Browse Articles

Lyophilized Listeria monocytogenes vaccines for storage at non-ultra-low temperatures and cancer therapy 2249461

Abstract Introduction Lyophilization (freeze-drying) is a common method of preparing bacteria for storage at higher temperatures with lyoprotectant and buffer selection greatly impacting viability. Listeria monocytogenes (LM) is a Gram-positive intracellular bacterium being investigated preclinically and clinically as an anticancer vaccine platform and is stored at ultra-low temperatures (ULT). ULT storage greatly limits LM therapy accessibility globally due to a lack of necessary storage equipment in many areas of the world. Methods Here, we investigated preparations of lyophilized (freeze-dried) LM for stability at cold-chain viable temperatures while maintaining the required infectivity for anticancer effect. Using a murine subcutaneous renal cell carcinoma model we further compared the antitumor efficacy of non-lyophilized versus lyophilized LM LLO-CD105A vaccine and the effects of lyophilization on immune recruitment to the tumor microenvironment. Results Through development of lyophilization media, we found that the choice of lyoprotectant and buffer dramatically impacts LM viability following lyophilization and during storage. Of those tested, the HEPES 2.5% sucrose medium (3mM HEPES 2.5% w/v sucrose) demonstrated the best viability while maintaining the infectivity and antitumor efficacy of LM LLO-CD105A vaccine in a subcutaneous renal cell carcinoma model. Lyophilized LM demonstrated near identical efficacy to non-lyophilized LM but resulted in higher myeloid-derived suppressor cell infiltration into the tumor microenvironment. Conclusion The methods described herein are easily adoptable in both laboratory and industry settings for the preparation of LM and stable storage at non-ultra-low temperatures. These methods address a critical need for translation of LM cancer therapies for global distribution and enable further development of novel LM therapeutic approaches. Funding Source Laura W. Bush Institute for Women’s Health, TTUHSC Office of Research and Innovation Topic Categories Technological Innovations in Immunology (TECH)

Mapping T cell phenotype with machine learning to optimize cancer immunotherapy 2307563

Abstract Introduction T cells play a central role in adaptive immune responses, enabling vertebrates to fight infections and eliminate cancer cells. Cancer immunotherapies, and in particular adoptive T-cell therapies, thus aim to harness their tumor-destroying capabilities to treat, or even cure, cancer patients. Despite recent advances in the development of these therapies, a major limitation remains our inability to predict, a priori, which tumor-infiltrating T cells will be best equipped to carry out anti-tumor immunity. Indeed, this multifactorial problem requires a model that integrates information on T cell receptor (TCR) specificity, tumor antigen abundance, and T cell activation history within the complex tumor microenvironment, yet such considerations are largely absent from current T cell selection strategies. Methods To address this gap, we are using a combination approach of high-throughput robotic multiplexing with computational and machine learning techniques to identify signatures of T cell phenotype that best predict response to tumor antigens. Specifically, we aim to study the roles of antigen presentation, TCR/antigen affinity, and inflammatory milieu on shaping T cell phenotype at the single-cell level, and training machine learning models to re-derive the activation history of T cells both in vivo and ex vivo. Results Preliminary results from in vitro co-culture data of T cells with cognate antigen-bearing splenocytes suggests that T cell antigen strength can indeed be back-calculated from single cell phenotype as measured by spectral flow cytometry. Conclusion With a validated model of tumor antigenicity in T cells, this research project aims to better inform T cell selection and optimal preparation strategies for adoptive T-cell therapies. Funding Source n/a Topic Categories Computational and Systems Immunology (COMP)

Human Apolipoprotein B Specific CD8 T Cells in Atherosclerosis 2260737

Abstract Introduction Atherosclerosis, contributing to ∼one-third of global deaths, is a chronic inflammatory disorder with autoimmune features. Atherosclerosis related antigen - apolipoprotein B (APOB) specific human CD4 T cells have been characterized, and CD8 T cell infiltration and oligoclonal expansion have been observed in human atherosclerotic lesions. Despite studies in mouse atherosclerosis models, APOB-specific CD8 T cells remain unexplored in humans. Methods Through in silico analysis, ∼0.1% of top-ranked APOB peptides predicted to bind HLA-A02:01 were selected. Using APOB peptide mesopools, PBMCs from HLA-A02:01+ donors underwent expansion-based restimulation, followed by flow cytometry. Responses to individual peptides were assessed via IFNγ ELISpot. Six immunodominant peptides were used to generate tetramers for tracking APOB-specific CD8+ T cells in human PBMCs. APOB tetramer-specific CD8 T cells from atherosclerosis patients were analyzed using single-cell RNA and TCR sequencing. Results APOB peptide stimulation induced the expression of activation markers (4-1BB, CD69, and CD25), proinflammatory cytokines (IFNγ and TNFα), and cytotoxic molecules (granzyme B and perforin) and enriched for effector memory phenotype in CD8 T cells. APOB epitopes that elicited the strongest IFNγ ELISpot response were mapped to 2 major antigenic regions in APOB. Tetramers detected APOB-specific circulating CD8 T cells in healthy humans and atherosclerosis patients. APOB-specific CD8 T cells from atherosclerotic humans were activated, as evidenced by high CD45RO and PD-1 expression. APOB-specific CD8 T cells had higher clonal expansion and lower diversity as compared to APOB non-specific CD8 T cells. Conclusion Human APOB harbors dominant HLA-I-restricted epitopes that trigger autoreactive CD8 T responses. Future assessment of transcriptomic and TCR data, followed by functional assays, will help characterize the nature and role of APOB-specific CD8 T cells in atherosclerosis. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)

Carbohydrate-Modified Virus-Like Particles as a Vaccine Platform for Leishmania braziliensis 2247392

Abstract Introduction Leishmania braziliensis is an important etiological agent of cutaneous leishmaniasis in the Americas, and it is associated with chronic lesions, tissue destruction, and severe morbidity. The toxicity, incomplete efficacy, and development of resistance associated with current chemotherapeutics emphasize the critical need for new immunoprophylactic approaches. Methods We tested the protective effect of a virus-like particle (VLP) platform that was recombinantly engineered to present the α-galactosyl (α-Gal) epitope, a carbohydrate recognized by natural anti-carbohydrate immune responses against L. braziliensis infection in mice. Mice were primed with empty Qβ VLPs (Qβ-WT), Qβ-αGal VLPs, or controls (PBS and naïve). Results Lesions were followed for 18 weeks post infection by footpad swelling. Qβ-αGal-vaccinated animals showed smaller lesion size than Qβ-WT and PBS controls, which presented swelling and severe pathology. Histological evaluation showed reduction in tissue damage in Qβ-αGal—immunized animals. Unvaccinated mice presented a much higher parasite load compared to Qβ-αGal—vaccinated animals. Spleen supernatant was analyzed, whose protection was correlated with the induction of Th1-related cytokines by increased IFN-γ and IL-12 in Qβ-αGal—immunized mice. These cytokines are essential for macrophage activation and parasite killing, implying that α-Gal—decorated VLPs induce a protective immune response. Qβ-WT and PBS showed little effect on cytokine induction, while lesions grew. Conclusion Our results provide proof of concept for the ability of α-Gal-conjugated VLP to induce a protective immune response against L. braziliensis infection, characterized by decreased lesion severity, parasite burden and enhanced Th1 responses. Results warrant further development of carbohydrate-based VLP vaccine as a potential approach against leishmaniasis. Studies will investigate antigen presentation optimization, the longevity of the immunity, and induction of cross-protection against Leishmania species. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

Tough, Ductile, and Strong Hard‐Soft Cementitious Composite Enabled by Multi‐Material Additive Manufacturing (Adv. Mater. 45/2026)

Unlocking Phthalonitrile‐Based Type I Photosensitizer Through D‐A Modulation to Promote Electron Transfer

ABSTRACT Type І photosensitizers (PSs) offer a promising strategy to overcome tumor hypoxia in photodynamic therapy (PDT) owing to their minimal oxygen dependence. However, their rational design remains elusive due to insufficient understanding of structure–property relationships. Herein, we leverage donor‐π bridge‐acceptor (D‐π‐A) conjugate modulation to design phthalonitrile‐based Type І PSs through the systematic regulation of four critical parameters, including Δ E ST , T 1 energy level, redox potential and steric hindrance, thereby optimizing efficient electron transfer pathway. These PSs exhibit aggregate‐induced Type І reactive oxygen species (ROS) generation, driven by favorable intermolecular electronic interactions. Among them, DTPCH 3 demonstrates the highest Type І ROS production, attributed to its minimal Δ E S1‐T2 and the most effective intermolecular electron transfer interactions. Upon encapsulation with amphiphilic polymer F127, DTPCH 3 nanoparticles (DTPCH 3 _NPs) retain efficient O 2 •− and HO• generation, resulting in potent cancer cell ablation and good hypoxic tolerance. In vivo studies further confirm significant tumor suppression by DTPCH 3 _NPs. Overall, this work establishes a molecular design strategy for Type I PSs, opening new avenues for the development of next‐generation PDT agents.

Experimental and computational evaluation of achieving RCCI combustion at low load in a dual-fuel light-duty diesel engine with biomethane surrogate

An AAV9-Based Strategy for the Treatment of Cervical Dysplasia 2259948

Abstract Introduction Cervical cancer is the fourth most commonly diagnosed cancer in women worldwide and in some countries is the most lethal cancer in women. Ninety-nine percent of cervical cancers are attributable to HPV infection, which has decreased dramatically with the introduction of HPV vaccines. However, the incidence of cervical cancer remains high in generations of women for whom the vaccine was not available as well as in unvaccinated women, as up to 40% of eligible individuals did not receive an HPV vaccination. There is a critical unmet need for noninvasive treatment options among these groups. Methods A hallmark of HPV-mediated oncogenesis is dysregulated E2F transcription factor activity. Leveraging this, we developed a rationally designed series of synthetic AAV transgene promoters for specific activity in HPV-positive cervical cancer cells, which can then be used to deliver therapeutic payloads to tumor tissue. Our panel of candidate promoter sequences was assessed for activity in HPV+ cervical cancer cell lines as well as normal epithelial cells for expression of a luciferase reporter, as determined by comparative expression in a dual-luciferase system. Results Testing our candidate promoters in a dual luciferase reporter assay we identified a construct with activity in multiple cell lines. This candidate achieved an 18.31-fold and a 4.45-fold increase in reporter expression in HPV16+ CaSki and SiHa cancer lines compared to normal controls, respectively. Further, it produced a 15.18-fold increase in the HPV18+ C4I line compared to normal controls. By modulating the levels of activating E2F family members in normal cells, we have shown a positive relationship between E2F levels and reporter expression. Conclusion Based upon our results in vitro we have developed a candidate AAV transgene construct for specific delivery of a therapeutic payload to cervical cancer cells in vivo. Funding Source T32 Postdoctoral Fellowship Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

The BMP-KLF3 circuit promotes resilient programming in CD8+ T cells during chronic infection and cancer 2257332

Abstract Introduction Cytotoxic CD8+ T cells progressively lose function and become terminally exhausted during chronic infections and cancer. We previously showed that chronic antigen exposure plus TGFβ1 signals drive epigenetically imprinted terminal exhaustion programs in CD8 T cells. In contrast, activating bone morphogenetic protein (BMP) signaling together with TGFβ1 receptor blockade restores functional programming and antitumor activity in CD8 T cells. Moreover, BMP activation recovers expression of the Krüppel-like factor 3 (KLF3) transcription factor, which is uniquely expressed in functional CD8 T cells during chronic infection and cancer. However, the role of BMP-KLF3 axis in reprogramming exhausted T cells remains unclear. Identifying programmable pathways that prevent terminal exhaustion is critical for improving the efficacy of T cell-based immunotherapies. Methods To assess the role of BMP-driven KLF3 in CD8 T cell programming, we co-adoptively transferred congenically distinct control and KLF3-overexpressing (OE) P14 CD8+ T cells into mice, followed by chronic LCMV infection or gp33-expressing melanoma. Functional, transcriptional, and epigenetic analyses were performed to identify the molecular circuits regulated by BMP-KLF3 pathway. Results We show that BMP activation prevented epigenetic silencing of KLF3 in chronically stimulated CD8 T cells. Importantly, induced expression of KLF3 (OE) reprogrammed exhausted T cells by enhancing their fitness and proliferation capacity during chronic infection, as well as promoting their cytotoxicity and tumor control in vivo. Conclusion Our findings reveal a novel molecular pathway in which BMP signaling drives KLF3, a key transcriptional mediator that programs resilient CD8+ T cell responses during chronic infection and cancer. Our findings reveal a promising approach to enhance responses to adoptive T cell-based immunotherapies. Funding Source NIH R01-AI170926, NIH T32-AI165391, OSU College of Medicine, OSU James CCC Funding Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Hippo pathway coactivator WWTR1 drives self-renewal in alveolar macrophages 2257963

Abstract Introduction It is well known that Alveolar macrophages (AMs) self-renew in the lung and do not depend on circulating monocytes to maintain their population, but the transcriptional mechanisms driving this process remain incompletely defined. We have investigated the role of WWTR1/TAZ, a Hippo pathway coactivator that regulates cell growth and organ size, in AMs self-renewal. Methods We generated inducible myeloid knockouts by crossing LysM-CreERT2 mice with Wwtr1^fl/fl mice to obtain LysM-CreERT2 Wwtr1^fl/fl (conditional KO) and littermate controls. Mice received tamoxifen diet for 14 days to induce recombination, then BrdU (2 mg/day, i.p.) for 5 consecutive days to assess AM proliferation. To profile chromatin accessibility, we performed ATAC-seq on AMs in bone-marrow chimeras (CD45.1↔CD45.2) generated after clodronate liposome depletion, comparing host-derived resident versus donor-derived recruited AMs. Results BrdU incorporation by AMs was significantly reduced in LysM-CreERT2 Wwtr1^fl/fl mice relative to wild-type/Cre-negative controls, indicating impaired self-renewal upon Wwtr1 loss in myeloid cells. ATAC-seq revealed that chromatin at the WWTR1/TAZ-associated regulatory region(s) was accessible in host-derived AMs but not accessible in donor-derived recruited AMs in chimeric lungs, consistent with a resident AM program linked to WWTR1 activity Conclusion These data identify WWTR1/TAZ as a key regulator for the long-term maintenance of the tissue-resident AM pool. Future studies are warranted to define WWTR1 target genes and test whether modulating this pathway can bolster AM-dependent lung immunity in disorders with defective AMs function. Funding Source R35 HL155458 (C.V.J.) Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Novel Roles of Splice-Derived Tumor Mirtrons in the Regulation of Macrophage Dichotomy 2310054

Abstract Introduction Background: Tumor-derived exosomes play key roles in cell-cell communication within the tumor microenvironment and have been shown to drive macrophage polarization towards an M2-like phenotype.We recently identified a novel class of splicing generated small RNAs, known as mirtrons, are highly packed in exosomes and potentially modulate macrophage plasticity. However, molecular mechanisms for this exosome-driven phenomenon are not completely clear. Methods Human macrophages (U937) were co-cultured with exosomes derived from human lung (H23), prostate (PC3), and a variety subtypes of breast cancer cell lines (T47D, SKBR3, and MDA-MB-468). Expression of mirtrons including miR-7110, -cv002, and -6756 in cancer cells and in exosomes was assessed by qPCR. Immunofluorescent imaging was used to assess exosomal small RNA uptake in macrophages and their intracellular localization. Genomic characterization of M2-associated mirtrons in association with exosome exposure was performed using ChIP-seq. Results All three mirtrons–particularly cv002–were consistently enriched in tumor-derived exosomes relative to their parent tumor cells across all cancer cell lines examined. However, mirtron expression levels in exosomes or the cytoplasm did not correlate with tumor aggressiveness in breast cancer. Immunofluorescence imaging demonstrated that mirtrons carried by tumor-derived exosomes are taken up by human monocyte U937 cells and are predominantly localized to the cytoplasm, consistent with the behavior of conventional miRNAs. Notably, a subset of mirtrons was also observed to translocate into the nucleus and interact with monocyte genomic DNA, as supported by ChIP-seq analyses. Importantly, these mirtron—DNA interactions were enriched at M2-associated gene loci, suggesting a novel regulatory mechanism underlying macrophage polarization. Conclusion Our results showed a novel mechanism through exosomal delivery of tumor derived mirtrons to manipulate M2-like macrophage polarization. Funding Source NIGMS (R16GM153648) Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Broad-spectrum antibiotics affect CCR4+ and CCR6+ Th17 and Treg recruitment to the gingiva 2259700

Abstract Introduction Susceptibility to periodontitis depends on the interplay between the oral plaque microbiota and host immune response. IL-17A-producing CD4 T cells (Th17) and regulatory T cells (Tregs) expressing either CCR4+ or CCR6+ are both present in human periodontitis. Oral vancomycin decreases intestinal priming of CCR6+ Th17, reducing their recruitment to the oral mucosa, which mitigates murine ligature-induced periodontitis (LIP). Yet, it is unclear how the oral microbiota affects the relative proportions of Th17 and Tregs. We hypothesized that broad-spectrum antibiotic treatment decreases the frequency of CCR4+ and CCR6+ Th17 cells to protect against LIP. Methods C57BL6 mice were pre-treated with (i) water alone or water containing (ii) broad-spectrum systemically absorbable antibiotics alternating weekly with non-absorbable antibiotics for 27 days. Periodontitis was induced with ligatures placed around upper molars concurrent with human oral pathobiont Porphyromonas gingivalis (Pg). In the gingival tissues, the frequencies of CCR4+, CCR6+, and CCR9+ CD4 T cells were assessed with flow cytometry, while CCL2, 17, 20, 22, or 25 mRNA expression was measured using qPCR. Results Broad-spectrum antibiotics reduced the frequency of both CCR4+ and CCR6+ Th17 and Tregs in the gingival tissues at 10 days after LIP induction. Broad-spectrum antibiotics reduced the frequency of CCR9+ Tregs at 10 days after LIP. CCR9+ Th17 cells were not detected. In the absence of antibiotic treatment, only CCL22 chemokine mRNA was elevated 10 days after LIP. Conclusion The protective effect of broad-spectrum antibiotics is associated with a reduction in CCR4+ and CCR6+ Th17 cells in the gingiva, despite a concurrent reduction in Tregs. A targeted reduction in Th17 cells may benefit human periodontitis irrespective of CCR4+ and CCR6+ Tregs. We speculate that broad-spectrum antibiotic treatment would reduce CCL22. Intestinal T cell priming drives CCR9+ Tregs but not Th17 cells to the gingiva. Funding Source NIH NIDCR NATL INST OF DENTAL 1R01DE030095-01A1 Topic Categories Cytokines and Chemokines and their Receptors (CCR)

Assessing Susceptibility of Cervical Explant Tissue to HIV-1 Infection Following Masculinizing Hormone Therapy 2250590

Abstract Introduction Masculinizing hormonal therapy, integral to gender-affirming care for transgender men (TGM), has immunomodulatory properties that heighten HIV-1 acquisition risk. Testosterone activates immune cells through androgen receptors (ARs), resulting in a cytokine shift and a defective innate immune response. The cervical mucosae are enriched with HIV-1 target cells inclusive of macrophages and CD4+ T cells that co-express ARs. The extent to which this impacts HIV-1 vulnerability in TGM remains unclear. Methods We investigated the impact of hormonal therapy on HIV-1 target cells, in an ex vivo model using cervical tissue explants (CETs). CETs from TGM vs CIS women were sectioned into 2 mm3 blocks for inoculation using Sub A and D T/F IMCs expressing snLuc or msCD24 reporters and monitored over 11 days. HIV-1 infection kinetics were measured using snLuc. HIV-1 infected and uninfected cells were characterized using a 28-color flow cytometry panel and Luminex Results CD4+ T cells were predominantly effector memory in both TGM (95.08% ± 0.54%) and CIS CETs (87.52% ± 0.10%), although CXCR4+CCR5+ co-expression was higher in TGM (28.71% ±0.23%) than CIS (6.79% ± 0.12%; p = 0.0006). The frequency of α4β7+CCR5+CXCR4+CD4Tem (p = 0.0103) and CD38+CD8+ T cells (p = 0.0066) was also higher in TGM. Similar trends were observed, in the TGM “M2” macrophages, although the expression of BST-2 and IFNy was downregulated in TGM. Interestingly, IFNα, CXCL10 and IL-1α, were elevated in TGM compared to CIS CETs culture supernatants. Collectively these findings alluded to increased HIV-1 acquisition risk, as shown by increased HIV-1 replication kinetics in TGM CETs compared to CIS CETs. Conclusion Taken together, our findings suggest increased susceptibility to HIV-1 infection, consistent with testosterone administration. Funding Source UAB Department of Medicine DREAM Council_ Reagan Durant Award Topic Categories Mucosal and Regional Immunology (MUC)

Enhancing Classroom Engagement through Mood Assessment of Students 2263296

Abstract Introduction Understanding and responding to students’ emotional states is becoming an increasingly important part of effective teaching and learning. Monitoring students’ moods provides teachers with essential insights into their readiness to learn, emotional health, and the classroom atmosphere. Conversely, positive emotional states can enhance motivation, creativity, and collaboration. By regularly checking students’ moods–through informal check-ins, digital tools, or observational techniques–educators can modify their teaching methods to meet students’ emotional and cognitive needs. Methods We at Chicago State University used appropriate surveillance methods, such as direct observation, digital monitoring, biometric sensors, and self-reporting tools. Results Results showed that assessing mood promotes a culture of empathy and psychological safety. When students feel recognized and understood, they are more likely to participate actively and take academic risks. This approach also helps identify students who may be struggling emotionally early on, allowing for timely interventions that support mental health and academic success. This summary highlights the importance of mood assessment in boosting classroom engagement and creating a supportive learning environment. Mood directly influences cognitive functions like attention, memory, and problem-solving. When students are anxious, disengaged, or emotionally upset, their ability to engage in classroom activities diminishes. Conclusion Adding mood checks into classroom routines takes little resources. Simple tools like mood meters, anonymous surveys, or reflective journaling can provide useful information. When used regularly, these tools help teachers adjust lesson speed, groupings, and behavior management on the spot. In summary, measuring students’ mood is not just a pastoral duty but an educational need. It links emotional health with academic achievement, creating a more inclusive, responsive, and engaging learning space. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)

Bacteria Ammoniphilus Associated with High Tertiary Lymphoid Structures in Lung Squamous Cell Carcinoma 2248408

Abstract Introduction Non—small cell lung cancer (NSCLC) is the leading cause of cancer death, and robust biomarkers predicting prognosis and/or efficacy of treatment are urgently needed. Tertiary lymphoid structures (TLSs) are recognized as predictors of response to immune checkpoint inhibitors. Tumor-resident bacteria have been shown to promotes TLSs formation. We hypothesized that bacteria in TLSs-high tumors could predict TLSs abundance and ICI responsiveness. Methods We reviewed resected cases of lung squamous cell carcinoma (LUSC) from a larger NSCLC cohort collected between 2013 and 2016. Of 39 cases, 24 met prespecified criteria. TLSs status was determined by PNAd+ high endothelial venule (HEV) immunohistochemistry. Twelve tumors were classified as TLSs-high and 12 as TLSs-low. Bacterial communities were profiled by 16S rRNA(V3—V4) sequencing and processed with QIIME2/DADA2. Differential taxa were identified with LEfSe (α =0.05, LDA>2.0). Functional profiles were inferred by PICRUSt2 and tested with STAMP (FDR-corrected q < 0.05). Results Bacteria specific to the TLS-high group were identified. Genus Ammoniphilus was enriched in TLS-high tumors (7/24; LDA 5.026; p = 0.033). Genus Ralstonia was more prevalent in adjacent normal tissues of TLS-high cases (8/24; LDA 4.367; p = 0.006). Functional analysis suggested that Ammoniphilus was involved in methionine, nucleotide, and energy metabolism pathways. Conclusion We identified Ammoniphilus as a first-in-LUSC candidate microbiome linked to TLSs formation. Its presence could indicate an immunologically “hot” tumor microenvironment and serve as a novel biomarker predicting response to immune checkpoint inhibitors. Funding Source n/a. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Mechanically Spatio‐Chimeric Fibrin Assembly Enables Vascular‐Integrated Muscle Reconstruction for Volumetric Muscle Loss Repair (Adv. Mater. 44/2026)

A taxonomically informed deep neural network with hierarchical regularization for microbiome data analysis

Spatiotemporal analysis of macrophages in neuroinflammatory lesions 2259964

Abstract Introduction During neuroinflammation such as multiple sclerosis (MS), macrophages infiltrate into the CNS and act as important drivers and regulators of inflammation. CNS-associated macrophages are highly plastic and perform diverse functions in neuroinflammation; they can mediate either pro- or anti-inflammatory effects. However, while it is becoming clear that multiple macrophage states exist, the origin and basis of the functional heterogeneity of macrophages is poorly understood. Methods To shed light on macrophage functional heterogeneity during neuroinflammation, we performed matched single-cell RNA-sequencing and spatial transcriptomics of immune infiltrates in evolving neuroinflammatory lesions in mice. Results We identified distinct macrophage subpopulations that appear at different stages of inflammation and express unique transcriptional profiles. By performing spatial co-localization and cell-cell-interaction analysis, we identified these different macrophage populations to mediate distinct functions in the neuroinflammatory lesions. To validate functional roles of specific macrophage cell-cell interactions we are performing CRISPR/Cas9 spatial screening. Conclusion We believe that our results improve our understanding of macrophage functional diversity and reveal fundamental processes underlying macrophage heterogeneity and, more importantly, have the potential to identify new biomarkers and actionable targets that could be exploited for targeted modulation of macrophages to improve the outcome of MS. Funding Source n/a Topic Categories Neuroimmunology (NEUR)

Subclinical disease detection and immune profiling in siblings of patients with Enthesitis-related arthritis category of juvenile idiopathic arthritis 2257529

Abstract Introduction Enthesitis related arthritis (ERA) category of juvenile arthritis is a form of juvenile spondylarthritis. In many immune-inflammatory disorders a subclinical phase precedes a clinical disease. First degree relatives either due to genetic predisposition or sharing of environment have a high recurrence risk for these diseases. No data is available in siblings of ERA patients. Methods 38 sibling-proband pairs were evaluated after consent. Clinical examination, X-ray LS-spine, MRI of SI-joints and MSUS for enthesitis were done to detect subclinical disease in siblings. Frequency of lymphocyte and monocyte subsets, IL-17 producing NK cells, NK-T cells, γδ T cells and CD4 lymphocytes, IL-6 and TNF-α producing monocytes was measured using FACS. Faecal calprotectin levels were measured using ELISA. Results 38 siblings of 36 proband (13.8±3.5 years) were included. 20 of 38 siblings were HLA-B27 positive. Among siblings, none had arthritis, 1 each had inflammatory back pain and uveitis. On radiological assessment, 5 showed evidence of sacroiliitis (Grade II-4, Grade IV-1) on X-ray while 5 siblings had bone marrow edema around SI joints (one quadrant-4, four quadrants-1) on MRI. On MSUS enthesitis was seen in 2 siblings (patellar tendon-1, Tendoachillies-1). All but one with abnormalities were HLA-B27 positive. In comparison to siblings, the probands had lower frequency of classical monocytes (p < 0.05), IL-6 producing monocytes (p < 0.01) and higher frequency of Th17 cells (p < 0.05). The faecal calprotectin levels were higher in probands (p < 0.01). Analysing only the HLA-B27 siblings since subclinical disease was seen in only HLA-B27 siblings, the frequency of IL-17 producing CD4 T cells and γδ T cells (p < 0.01) and faecal calprotectin levels (p < 0.01) were higher in probands when compared with B27 positive siblings. Conclusion Subclinical disease is seen in subset of HLA-B27 siblings of patients with ERA. Gut inflammation and a pro-inflammatory state probably results in clinical disease in probands. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)

CD25 expressed on keratinocytes sustains CD8+ T cell homeostasis and promotes anti-tumor responses 2258211

Abstract Introduction CD8+ T cells are crucial for anti-tumor immunity, with IL-2 signaling essential for maintaining their homeostasis. While IL-2 typically signals through cis-presentation via the IL-2R alpha, beta, and gamma subunits, trans-presentation by IL-2R alpha (CD25)-expressing antigen-presenting cells is also important. Notably, we observed CD25 expression on keratinocytes and investigated its role in maintaining CD8+ T cell homeostasis and enhancing anti-tumor responses. Methods Keratinocyte-specific CD25-deficient (K14creCD25fl/fl) mice were generated using the LoxP-Cre system. Under steady-state conditions, CD8+ T cell homeostasis was assessed in skin and secondary lymphoid organs between wild-type (WT) and K14creCD25fl/fl mice. Anti-tumor responses were assessed in a B16 melanoma model, evaluating tumor progression, CD8+ T cell infiltration, effector function. Co-culture experiments examined the direct effects of keratinocyte-derived CD25 on CD8+ T cell. Results Compared to WT mice, K14creCD25fl/fl mice exhibited significantly reduced resident memory CD8+ T cells in dorsal skin and decreased CD8+ T cell homeostasis in secondary lymphoid organs, especially peripheral lymph nodes under steady-state conditions. Co-culture experiments confirmed that CD8+ T cells showed impaired homeostasis when cultured with CD25-deficient keratinocytes. In the B16 melanoma model, K14creCD25fl/fl mice exhibited accelerated tumor progression with reduced CD8+ T cell infiltration into the tumor microenvironment, decreased production of cytotoxic molecules, and suppressed proliferation. Conclusion Keratinocyte-expressed CD25 plays a critical role in maintaining CD8+ T cell homeostasis under steady-state conditions and significantly enhances anti-tumor immunity. These findings reveal a novel mechanism by which skin keratinocytes contribute to systemic immune surveillance through IL-2 trans-presentation, providing new insight into the skin immune system and potential therapeutic targets for enhancing anti-tumor responses. Funding Source This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : RS-2024-00440637). Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)