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Evaluation of Vesiculab Vesi-Ref CD63- mNeonGreen (mNG) Extracellular Vesicles (EV) Reference Standard on the CytoFLEX nano Flow Cytometer 2306506
Abstract Introduction Accurate and reproducible characterization of EVs by nanoscale flow cytometry requires both robust reference materials and carefully controlled experimental conditions. This study evaluated the performance of the Vesiculab Vesi-Ref CD63-mNG EV reference standard using the Beckman Coulter Life Sciences CytoFLEX nano Flow Cytometer. Methods Our experimental design involved determining the optimal dilution of unstained Vesi-Ref CD63-mNG EVs through serial dilution in 20-nm-filtered PBS. This step aimed to achieve optimal target event rates (1000—2000 events per second) and minimize swarming by monitoring VSSC1-H scatter profiles. Concurrently, CD63-Alexa Fluor 647 antibody titrations (0.25-2.0 µg/mL) were performed to assess antibody aggregation and background signal. Subsequently, stained Vesi-Ref CD63-mNG EVs were acquired to evaluate dual fluorescence expression (mNG and CD63-AF647). Results Unstained Vesi-Ref CD63-mNG EVs showed optimal acquisition at 1:1,000 dilution, yielding approximately 2,164 events per second (EPS) and distinct separation from instrument noise. The mNG fluorescent population was detected in the B531-H channel and exhibited two discernible size populations based on VSSC1-H gating. Antibody-only controls for CD63-AF647 indicated minimal aggregation across all tested concentrations, with lower concentrations (0.25 µg/mL and 0.5 µg/mL) showing reduced background in the R670-H channel. Staining of Vesi-Ref CD63-mNG EVs with CD63-AF647 consistently resulted in approximately 72% dual-positive events (mNG and CD63-AF647) across all antibody concentrations, alongside a minor (3—4%) population positive for CD63-AF647 only (0.4%). Conclusion These findings confirm the suitability of the Vesi-Ref CD63-mNG EV reference standard as a positive control for EV characterization in nanoscale flow cytometry, supporting the validation of antibody-staining protocols due to its distinct fluorescent and scatter properties. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)
Il22 regulatory elements shape inflammatory responses in Imiquimod-induced psoriasis 2258410
Abstract Introduction IL22 is a type III cytokine produced by innate and adaptive lymphocytes that is essential for maintaining tissue homeostasis and establishing a defense against extracellular pathogens. Dysregulation of IL22 contributes to the development of immune-mediated diseases such as psoriasis. Methods We identified two conserved enhancer elements (E22-1 and E22-2) for Il22 by a CRISPR interference (CRISPRi) screen. We additionally identified a superenhancer for Il22 (SE22) using H2K27ac chromatin immunoprecipitation sequencing (ChIP-Seq) of human tonsillar ILC3s, which is also conserved in mouse. Results Knockout mice for SE22 or E22-1 resulted in partial loss of IL-22 expression in ILC3s as well as αβ T, iNKT, and γδ T cells. In contrast, deletion of E22-2 impaired IL-22 expression only in ILC3s. We demonstrate that the enhancer elements are bound by the RUNX family and RORC transcription factors in a site dependent manner resulting in cell type specific control of IL22 expression in ILC3s by E22-2. We further demonstrate that the partial loss of IL22 in each of the knockout strains protects from development of imiquimod induced psoriasis through a decrease in Il22 but not Il17a or Il17f. Conclusion Further characterization of cis-regulatory elements of Il22 may provide key targets for epigenetic therapy to treat autoimmune disease. Funding Source NIH 5R01AI134035 and NIH 5T32AI165391 Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Integrated Multi-Omic Approach Using Spatial Transcriptomics and Proteomics Reveals an In-Depth View of Localized Immune Response in Renal Cancer 2259256
Abstract Introduction Clear cell renal cell carcinoma (ccRCC) is a highly heterogeneous cancer with complex tumor and immune microenvironment interactions influencing progression and therapy response. Advances in spatial technologies now allow for simultaneous spatial mapping of transcriptomic and proteomic features from patient tissue and phenotyping of blood cells, enabling detailed assessment of disease biology. This study highlights a multi-omic approach to characterizing systemic immune responses, tumor microenvironment niches, immune states and tumor heterogeneity in ccRCC patient samples. Methods We profiled ccRCC patient samples using peripheral blood mononuclear cells (PBMC) and tumor-derived cells (TDCs) by CyTOF™ technology with a 50-plus-antibody panel. Matched formalin-fixed tumor tissue was examined by a spatial transcriptomic platform, hematoxylin and eosin (H&E) assessment, and Imaging Mass Cytometry™ (IMC™) technology with a 43-marker panel sequentially on the same tissue section. These datasets were analyzed to explore immune and tumor signatures linked to disease states. Results We demonstrate compatibility of IMC workflows with H&E and spatial transcriptomic modalities. IMC technology identified spatial distribution and activation states of immune and tumor cells in ccRCC. Combining spatial transcriptomics and proteomics enabled detailed phenotyping of tumor metabolic and signaling states and immune cytokine and transcription factor expression. Comparison of PBMC and TDC composition provided insights into systemic and localized immune responses. Conclusion This study highlights the power of spatial multi-omic profiling to unravel the immune and oncologic landscape of ccRCC with high resolution. Identification of spatially defined immune cell states and niches offered insights into tumor immune evasion and resistance mechanisms. These findings pave the way for spatially informed biomarkers and potential precision therapies for ccRCC. For Research Use Only. Not for use in diagnostic procedures. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Dual‐Interfacial Engineering Enabled by a Zn <sup>2+</sup> ‐Induced Self‐Assembled Hydrogel Electrolyte Toward Flexible On‐Body Zinc‐Iodine Batteries
ABSTRACT Aqueous zinc‐iodine batteries (AZIBs) are promising candidates for flexible electronics owing to their intrinsic safety. Nevertheless, their practical deployment is impeded by severe interfacial challenges, including polyiodide shuttling, zinc (Zn) dendrite growth, and parasitic reactions. Herein, a Zn 2 + ‐induced molecular self‐assembly strategy based on natural glycyrrhizic acid (Gl) is proposed to construct a robust dual‐network hydrogel electrolyte. The self‐assembled Gl nanofibrous network can simultaneously and effectively regulate the dual electrode/electrolyte interfaces. At the anode interface, the Gl nanofibrous network adsorbed on the Zn surface enables homoepitaxial Zn plating, promoting uniform Zn nucleation and growth. At the cathode interface, the p – π conjugated structure and nanofibrous network of Gl cooperatively immobilize polyiodides, effectively suppressing the shuttle effect. Consequently, the Zn||Zn symmetric cell exhibits excellent cycling stability over 1900 h at 1 mA cm −2 with 1 mAh cm −2 . The Zn||I 2 pouch cell achieves a long lifespan with a high‐capacity retention of 78.1% and a low N/P ratio of 1.93. Moreover, leveraging the high biocompatibility and flexibility of this self‐assembled hydrogel, an on‐body transcranial direct current stimulation (tDCS) device integrated with the Zn||I 2 battery delivers a stable 1.5 mA current for therapeutic neuromodulation, demonstrating a feasible pathway for flexible on‐body AZIBs.
Identification of microglial immunomodulatory compounds by high-throughput proteomics: insights from quantification of 1000 proteins in &gt; 6,500 samples 2259191
Abstract Introduction Microglia are critical for maintaining neural homeostasis. However, under pathological conditions, they can adopt a chronically pro-inflammatory phenotype contributing to neurodegenerative diseases including Alzheimer’s, Parkinson’s, and multiple sclerosis (MS). Identifying potential therapies requires capturing the breath of inflammatory proteins produced by microglia, and signals of potential toxicities to minimize adverse events (AEs). Methods The high cost of capturing these diverse signals at scale limits typical drug discovery efforts to simple low-plex readouts. To address this gap, we previously described the Nomic platform, a proteomics tool capable of quantifying &gt;1000 proteins simultaneously. Here, we leverage the Nomic’s Omni 1000 to screen 510 bioactive small molecules in microglia stimulated with LPS, generating &gt;6,500 samples and &gt;6.5 million data points. Results LPS led to expected increased expression of TNF-alpha, IL-1β, and IL-6, as well as ISG15 and IL-12 p40, indicators of chronic inflammation associated with neurodegenerative diseases. Approximately ⅓ of the compounds screened blocked inflammation, of which ¼ presented clear signs of toxicity, characterized by leaking of intracellular content into the supernatant. To further assess potential toxicities, we treated cardiomyocytes and hepatocytes with these compounds, identifying expected and novel drugs with promising properties. For example, Berberine (BBR) is being developed to revert M1 microglia to M2 by blocking NF-κB signaling. We observed significantly reduced IL-12 p40, IL-10, and CXCL6 upon addition of BBR, with minimal signs of toxicities. Methylprednisolone was similarly anti-inflammatory, but paradoxically led to increased SAA and IL-6, consistent with clinical reports of hepatic AEs with high MP exposure. Conclusion Our results demonstrate the value of high-throughput proteomics to simultaneously identify immunomodulatory compounds and characterize their efficacy and safety profile. Funding Source n/a Topic Categories Neuroimmunology (NEUR)
Losing Their Grip: Aging Weakens Treg TCR Catch Bonds Under Force 2260253
Abstract Introduction Aging is associated with a decline in regulatory T cell (Treg) function contributing to the increased severity of autoimmune disease in older individuals. Our data indicate that Tregs from middle-aged mice display reduced suppressive function in experimental autoimmune encephalomyelitis (EAE). In aged mice we found diminished force-dependent suppression via trogocytosis of peptide:MHC complexes. These functional decreases correlated with a reduction in the bond lifetime under force in aged mice compared to their young counterparts. This data suggests that age-related biophysical changes in TCR binding kinetics drive the impaired function of Tregs. To test this, we examined the bond lifetime under force in myelin oligodendrocyte glycoprotein (MOG) specific Tregs in aged C57BL/6 and NOD mice. We found a reduction in bond lifetime under force in aged Tregs. We hypothesized the reduction in bond lifetime was due to changes in membrane rigidity and our findings demonstrate that aged Tregs exhibit increased rigidity. This alters membrane dynamics and reduces the stability of TCR:pMHC interactions, decreasing the capacity for aged Tregs to perform force mediated suppression via trogocytosis. In addition we show this lowers antigen specific Treg function and leads to earlier EAE onset and increased severity. Together these studies are the first to show how aging alters Treg TCR:pMHC binding kinetics and will allow for exploration of therapeutic strategies to enhance immune tolerance in older individuals with autoimmune disease. Methods Results Conclusion Funding Source 5R01AI169835-03 Topic Categories Basic Autoimmunity (BA)
Identifying Macrophages as Peripheral Transporters of JC Polyomavirus 2258486
Abstract Introduction JC polyomavirus (JCPyV) is a DNA virus that can cause progressive multifocal leukoencephalopathy (PML) upon reactivation. PML is a rare but often fatal demyelinating disease of the central nervous system (CNS) characterized by motor dysfunction, speech impairment, and seizures. Currently, there is no cure, but treatment relies on boosting host immune responses. JCPyV typically establishes a benign infection in the kidney tubule epithelium. Viral reactivation may occur under severe immunosuppression such as radiation, HIV infection, organ transplantation, or immunosuppressive drug therapy. While JCPyV infection of astrocytes and oligodendrocytes is well established, mechanisms of viral entry into the CNS remain unclear. Macrophages are key candidates as peripheral immune cells capable of trafficking JCPyV to the CNS. We hypothesize that monocytes and macrophages are targets of JCPyV infection and may elucidate how the virus travels from the kidney to the CNS. Methods Human monocytes were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) using CD14+ bead isolation and differentiated into macrophages with human M-CSF in vitro. Digital polymerase chain reaction (dPCR) was performed to verify JCPyV infection in monocytes and macrophages. Fixed and sectioned PML patient brain tissues were stained by immunohistochemistry to assess macrophage infection in vivo. Results Preliminary dPCR data reveal JCPyV infection of both monocytes and macrophages ex vivo, with robust detection of two distinct viral probes. PML brain tissue also showed JCPyV-positive macrophages; however, further studies are needed to determine whether these cells migrated from the periphery or are brain resident. Conclusion These findings suggest that human monocytes and macrophages can support JCPyV infection and may act as cellular vehicles for viral dissemination to the CNS. Understanding macrophage-mediated viral transport could provide key insights into PML pathogenesis and identify new therapeutic targets. Funding Source R01NS116278 Topic Categories Viral Immunology (VIR)
Human NK Cell Enrichment and Expansion Using Defined GMP Reagents: Functional and Phenotypic Outcomes 2252995
Abstract Introduction Natural killer (NK) cells are a key component of the innate immune system and chimeric antigen receptor NK cells have shown promise in adoptive cell therapy. NK cell therapies require highly controlled expansion and deep characterization. Here we evaluated Cell-ViveTM GMP reagents for utility in NK cell isolation, activation, culture and characterization. Methods NK cells were isolated from leukopak mononuclear cells using two methods: (1) positive selection via sequential T cell depletion and NK cell enrichment with GMP anti-CD3 and anti-CD56 magnetic beads, and (2) negative selection using a GMP NK cell isolation kit. Following isolation, NK cells were activated using GMP anti-NKp46/CD2 magnetic beads and expanded 14 days in vitro under serum-free, xeno-free conditions supplemented with GMP-grade recombinant human IL-2 and IL-15. Results Expanded NK cells were assessed for viability, expansion, surface marker expression, cytokine production, and cytotoxicity. Both isolation strategies yielded highly viable NK cell populations, with different purity and expansion kinetic profiles. Functional analyses demonstrated effective cytokine secretion and target cell killing capacity post-expansion. Conclusion Our results provide comprehensive evaluation of NK cell enrichment and characterization using two different GMP-grade isolation kits. Further, we demonstrate the capability of the Cell-ViveTM GMP portfolio to support and develop sophisticated ex vivo processing workflows for NK cell-based therapies. Funding Source N/A Topic Categories Technological Innovations in Immunology (TECH)
A Novel Hybrid Biological-Biomaterial Vector for Antigen Delivery and Human Dendritic Cell Maturation 2306812
Abstract Introduction The use of antigen (Ag) presenting dendritic cells (DC) strategy can prompt effective and personalized anti-tumor immune responses with little immune-mediated toxicity. However, current formulations have resulted in clinical objective responses amongst only a small subset of patients. Current strategies rely on cytokine-based maturation cocktails that result in heterogenous maturation of DCs as measured by costimulatory molecule expression. We propose a novel hybrid vector (HV) that utilizes a bacterial core encased in a polymeric shell to deliver plasmid DNA (pDNA)-encoded Ag, resulting in simultaneous delivery of maturation stimuli and target Ag to be durably expressed within DC. We hypothesize that this method will improve DC maturation and resultant Ag-specific T cell expansion, thereby improving antitumor responses. Methods To evaluate the effect of the HV, human monocytes isolated from whole blood were differentiated for 7 days with GM-CSF and IL-4 into monocyte-derived DC and subsequently co-cultured with HV constructs or a maturation cytokine cocktail of IL-1β, TNFα, IL-6, and PGE2. Following stimulation, DC were directly analyzed via flow cytometry or co-cultured with T cells before analysis. Results HV-conditioned DC showed a 1.9-fold increase in CD80 expression and a 2.7-fold increase in CD83 expression after 48 hours compared to standard DC differentiation protocols. Using plasmid-encoded fluorescent reporters, expression of pDNA-encoded protein was detected via imaging flow cytometry and fluorescent microscopy only in HV-conditioned DC, suggesting successful pDNA translocation and expression within DC. DC conditioned with the HV encoding MART-1 Ag promoted a 4-fold increase in the expansion of MART-1-specific T cells when co-cultured compared to a plasmid control. Conclusion The HV provides a novel and highly modifiable platform for both enhancing DC maturation and Ag presentation while also offering mechanistic insight into both DC biology and resultant immune responses. Funding Source NIH R01 CA270155 Topic Categories Vaccines and Immunotherapy (VAC)
Type 2 innate lymphoid cells play a diverse role in breast cancer development and progression 2258430
Abstract Introduction The tumor microenvironment (TME) is a crucial regulator of breast cancer (BC) development. It comprises acellular and cellular factors, including immune cells like type 2 innate lymphoid cells (ILC2s). We aim to understand the role of ILC2s in the BC TME and whether they can be therapeutically targeted. Methods To reveal the ILC2 phenotype in BC, we used the polyoma middle T antigen transgenic mouse model, which mimics luminal BC development. Results Single-cell (sc)RNA sequencing and flow cytometry experiments showed that ILC2s (expressing Gata3, Rora, Il2ra, and Il7ra) accumulated in the mammary fat pad (MFP) during BC development, and ILC2s phenotypically changed during tumor development by increasing PD-1 and Neuropilin-1 expression. Next, we tested if ILC2s directly regulate cancer cell growth in vitro. Co-culturing the BC cell line ‘VO’ with healthy MFP or tumor ILC2s for 48 hours increased the number of viable VO cells. To assess the functional relevance of ILC2s in BC in vivo, we co-injected ILC2s and VO cells orthotopically into FVB mice and measured the tumor growth. While 85% of the mice co-injected with VO cells and ILC2s developed a tumor, 50% of the VO-only recipients rejected the tumor cells. ILC2 co-injection increased infiltration of Gr-1+CD11b+ neutrophils, which could inhibit anti-cancer immunity. Injecting the breast cancer cell line ‘Py8119’ into the healthy MFP of control and ILC2-deficient mice resulted in similar findings, with smaller tumors in mice lacking ILC2s. Conversely, when we injected the ILC2-activator IL-33 into Py8119 tumor-bearing mice, the tumors shrank massively and were full of ILC2s. These results indicate distinct ILC2 states, making ILC2s context-dependent, either pro- or anti-tumorigenic. Conclusion We showed that ILC2s accumulate, undergo phenotypic changes, and promote an immunoinhibitory environment during BC development. Notably, we can modify the ILC2 phenotype, making them anti-tumorigenic and a promising target for new therapeutic approaches. Funding Source Hewitt Foundation for Biomedical Research Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Antigen processing of Mycobacterium bovis BCG vaccine substrains differs in mouse dendritic cells 2259586
Abstract Introduction The BCG vaccine given at birth varies in its protective efficacy against tuberculosis that kills millions of people. We hypothesized that this could be due to a heterogeneity among BCG sub strains. Therefore, we examined their ex vivo immunogenicity using dendritic cells. Methods The DC.2.4 cell line derived from C57BL/6 mice were infected with Pasteur, Copenhagen, Japan, Moreau, and Russian strains, followed by an assay for their ability to present Antigen85B-derived peptide to CD4 T cells triggering IL-2; RNAseq analysis for gene expression; and proteomics. Results 1) Antigen presentation by Copenhagen infected DCs to T cells was better than others. 2) RNAseq data analyzed using KEGG, GO and Reactome analyses indicated that Copenhagen infected DCs showed a better enrichment of genes Involved in antigen processing, autophagy and cytokine-chemokine modules compared to others. 3) Proteomics confirmed that the antigen processing machinery (Cathepsins, MHC and autophagy-mediating proteins) was differentially upregulated; Copenhagen showed the greatest enrichment followed by Russian, Moreau, Pasteur and Japan. Conclusion We propose that BCG sub strains differ in DC-dependent T cell activation, likely to affect protection against tuberculosis. Funding Source NIH NIAID R01-AI-I87440 Topic Categories Vaccines and Immunotherapy (VAC)
Dual‐Structural‐Unit‐Designed Bismuth Titanium Oxides: −70 to 80°C Ultra‐Wide Temperature Adaptability and 24 000‐Cycle Stability for High‐Rate Sodium‐Ion Storage
ABSTRACT Sodium‐ion batteries are naturally suitable for large‐scale energy storage due to the low cost, yet the high‐performance anode material remains a key challenge. Alloy‐type bismuth (Bi)‐based anodes have high theoretical capacity and an appropriate operating voltage plateau, but the main obstacles are cycle life, rate, and wide operating temperature. This work proposes an innovative design of Bi‐based anode materials, where Bi serves as the sodium storage unit and is integrated with a robust Ti‐O framework of excellent Na + diffusion ability. Different compounds and synthesis/modified methods are systematically investigated to optimize crystal structure, size, morphology, and conductivity. The optimal Bi 4 Ti 3 O 12 −x delivers a reversible capacity of 330 mAh g −1 at 0.5 C, 132 mAh g −1 at 150 C, and 70 mAh g −1 even at 250 C. Besides, it can operate under an ultra‐wide temperature range from −70°C to 80°C. A reversible charge/discharge capacity of 151 mAh g −1 at −55°C is achieved. The capacity retention is 97% (25°C, 20 C) and 61% (−40°C, 5 C) over 24 000 cycles. The proposed design of incorporating alloy materials as sodium storage units into the stable host framework unit is expected to complement the effective strategy of practical low‐temperature fast‐charging electrodes.
Olfactory Receptor 2 Signaling in Monocytes Facilitates Chemotactic Migration to Atherosclerotic Aorta Due to Octanal from Dysfunctional Endothelium 2259390
Abstract Introduction Atherosclerosis is a chronic inflammatory disease of the arterial wall initiated by monocyte recruitment and foamy macrophage accumulation. Human and mouse vascular macrophages, mouse bone marrow-derived macrophages (BMDMs), and monocytes express several Olfactory Receptors (ORs), including Olfr2 and its human orthologue OR6A2. Although, its role in monocytes is unknown. Ligation of Olfr2/OR6A2 with their ligand, Octanal, activates the NLRP3 inflammasome pathway and exacerbates atherosclerosis, while Olfr2 KO mice reduces atherosclerosis progression in vivo. Additionally, the original source of Octanal is not yet defined. Due to Endothelial Dysfunction (ED) being one of the first major steps in atherosclerosis initiation, we propose that Octanal is produced by lipid peroxidation by a dysfunctional endothelium, specifically through the ferroptosis pathway. Methods We employed Bulk RNAseq to characterize the potential functions of Olfr2 which revealed that Olfr2 may behave as a chemotactic receptor for monocytes. Thus, migration assays were conducted using mouse monocytes isolated from the bone marrow to test migration significance with and without Olfr2. To test if Octanal is derived from ED, we treated Human Aortic Endothelial Cells (HAECs) with Palmitic Acid, which is a major fatty acid precursor, and used that supernatant to test if monocytes would migrate. Results Bulk RNAseq identified Olfr2/OR6A2 protein expression in about 20% of monocytes, with expression increasing with disease in humans and mice. It also may enhance the chemotactic function of monocytes. Migration assays revealed that monocytes significantly migrate in response to Octanal, a response ablated in Olfr2 KO monocytes. We verified further with human monocytic cell lines, THP1 and Mono-Mac 6. Conclusion These experiments suggest that Octanal derived from ED could drive Olfr2-mediated chemotaxis of monocytes and macrophages, highlighting Olfr2 as a possible previously unrecognized chemotactic receptor and therapeutic target. Funding Source n/a Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)
Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity 2260408
Abstract Introduction Glioblastoma (GBM) is an aggressive primary brain cancer with minimal response to current therapies. An immunosuppressive tumor microenvironment limits immunotherapeutic efficacy. Astrocytes are the most abundant glial cells in the central nervous system and play important roles in locally regulating immune responses. However, in the context of GBM little is known about mechanisms regulating astrocytes or their roles in the modulating anti-tumor immune responses. Methods We used single-cell and bulk RNA sequencing of human GBM specimens and murine preclinical models, multiplexed immunofluorescence, in vivo cell-specific genetic perturbations via CRISPR and in vitro mouse and human experimental astrocyte and GBM models to address this gap in knowledge. Results We identified an astrocyte subset which limits T cell anti-tumor responses by inducing T cell apoptosis via TRAIL, a death receptor ligand. Further, we identified IL-11 produced by GBM cells as a driver of STAT3 signaling in this astrocyte subset and a regulator of TRAIL expression. Astrocyte STAT3 signaling and TRAIL expression were correlated with more aggressive tumor progression and decreased survival in GBM patients. Following in vivo genetic inactivation of TRAIL or of the IL-11 receptor in astrocytes we observed extended survival in GBM mouse models, alongside enhanced T cell and macrophage responses. Finally, we engineered an oncolytic HSV-1 virus to express a TRAIL-blocking single-chain antibody in the tumor microenvironment. TRAIL blockade enhanced therapeutic effect of oncolytic viruses, extended survival and enhanced tumor-specific immunity in preclinical models of GBM. Conclusion In summary, we establish that IL-11—STAT3 signaling drives astrocytes to suppress GBM-specific immune responses by inducing TRAIL-dependent T cell apoptosis, and engineered a therapeutic strategy leveraging oncolytic viruses as delivery vectors to target this mechanism of astrocyte-driven immunosuppression in the tumor microenvironment. Funding Source This work was supported by grants NS102807, ES02530, ES029136, AI126880 from the NIH; RG4111A1 and JF2161-A-5 from the NMSS; RSG-14-198-01-LIB from the American Cancer Society; and PA-1604-08459 from the International Progressive MS Alliance. This work was further supported by a scholarship from the German Academic Exchange Service (DAAD). Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Characterization of the B1 Cell Repertoire in Sickle Cell Disease Pre- and Post-Pneumococcal Vaccination 2258731
Abstract Introduction Rationale: Patients with sickle cell disease (SCD) are 600 times more susceptible to S. pneumoniae infections than the general population. This increased risk is mainly attributed to the absence of a functional spleen in SCD patients. However, those with SCD remain more susceptible than those without a functional spleen for reasons unrelated to SCD. Although vaccination has helped counteract this increased risk, pneumococcal infection remains a significant threat to patients with SCD. CD5+B1a cells produce natural antibodies that provide immediate protection against pneumococcal infections, whereas CD5-B1b cells produce protective adaptive antibodies in response to pneumococcal vaccination. Methods We examined the peritoneal and splenic CD5+B1a and CD5-B1b cell repertoires pre- and post-vaccination with Pneumovax23 (PPSV23) in mice with sickle cell disease (Townes model) and compared them with healthy controls. PPSV23 is a native vaccine that covers 23 pneumococcal serotypes and elicits a T-cell-independent response. CD5+B1a and CD5-B1b cells were single-cell sorted from mice with SCD and healthy controls (wild type) pre- and 14 days post-PPSV23 vaccination. The variable heavy chain (IgM) was amplified by PCR and sequenced. Results Single-cell BCR repertoire analysis demonstrated significant differences in VH use (notably VH11 and VH12) and the number of N-region additions (more N-additions pre- and post-vaccination in SCD mice) between wild-type and SCD mice. In mice with SCD, B1 cells over-utilized different identical CDR-H3 sequences compared to wild-type mice. Conclusion Our results revealed significant differences in the pre- and post-PPSV23 vaccination repertoire of B1 cells in SCD mice compared to healthy mice, offering insights into the effectiveness of pneumococcal vaccination in SCD. These results provide a rationale for optimizing vaccine strategies and developing targeted immunotherapies to enhance immune protection in patients with SCD. Funding Source Research reported was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R03AI123875. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Personalized Tumor Membrane Vesicle Vaccine Immunotherapy for Head and Neck Squamous Cell Carcinoma (HNSCC) 2253480
Abstract Introduction Head and neck cancer exhibits a high degree of heterogeneity, with lack of common therapeutic targets. We have developed an autologous therapeutic cancer vaccine approach using tumor-membrane vesicles (TMVs) prepared from surgically removed tumor tissue incorporated with glycolipid-anchored immunostimulatory molecules with B7.1 and IL-12. We hypothesize that tumors resistant to immune checkpoint inhibitor therapy (cold tumors) will be turned into hot tumor by TMV vaccine and induce response to anti-PD-1 antibody therapy in advanced metastatic HNSCC patients. Our phase 1 clinical trial (NCT06868433) will evaluate a novel personalized immunotherapy approach using TMV prepared from the patients’ own surgically excised tumor tissue and modifying them by attaching GPI-B7-1 and GPI-IL-12 as immune boosting molecules. Methods A total of 10 HNSCC tumor specimens were collected (0.29-1.23 grams) and graded for cellularity by a pathologist. Tumor samples were then homogenized, and homogenate was ultracentrifuged over a sucrose gradient to enrich TMV. TMV preparations were incorporated with GPI-B7-1 and GPI-IL-12 to generate the TMV vaccine. We used established murine oral cancer models to investigate the protective anti-tumor immune response of TMV vaccine in combination with anti-PD1 antibody therapy. Spleens were flash frozen in liquid nitrogen and stored in -80oC for RNA-seq analysis. Results TMV yields were 2.5 — 5 mg/gram of tissue from patient samples. Further refinement of the TMV production process yielded up to 6 mg of TMV/gram of tumor. We have just begun enrolling patients in the phase 1 trial and banking tumor for vaccine production if they ever become eligible to receive the vaccine to treat recurrent/metastatic disease. RNA-seq data from mouse model suggests that the TMV vaccine showed synergistic activity when combined with anti-PD1 antibody, with enhanced T cell responses. Conclusion TMV vaccine synergizes with anti-PD1 in inducing anti-tumor responses. Funding Source Supported by NIH/NCI 1R01CA262123-01A1 to DMS & PS. Topic Categories Vaccines and Immunotherapy (VAC)
Multi-omic analysis of long COVID patients reveals obesity-associated metabolic dysfunction and inflammation in adipose-residing immune cells 2334840
Abstract Introduction Long COVID is a chronic disease with potentially debilitating symptoms that affects millions of patients worldwide. Affected individuals often suffer from chronic inflammation and metabolic dysfunction, which may drive the chronic fatigue and brain fog characteristic of long COVID. Given the severity of symptoms and the high number of affected patients, understanding the causes and risk factors of this condition is crucial. Obesity is a known comorbidity for long COVID and drives immune and metabolic dysregulation. Thus, we hypothesized that obesity would exacerbate the underlying processes that contribute to long COVID. Methods To address this hypothesis, we performed multi-omic analyses on a cohort of patients with and without long COVID (LC+/-. These patients were then analyzed across BMI ranges (20-62). We performed serum proteomics and metabolomics. scRNAseq was also performed to determine the transcriptional profile of PBMCs and immune cells derived from subcutaneous adipose tissue in these patients. Results Our analysis revealed elevated levels of pro-inflammatory molecules in the serum of LC+ individuals. Furthermore, we observed enrichment of inflammatory gene signatures in a subset of PBMCs in LC+ patients and evidence of metabolic dysfunction. We next examined the adipose-residing immune cells where we observed widespread enrichment of inflammatory and anti-viral signatures in the LC+ individuals. Of note, the enrichment and breadth of these signatures correlated with patient BMI, as LC+ patients with higher BMIs appeared to have more widespread anti-viral signatures and metabolic dysfunction. Conclusion Taken together our data indicate that obesity exacerbates the immune and metabolic dysfunction that are hallmarks of long COVID. Given that patients with obesity have greater adiposity these data suggest that targeting adiposity may be an effective strategy in treatment of long COVID. Indeed, preliminary data suggests that GLP-1 agonists can provide relief for long COVID patients. Funding Source NIH RECOVER initiative Topic Categories Computational and Systems Immunology (COMP)
Developing Novel Bispecific T-cell Engager Therapy by Concurrent Targeting GARP on Cancer Cells and Regulatory T cells 2258672
Abstract Introduction While cancer immunotherapies have transformed treatment outcomes, most patients fail to respond primarily due to immune resistance mechanisms. Targeting tumor antigens along with suppressive factors may overcome such barriers. Glycoprotein A repetitions predominant (GARP) is a cell surface receptor that anchors latent transforming growth factor beta and contributes to immunosuppression, with its expression mostly to regulatory T cells (Tregs), cancer cells, and platelets. This study examines the therapeutic impact of engaging effector T cells to target GARP-expressing tumor cells along with Tregs, utilizing our unique anti-GARP antibody (PIIO-1) that targets tumors and Tregs but spares platelets. Methods We developed an anti-human GARP/CD3 bispecific T-cell Engager (BiTCE) with a tandem single-chain variable fragment construct. Human T cells armed with BiTCE were co-cultured with GARP+ tumor cells to assess cytotoxicity. In vivo efficacy and safety were evaluated using both immunocompromised and immunocompetent preclinical models that represent highly refractory diffuse large B-cell lymphoma, acute lymphoblastic leukemia, and glioblastoma. Importantly, we utilized systemic adeno-associated virus-mediated BiTCE delivery in vivo to overcome the pharmacokinetic challenges of short half-life of the typical BiTCE proteins without Fc. Results We demonstrate that elevated GARP expression in human tumors correlates with poor survival and higher relapse rates after first-line therapies. Our anti-GARP/CD3 BiTCE shows strong in vitro and in vivo efficacy against both GARP-expressing hematologic and solid tumor models without significant toxicity. Conclusion Our study provides a novel BiTCE therapeutic platform that enables us to achieve long-term tumor control and overcome tumor resistance by engaging CD3 on effector T cells to target GARP-expressing cancer and Tregs. The Anti-GARP/CD3 platform therefore holds the promise as a novel therapy of cancer immunotherapy worthy for clinical translation. Funding Source NIH R01 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
The Role of NK Cells in Cutaneous Wound Healing 2259529
Abstract Introduction Chronic poor wound healing is a significant global health burden, contributing to morbidity and mortality. Natural killer (NK) cells are innate lymphoid cells that possess both cytotoxic and infection-clearing roles. In the context of tissue repair, NK cells have been shown to have interactions with endothelial cells in the modulation of fibrosis and in the process of re-epithelialization. Consequently, NK cells may have the potential to influence the process of wound healing. The few studies that have examined NK cells’ role in wound healing have generated conflicting results, with one study reporting that NK cell ablation accelerates wound closure while another stated that NK cells have a positive role by limiting the acute inflammatory reaction to wounding. We investigate the role of NK cells as regulators of wound healing using multiple mouse models of skin injury. Methods Our data established the kinetics of NK cell recruitment in the wound with flow cytometry and characterized a unique phenotype that wound NK cells possess. Two mouse models of wound closure, a dorsal punch biopsy and tail-excisional wound, were used in conjunction with a NK-depleting antibody to measure the rate of wound closure in the absence of NK cells. Results The tail-excisional model, which heals primarily by re- epithelialization, demonstrated that wound closure is slower in the absence of NK cells. However, in the punch biopsy model, which relies on the contraction of skin to heal, NK depletion had no significant impact on the rate of wound closure. Using a model that allowed for the analysis of wound cells and fluid, we found that the growth factors EGF and VEGF were increased in the absence of NK cells. RNAseq data of NK-depleted mice showed the overexpression of collagen production genes related to skin and vasculature development. Conclusion This work contributes to the wound healing field by furthering insights regarding NK cells’ influence on wound closure, re-epithelialization, and vascularization. Funding Source Defense Advanced Research Projects Agency (DARPA), Dean’s Areas of Emerging New Science Award (Brown U.), NHLBI R01HL126887, Carney Institute Innovation Awards, R01HL165259-01A1 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)
Heterogeneous Indirect Redox Electrocatalysis in Electrolyte
ABSTRACT Heterogeneous indirect electrocatalysis has emerged as a promising electrochemical transformation technology, demonstrating broad application potential in diverse molecular conversions. By utilizing heterogeneous redox mediators as electron shuttles or catalysts to promote efficient electron transfer and substrate activation, this approach effectively addresses critical challenges inherent in conventional electrolysis technologies such as high overpotential, low selectivity, electrode deactivation, and difficulty in catalyst recovery. Although novel indirect electrocatalysis systems mediated by heterogeneous redox media have been continuously developed, related comprehensive reviews are still rarely reported. Therefore, this review comprehensively summarizes the design concepts, system compositions (e.g., reactor, electrode, electrolyte and redox mediators), the type of heterogeneous redox mediators (e.g., foams, polymers, COFs, MOFs and POMOFs), and relevant catalytic applications of heterogeneous indirect electrocatalysis. Particular emphasis is placed on discussing the selection criteria, reaction mechanism and performance optimization strategies. Meanwhile, the characteristics, challenges, and perspectives of heterogeneous indirect electrocatalysis are reviewed, and their unsolved problems and possible developments are discussed.