimulations of B6.Nur77GFP and B6.Nur77GFP.SAP-/- thymocytes with anti-TCRδ revealed increased GFP expression in B6.Nur77GFP.SAP-/- γδ T cells. Conclusion All together, these data suggest that the SLAM/SAP signaling pathway can modulate the TCR signal strength of thymic γδ T cells. However, it may utilize fundamentally different modes of action in cells destined for the γδT17 and γδT1 effector fate, where SAP is co-stimulatory during γδT17 development, but is inhibitory during γδT1 development. Funding Source n/a Topic Categories Hematopoiesis and Immune System Development (HEM)

Bacterial toxins influence tissue resident memory T cell formation in the lungs 2256485

Abstract Introduction Tissue-resident adaptive immunity is crucial for protection against diverse respiratory pathogens. Indeed, lung-resident memory CD4+ T (TRM) and B (BRM) cells that develop following Streptococcus pneumoniae (Spn) experience are cross-protective against different serotypes of Spn, identifying these cells as key vaccine targets. Despite their significance for human health, the cues that mediate anti-Spn CD4+ TRM cell formation are unclear. Whether human pathobionts like Spn can modulate establishment of tissue-resident CD4+ TRM cells using virulence factors is unexplored. Methods Using a mouse model that uncouples CD4+ TRM cell establishment in the lungs from their initial priming in the lymph nodes, we asked if expression of the Spn pore-forming toxin pneumolysin (Ply) alters the lung-resident adaptive immune landscape. Multispectral flow cytometry was used to analyze TRM cell formation at different timepoints post infection. Results To our surprise, we found that Ply supports, rather than evades, greater CD4+ and CD8+ TRM cell establishment in the lung. The adaptive immune composition of the systemic circulation remained similar independent of the toxin, suggesting a more lung-specific immunomodulation by Ply. We also find that pneumolysin promotes recruitment of CD4+ TRM cell precursors to the lung and aids their survival locally. Our ongoing studies are focused on elucidating the molecular mechanisms driving this enhanced recruitment and survival leading up to TRM establishment. Conclusion Our studies uncover an unexpected role for bacterial toxins in instructing TRM cell establishment within non-lymphoid tissues and have implications for development of the next generation CD4+ TRM cell-directed anti-bacterial vaccines. Funding Source NIH Topic Categories Mucosal and Regional Immunology (MUC)

Epithelial tissue factor drives pneumococcus-mediated necrosuppurative pneumonia 2257285

Abstract Introduction Pulmonary infections induce heterogeneous lung immune responses, resulting in multiple pneumonia histopathology sub-phenotypes. Necrosuppurative pneumonia is characterized by alveolar necrosis, neutrophils, edema, and airspace fibrin accumulation, observed in human autopsies and S. pneumoniae (Sp)-infected mice. We endeavor to elucidate innate immunity mechanisms leading to the necrosuppurative sub-phenotype of pneumonia. Methods We compared outcomes of Sp pneumonia in wild-type (WT) mice and those with tissue factor (TF) inducibly deleted from lung epithelial cells. Results In WT mice, fibrinogen mRNA was induced in the liver and elevated in the blood during pneumonia, while TF mRNA was strongest in lung epithelial cells. Bacteria grew inexorably in the lungs and caused bacteremia, which was not affected by deletion of TF from the lung epithelium. However, deletion of lung epithelial cell TF reduced alveolar fibrin deposition and neutrophil recruitment. While Sp infection caused alveolar necrosis in WT mice, we instead observed type II epithelial cell hyperplasia when TF was deleted from the lung epithelium. Conclusion The airspace fibrin, neutrophil accumulation, and epithelial necrosis that are defining features of necrosuppurative pneumonia caused by severe pneumococcal infection all depend on TF produced by lung epithelial cells. Funding Source NIH-NHLBI Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Incoming medical students need tools to develop skills for critical thinking and become life-long learners to improve their overall performance 2260242

Abstract Introduction In the last few years, the performance of medical students on the Step 1 exam has decreased at the national level. There are various reasons that can explain this outcome, 1) medical students do not use effective metacognitive strategies and are unable to recall information optimally, 2) the covid pandemic negatively impacted student’s ability to learn, 3) student’s demographic has changed, 4) and students lack skills for critical thinking. Methods To address this issue, we conducted a survey with M2 and M4 students to assess their needs and discuss options and potential barriers to address those needs. We also determined actionable steps through a focus group with M4 students facilitating Problem-based learning (PBL) and meeting with individuals who train students involved in medical education. Results We found that M2 and M4 students wished they had received some training to help them develop skills for critical thinking to perform better during PBL, clerkship courses and clinical rotations. 80% of M2 students and 70 % of M4 students believed that 2 sessions with a M4 and a small number of incoming students would be helpful to discuss development of skills for critical thinking and effective metacognitive strategies. The focused group proposed a plan for those sessions including the number of sessions, their content and the optimal time frame for training the M4 students facilitating those sessions. We also identified a plan to centralize the training and standardize the curriculum for the different groups of medical students involved in medical education, including the potential individuals who would provide this training. Conclusion By taking advantage of PBL groups, it may be possible to hold a few sessions facilitated by senior medical students who have been trained appropriately to help with the acquisition of very valuable skills. This strategy may enhance the performance of incoming medical students in all aspects of their medical education including Step 1 exam. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)

Stable Protein‐Based G‐Quadruplex‐Derived Supramolecular Bioinks as Tunable ECM‐Mimetic Constructs Assembled by Combining Non‐Covalent and Covalent Strategies (Adv. Mater. 44/2026)

Solvent‐Guided Chiral Assemblies and Chemical Doping of OEG‐Functionalized Conjugated Polymers

ABSTRACT Chiral assembly of achiral conjugated polymers has emerged as a new avenue to promote (opto)electronic properties and control the angular momentum of charge carriers and photons. Solvent plays a key role in chiral emergence through the lyotropic liquid crystal (LLC) assembly pathway, yet rules underpinning solvent effect remain elusive. Here, we establish a unifying framework linking solvent quality to aggregate structure and supramolecular chirality. Using redox active conjugated polymers as model systems, we reveal two types of solution aggregation: (1) β1 aggregation, defined by fibrillar structures with weak interchain coupling and torsional backbone, promotes LLC‐mediated chiral assemblies via formation of helical fibers; (2) β2 aggregation, characterized by stacked fibrils with strong interchain coupling and reduced backbone torsion, suppresses chirality. Temperature‐dependent studies show that β2 aggregates revert to β1 upon heating, thus restoring chiral assemblies. Extending to 6 conjugated polymers across 37 polymer–solvent–temperature combinations, we demonstrate the universal link between aggregate type and chiral emergence. Further, chemical doping of chiral films derived from β1 aggregates elevates electrical conductivity by up to 100‐fold across nine polymer–solvent pairs spanning a wide range of processing conditions. These findings reveal general principles for solvent‐guided chiral assembly enabling high‐performance chiral electronics, optoelectronics and spintronics.

Processing-Optimized Multi-Epitope mRNA Vaccine Enhances HIV-1—Specific CD8+ T Cells 2258806

Abstract Introduction Despite ART, HIV-1 persists in latency and rapidly rebounds after interruption. A functional cure will require potent CD8+ T cells that recognize and eliminate latent reservoirs. mRNA vaccines enable rapid, flexible, multi-epitope design. We tested an mRNA encoding tandem CD8+ epitopes with flanking sequences optimized for antigen processing/MHC-I presentation in mice and human PBMCs and assessed co-stimulation by a TLR8 ligand (TLR8L). Methods We generated mRNAs carrying model epitopes either directly concatenated or appended with flanking regions designed to favor proteasomal generation. HLA-A*02:01 transgenic mice were immunized, and antigen-specific CD8+ T-cell responses and multifunctionality were quantified by ELISpot and flow cytometry. We then established a 10-day in vitro boosting assay using PBMCs from people with HIV-1 cultured with FLT3L and exposed to mRNA encoding tandem HIV-1 epitopes. Epitope-specific CD8+ T-cell frequencies were measured by HLA-tetramers, and cytotoxic potential was assessed by granzyme B and perforin expression. Results In HLA-A*02:01 mice, the flanking-optimized tandem design increased multifunctional CD8+ responses co-producing CD107a/IFN-γ/TNF. In PBMCs from people with HIV-1, the HIV epitope—tandem mRNA increased epitope-specific CD8+ T-cell frequency 3.4-fold versus no vaccine (p = 0.0137); addition of TLR8L further raised this to 4.7-fold (p = 0.0273). The proportion of cells co-expressing granzyme B and perforin rose from 40.8% to 80.5% with TLR8L (p = 0.0137). Conclusion An mRNA vaccine encoding tandem CD8+ T-cell epitopes with optimized flanking sequences elicited quantitatively and qualitatively superior epitope-specific CD8+ responses in HLA-A*02:01 mice and in PBMCs from people with HIV-1. TLR8L further enhanced these responses. This platform supports development of therapeutic mRNA vaccines aimed at strengthening CD8+ T-cell immunity toward a functional cure of HIV-1. Funding Source Japan Society for the Promotion of Science Grant-in-Aid for Early-Career Scientists Topic Categories Vaccines and Immunotherapy (VAC)

Nanoscale artificial antigen presenting cells to expand human antigen-specific cytotoxic CD4 T cells for immunotherapy 2258982

Abstract Introduction CD4 T cells play key roles in immune modulation and cancer immunity. Increasing evidence shows they also exhibit cytotoxicity and can be harnessed for cancer immunotherapy. However, methods to reliably expand antigen-specific CD4 T cells remain limited. Here, we developed a nanoscale platform to expand antigen-specific CD4 T cells with cytotoxicity against cognate target cells. Methods We generated nanoscale artificial antigen-presenting cells (aAPCs) composed of iron-dextran nanoparticles conjugated with peptide-MHC II dimers and anti-CD28 antibodies to provide antigen-specific and co-stimulatory signals. HLA-DP4 dimers were loaded with the DP4-restricted tetanus toxoid p30 (947—967) or HSV (283—302). CD4 T cells isolated from healthy donor PBMCs were co-cultured with aAPCs and cytokines for 14 days. Antigen-specific populations were quantified by tetramer staining and analyzed by flow cytometry, bulk RNA-seq, and TCR-seq to assess phenotype, transcriptome, and clonality. Results p30-specific cells expanded 1000-fold and HSV-specific cells expanded 100-fold by day 14. The most robust donor reached over 75% antigen-specific cells within the CD4 compartment. The expanded cells included stem cell memory, central memory, and effector memory subsets and produced TNF-α, IFN-γ, IL-2, granzyme B, and perforin. Functionally, the expanded CD4 T cells exhibited antigen-specific cytotoxicity, killing nearly 90% of cognate targets at a CD4:target ratio of 1. Blocking assays demonstrated that cytotoxicity required HLA-II recognition and granzyme B activity. Transcriptomic profiling revealed a Th1-skewed, NK-like cytotoxic phenotype, while TCR sequencing confirmed antigen-driven clonal selection. Conclusion Together, our data show that nanoscale aAPCs reliably expand functional antigen-specific CD4 T cells from human PBMCs, providing both mechanistic insight into cytotoxic CD4 biology and a translational foundation for antigen-specific cell immunotherapy. Funding Source PhRMA foundation, NIH, Taiwan Government Funding for Study Abroad Topic Categories Technological Innovations in Immunology (TECH)

EPS-3903 Is a Potent and Selective Oral STAT6 Inhibitor that Blocks Th2 Inflammation in an Ovalbumin Asthma Mouse Model 2253997

Abstract Introduction STAT6 is a key transcription factor central to type 2 inflammation and activated by interleukins IL-4 and IL-13. Inhibition of the IL-4/IL-13 pathway is clinically validated for the treatment of asthma. Our aim is to develop an oral small molecule that selectively inhibits activation of STAT6 for the treatment of allergic diseases. Here, we evaluate EPS-3903 in the ovalbumin (OVA) asthma mouse model, characterized by eosinophilic infiltration, IgE production, and Th2 inflammation, mimicking key features of the human disease. Methods In vitro EPS-3903 potency was assessed in cellular assays measuring phosphorylated STAT6 and TARC secretion. Ex vivo target engagement was evaluated in IL-4-stimated whole blood from mice orally dosed with EPS-3903. In vivo efficacy was assessed in the OVA asthma model in mice administered with EPS-3903 or an anti-mouse IL-4/IL-13 (control) in a therapeutic setting. Inhibition of Th2 markers of inflammation was evaluated in lung, bronchio-alveolar lavage fluid (BALF), and serum. Results EPS-3903 inhibited IL-4-stimulated pSTAT6 in mouse splenocytes, with an IC50 of 9 nM. In whole blood collected from mice treated with EPS-3903, pSTAT6 was suppressed >90% for 24h. In the OVA asthma model, EPS-3903 robustly suppressed pSTAT6 in lung tissue without degrading total STAT6, leading to decreases in OVA-induced Th2 inflammation, including lung TARC and eotaxin, serum OVA-IgE, and BALF eosinophils. Histological improvements comparable to anti-mouse IL-4/IL-13 were observed with reductions in both inflammation and goblet cell hyperplasia. Conclusion EPS-3903 is an orally bioavailable and potent allosteric inhibitor of STAT6 with robust efficacy in a mouse model of allergic asthma. EPS-3903 may offer therapeutic potential for the treatment of allergic diseases such as asthma and atopic dermatitis. Funding Source n/a Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Sex Differences in Hematological and Immunological Features of Sarcoidosis 2307192

Abstract Introduction Sarcoidosis is a systemic granulomatous disease marked by immune dysregulation. While sex-related differences in clinical features are known, hematological and immunological differences remain poorly defined. This study aimed to assess immune and hematological parameters in adults with sarcoidosis, focusing on sex-based differences. Methods This retrospective cohort study evaluated 74 patients with sarcoidosis treated at Hacettepe University Hospitals. Demographic characteristics, comorbidities, and laboratory data, including complete blood count, immunoglobulin levels (IgA, IgG, IgM, IgE), and lymphocyte subsets–were analyzed. Results were classified according to standard reference ranges, and group comparisons were performed using the t-test. Results Female/male ratio was 50/24 (32.4/67.6%). Concomitant autoimmune diseases were present in 24.3% of patients, most commonly Hashimoto thyroiditis and Sjögren syndrome. Hematological abnormalities were frequent: anemia was observed in 62.5%. High leukocyte levels were found in in 67.6% of the patients (above 10800 cells/µL). Neutrophil levels were high in 63.4% of patients (above 7000 neutrophils/uL). Lymphopenia was detected in 31.0%. Immunological assessment showed that IgM deficiency was the most common immunoglobulin abnormality (33.9%), followed by IgA deficiency (26%). Reduced CD19+ B-cell counts were observed in 44.4% of patients. Significant sex-based differences were identified: female patients had higher CD8+ T-cell counts (p = 0.029) and IgE levels (p = 0.034), while male patients had lower total lymphocyte counts compared to females (p = 0.013). Conclusion Sarcoidosis is associated with frequent hematological and immunological abnormalities. Distinct sex-specific immune patterns, particularly higher CD8+ T-cell and IgE levels in females, may contribute to disease heterogeneity. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)

Lymphocytic Variant Hypereosinophilic Syndrome is Composed of pathogenic CD4+ T cells with Features Distinct from those in T Cell Lymphoma 2300510

Abstract Introduction Lymphocytic variant hypereosinophilic syndrome (LHES) is an eosinophilic disorder characterized by phenotypically aberrant memory CD4+ T lymphocytes that secrete type 2 cytokines, with patients at an increased risk of progression to lymphoma. The most common aberrant phenotype, CD3loCD4+, is also seen in some types of T cell lymphoma, including angioimmunoblastic T cell lymphoma (AITL). Methods To increase our immunological understanding of LHES and its relationship to T cell lymphoma, we performed CITE-seq with VDJ sequencing on CD4+ T cells from 15 patients, 12 with LHES and 3 with angioimmunoblastic T cell lymphoma (AITL). Results Cell annotation and TCR clonotypic analysis confirmed hyperexpanded CD3lo clonal populations in all 15 patients and identified clonally expanded CD4+ regulatory T cells in LHES but not AITL. Clonal expansion of cytotoxic CD4+ T cells was also seen in some patients. CD3lo cells were comprised of a single expanded clone in 7 patients with LHES but were oligoclonal in the remaining patients. CD3lo cells in LHES showed increased expression of Th2 receptor genes involved in effector responses (PTGDR2, IL17RB, IL9R, IL5RA) with variable expression of Th2 cytokines. Strikingly, many subjects had prominent adhesion/tissue signatures (MXRA7, CLU, DNM3, SEMA5A, PDLIM1, CCR4, CCR8), indicative of tissue residence. Three LHES patients had a mixed Th2/IL9R phenotype and one patient had cytotoxic Th2 cells. Analyses of clones from AITL subjects revealed effector programs distinct from Th2 signaling, indicating a development pathway distinct from that in LHES. Conclusion In summary, LHES is characterized by clonal expansion of multiple CD4+ T cell lineages. The expression profile of CD3loCD4+ cells in LHES is consistent with a direct role for lymphocytes in LHES tissue pathology, beyond promoting eosinophilia. Further, RNA expression profiling distinguishes CD3lo T cells in LHES from those in AITL. Funding Source NIH Intramural funding Topic Categories Immune Mechanisms of Human Disease (HUM)

The phenomenon of Fear of Missing Out (FOMO) has emerged as a significant psychological and behavioral challenge in contemporary education 2257142

Abstract Introduction FOMO, which is the widespread worry that others are experiencing rewarding moments that one is missing, is intensified by the constant connectivity and social comparison facilitated by digital platforms. For students, this often manifests as compulsive checking of social media, messaging apps, and online communities during class, resulting in broken concentration and reduced academic engagement. Methods We at Chicago State University 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. Used tools such as LMS analytics, screen-monitoring software, and video analysis platforms to collect data. Analyzed the data using qualitative or quantitative methods. Results We examined how FOMO affects teaching and learning, showing how it disrupts focus, decreases intrinsic motivation, and leads to academic struggles. The widespread use of digital devices in classrooms and remote learning environments has blurred the lines between academic and social worlds, making it harder for students to stay present and attentive. Additionally, FOMO caused increased anxiety, sleep issues, and lowered well-being, all of which negatively affect students’ ability to learn effectively. Educators face the dual challenge of engaging students while effectively incorporating technology into teaching. Strategies like digital mindfulness training, clear technology policies, and interactive learning activities have proven effective in reducing FOMO. Conclusion Understanding FOMO’s impact on learning is essential for creating effective educational approaches that mirror students’ digital realities. By addressing the root causes of digital distraction and promoting mindful engagement, educators can cultivate more focused and supportive learning environments that enable students to succeed both academically and personally. Funding Source N/a Topic Categories Immunology Education and Communication (EDU)

The regulatory subunit ppp2r5a of protein phosphatase 2A selectively regulates spleen B-1 cell proliferation and accumulation 2257493

Abstract Introduction B-1 cells are a fetal/neonatal-derived B cell population located in small frequencies in the spleen. They are also the predominant B cell subset in the body cavities of common laboratory mice. B-1 cells differ from conventional (B-2) cells in that they maintain their numbers throughout life not by bone marrow replenishment but rather by “self-renewal”, a poorly understood process in which B-1 cells divide following unknown stimuli. Using cell population data obtained in an unbiased phenotypic screen, conducted by flow cytometry of 56 cell populations in 640 mouse lines each carrying a single gene deletion, we identified ppp2r5a as a candidate gene required for the selective control of B-1 cell maintenance and/or generation. Ppp2r5a encodes a regulatory subunit of protein phosphatase PP2A; a phosphase known to participate in cell activation, proliferation and cell death through its function in the WNT/Beta-catenin pathway. Here we tested the hypothesis that PP2A regulation is necessary for B-1 cell self-renewal. Methods Flow cytometry, in vitro cell culture, ELISA assy. Results Ppp2r5a-/- mice showed significantly lower frequencies of B-1 cells and B-1 derived plasma cells (PC) in spleen and BM, respectively, but not the body cavities. Consistent with B-1 cell’s role as producers of natural antibodies, mice had lower concentrations of serum IgM, IgG and IgG3 compared to wild type controls. Compared to B-1 cells from wild type mice, Ppp2r5a-/- B-1 cells showed increased proliferation in response to TLR4 agonist stimulation, but also increased cell death, as assessed by flow cytometric staining for Caspase 3 and 7. In addition, Ppp2r5a-/- B-1 cells showed dysregulated expression of mitochondria and Beta-catenin. Conclusion Together, these data implicate the WNT/ Beta-catenin pathway as an important regulator of B-1 cell dynamics. Funding Source NIHR21AI172208 (NB) and NIH UM1 OD023221 (KCKL) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Design and Evaluation of a Novel Recombinant Antigen for a T Cell-Based Vaccine Against Valley Fever 2260372

Abstract Introduction Valley Fever (VF) is a potentially fatal fungal disease caused by Coccidioides; however, no clinical vaccine exists. We previously created a recombinant Coccidioides antigen, rCpa1, with DRB1*04:01 (DR4) epitopes, but it is poorly expressed in E. coli. To enhance solubility and broaden HLA coverage, we removed 25 amino acids and incorporated three new DRB1*03:01 (DR3) epitopes, generating the rCpa5 antigen. We formulated rCpa5 with glucan-chitin particles (GCPs) as an adjuvant/delivery system. We hypothesize that GCP-rCpa5 can protect against VF by activating Th1/Th17 responses. Methods C57BL/6 (BL/6), DR4, and DR3 transgenic mice were vaccinated subcutaneously three times with GCP-rCpa5, and T cell epitopes were mapped by IFN-γ recall assays. Vaccinated and control mice were challenged intranasally to determine efficacy by survival (45 DPC) and fungal burden at 7 and 14 DPC. Lung T cell responses were profiled by intracellular cytokine assays. Additionally intramuscular and oropharyngeal vaccination routes were compared to identify the optimal route. Results The yield of the rCpa5 antigen was improved over 100-fold compared to rCpa1. We identified 14, 12, and 9 novel T cell epitopes within rCpa5 for BL/6, DR4, and DR3 mice, respectively. GCP-rCpa5 vaccination conferred 90% survival for BL/6 mice versus 10% in controls. Vaccinated DR4 mice showed a significant reduction in fungal burden in the lungs and spleen, with elevated Th1/Th17 responses compared to control mice. Comparable fungal clearance was observed across all three vaccination routes. Conclusion The rCpa5 antigen contains multiple epitopes for prevalent human MHC-II alleles and the GCP-rCpa5 vaccine is effective against Coccidioides infection for laboratory and humanized mice. Vaccine-induced protection is associated with mixed Th1/Th17 responses elicited by subcutaneous, oropharyngeal, and intramuscular vaccination. These data support that GCP-rCpa5 is protective and worthy of further development for clinical application. Funding Source NIH U19AI166761, NIAID-R01AI35005, 75N93019C00064BAA Topic Categories Vaccines and Immunotherapy (VAC)