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Bispecific T Cell Engagers Sensitize the Glioblastoma Tumor Microenvironment to Immune Checkpoint Blockade Responsiveness 2261116
Abstract Introduction Glioblastoma (GBM) is the most aggressive brain tumor, accounting for ∼48% of all brain malignancies in children. Despite aggressive treatments, median survival remains only 12—15 months, with < 5% patients surviving beyond 5 years. Although several immunotherapies are currently in clinical trials, none have yet been approved. Locoregional treatment with personalized CAR-T cells has shown modest success. Moreover, immune checkpoint blockade (ICB) therapies are largely ineffective in GBM. Here, we explored Bispecific T cell engagers (BTE) — off-the-shelf therapeutics that connect pre-existing endogenous T cells with tumor targets — as a potential new immunotherapy to sensitize ICB responsiveness in GBM. Methods We used CD3:B7H3 BTEs to engage endogenous T cells through CD3 and tumor targets through B7H3, a tumor-associated antigen highly expressed on GBM. Preclinical immunocompetent murine models of syngeneic orthotopic immunologically distinct “hot” and “cold” GBM were treated intracranially with BTEs to bypass the challenge posed by blood-brain-barrier. Results BTE treatment led to robust expansion and effector differentiation of CD8 T cells, with increased effector T cell:Treg ratios and early tumor control. However, tumor control was not sustained, and correlated with loss of effector CD8 T cells and upregulation of PDL1 in the TME myeloid compartment. Converting this immune evasion mechanism into a tumor vulnerability, combination BTE therapy with ICB significantly enhanced tumor control, with improved 2X survival in 50% of the cohort. Tumor regression was associated with complete remodeling of the TME from immunosuppressive to more immunogenic with increased CD8 T cells, decreased Tregs, decreased myeloid-derived suppressor cells and increased activated microglia. Conclusion These studies set the groundwork for BTE immunotherapy in treatment refractory GBM and pose a unique window of opportunity for established ICB immunotherapies to synergize for long-term remission and overall survival. Funding Source This work was supported by the National Institutes of Health (National Cancer Institute), Pediatric Cancer Research Foundation and the Mark Foundation Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
The transcription factor IRF8 promotes the survival of differentiated effector CD8 T cells 2254435
Abstract Introduction CD8+ T cells depend on IL-2 signals to drive effector differentiation and sustain clonal expansion, yet how IL-2—responsive transcription factors coordinate these outcomes remains incompletely defined. The transcription factor IRF8, previously known for roles in myeloid and B-cell lineage reinforcement, is highly sensitive to IL-2 signals in CD8+ T cells. We hypothesized that IRF8 integrates IL-2—dependent transcriptional programs to support effector differentiation in CD8+ T cells. Methods Equal numbers of congenically marked WT and IRF8-deficient cells (generated by conditional deletion or CRISPR/Cas9 editing) were co-transferred into recipients that were either given acute (Listeria-OVA) or chronic (B16-OVA, MC38-OVA bearing hosts) antigen challenges. Effector differentiation, persistence, and function were evaluated by flow cytometry Results While IRF8 expression is minimal at the peak of the acute response, CTLs generated during Listeria-OVA infection induce IRF8 when cultured in the presence of IL-2 in a dose-dependent manner. IRF8-deficient cells form fewer SLECs at day 7 p.i., and exhibit a stark defect in persistence during the contraction phase. In chronic antigen models, significantly fewer IRF8-deficient cells are recoverable from the tumor, tumor-draining lymph node, and spleens of B16-OVA and MC38-OVA bearing mice compared to WT. This finding is supported by higher levels of the pro-apoptotic protein Bim in these cells. Conversely, forced overexpression of IRF8 in adoptively transferred cells in this model provides a competitive survival advantage indicated by greater numbers and lower expression of Bim over WT. Conclusion These findings position IRF8 as a cytokine-sensitive transcription factor that orchestrates the transcriptional control of apoptotic restraint, promoting sustained fitness and survival in effector CD8 T cells. Our work highlights the potential for overexpression of IRF8 during adoptive cell therapy regimens as a strategy for enhancing their long-term persistence. Funding Source CU School of Medicine Startup Award NIH R01A01043957 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
High-resolution spatial mapping of cell state and lineage dynamics in the tumor microenvironment with PEtracer 2302205
Abstract Introduction The tumor microenvironment is a complex ecosystem of cancer, immune, and stromal cells. Within this environment, cancer progression is coordinated by both cell-intrinsic and -extrinsic factors. Targeting signaling interactions between cancer and immune cells has proven to be an effective therapeutic strategy, as evidenced by the remarkable success of cancer immunotherapies. However, clinical responses vary across patients and tumor types. Developing effective cancer therapies and defining critical windows for clinical intervention requires a detailed understanding of the phenotypic and spatiotemporal dynamics of the cell types that make up the tumor microenvironment. Methods To enable high-resolution reconstruction of cell fate decisions in the tumor microenvironment, we developed PEtracer, a prime editing-based lineage tracing system compatible with single-cell sequencing and multimodal imaging, allowing for joint analysis of cell state and lineage in dissociated cells or in intact tissues with high spatial resolution. Results Using PEtracer coupled with single-cell and spatial transcriptomic profiling in syngeneic mouse models of cancer, we reconstructed the growth of individually seeded tumors in vivo, uncovered distinct modules of cell-intrinsic and -extrinsic factors that coordinate tumor growth, and mapped the recruitment and differentiation dynamics of tumor-infiltrating immune cells. Conclusion PEtracer enables high-resolution reconstruction of cell state and lineage dynamics within the tumor microenvironment in vivo. By leveraging prime editing-based lineage recording and multimodal imaging, PEtracer allows systematic dissection of how tumor, immune, and stromal cells evolve during cancer progression and therapeutic response. The resulting spatially resolved multimodal datasets provide a foundation for predictive models of tumor microenvironment dynamics. Funding Source National Institutes of Health Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
N. brasiliensis priming in three different inbred strains of mice provides protection to influenza A virus challenge in a strain dependent manner 2307446
Abstract Introduction N. brasiliensis (Nb) is a helminth that induces extensive type 2 remodeling in the lung due to its migration route through the host. Recent studies have investigated how exposure to helminths alters the immune response to challenge viral infection, but genetic variation is rarely studied. The aim of this study was to investigate how genetic variation impacts helminth-induced remodeling and its effects on subsequent viral challenge utilizing influenza A virus. Methods Females of three different inbred strains of mice were used in this study: BALB/c, C57BL/6, and B10.BR, and were primed with 500 Nb larvae subcutaneously on day -28 and allowed to recover naturally before being infected with influenza A/PR/8 virus on day 0. Mice were monitored for 10 days for weight loss and survival. Mice were also taken down on day 0 following priming to analyze the lung environment. Lung lobes were taken for histopathology, a multiplex cytokine and chemokine panel, and spectral flow cytometry. Results Priming with Nb was found to improve weight loss and survival following challenge with influenza A/PR/8 virus. However, there was strain-specific protection, namely BALB/c mice recovered much slower than C57BL/6 and B10.BR mice. Primed only mice showed varied lung environments that were distinct from each other. Particularly, BALB/c mice had decreased alternatively activated macrophages and dendritic cells, and increased Ly6G+ macrophages from spectral flow cytometry data. The histopathology also showed they had decreased inflammation and increased emphysema-like characteristics compared to the C57BL/6 and B10.BR mice. Conclusion Based on our study, the extent of protection to influenza A challenge infection in Nb-primed mice is dependent on genotype. These results expand our understanding on how the contribution of an individual’s infection history and genetics can influence the response to subsequent viral infection. Funding Source 1ZIAAI001386-01 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
SP2H selectively depletes Stimulator of Interferon Genes (STING), reducing inflammation and improving survival in Trex1-/- mice 2255319
Abstract Introduction STING contributes to the pathogenesis of autoimmune diseases, e.g. systemic sclerosis (SSc) and lupus (SLE). SP2H is a targeted STING degrader that depletes STING in cell lines and human fibroblasts (HFs), potently inhibiting interferon (IFN)-beta and chemokine expression induced by STING agonists, e.g. diABZI. Methods Proteomic analyses were performed in primary HFs after 26h exposure to 1 micromolar SP2H with and without exposure to 0.3 micromolar diABZI for the final 6h of incubation. C57Bl/6 and Trex1-/- mice were treated with SP2H subcutaneously (sc), either with single doses or once-daily in repeat dose studies. Trex1-/- mice were randomized by sex, body weight, serum anti-double stranded DNA antibody titre and serum lactate dehydrogenase level before the initiation of treatment at age 6 weeks. Chemokines and cytokines in mouse serum samples were quantified by ELISA. Results HFs incubated with SP2H showed selective STING depletion (>95%) and potent inhibition of diABZI-induced inflammatory protein expression, e.g. ISG15 and COX-2. When given to mice 16h before an iv dose of diABZI, a single sc dose of 3mg/kg SP2H resulted in 1.6-, 2.3-, 2.7- and >1.2log10 reductions in serum CXCL10, CCL2, IL-6, and IFN-beta (all p < 0.01). Immunohistochemistry with AI-assisted quantification showed large and significant reductions in STING levels in lung, kidney and other tissues from mice treated for 14d with SP2H compared to controls. Treatment of Trex1-/- mice with 0.3 or 3mg/kg SP2H sc qd for 8 weeks was well tolerated and improved overall survival compared to control (Kaplan-Meier, p = 0.0494), with 100% survival at the 3mg/kg dose. This dose lowered serum CXCL10 and TNF-alpha by > 4.5-fold compared to the control arm and significantly reduced inflammatory gene expression (Cxcl10, Tnfa, Ifnb, Isg15, Ifi44) in the heart at the end of treatment. Conclusion Given its excellent biological and safety profile, SP2H is being developed for diseases involving pathological STING activation including SSc and SLE. Funding Source Sulis Therapeutics Topic Categories Therapeutic Approaches to Autoimmunity (THER)
DNA‐Regulated Catalytic‐Plasmonic Colocalization Enables Synergistic Signal Amplification in SERS and Nanozyme‐Based Sensors
ABSTRACT The critical need for accessible disease monitoring underscores the urgency of developing advanced point‐of‐care testing (POCT). We present a DNA‐regulated catalytic‐plasmonic colocalization‐based synergy coupling mechanism that resolved the spatiotemporal disjunction commonly present in conventional SERS‐catalysis systems through programmed spatial confinement. Rolling circle amplification‐derived DNA nanocages were employed as programmable spatial regulators to precisely position Au@Pt nanozymes within plasmonic hotspots via base pair encoded hybridization, thereby enforcing spatial and temporal consistency between catalytic generation of Raman‐active species and electromagnetic field enhancement. This strategy integrated coordinated interface, pore, and interlayer confinement, enabling cross‐scale signal amplification from molecular to microscale levels. As a result, it yielded exceptional SERS enhancement (an approximate 41‐fold versus controls), sensitivity (10 2 exosomes µL −1 ), and reproducibility (6.5% RSD). To translate this mechanism into practical application, a portable dual‐modal detection platform with potential point‐of‐care applicability was developed that preserved catalytic‐plasmonic colocalization during both colorimetric screening and SERS quantification. The device achieved radical miniaturization (95% volume, 91% weight reduction) and cost‐efficiency (90% reduction vs. commercial systems). By coupling programmable nanomaterial design with customizable device engineering, we established a robust paradigm for next‐generation POCT, providing a promising platform for biomedical detection, environmental surveillance, and food safety monitoring.
Ori-TTV particles enable potent in vivo CAR-T cell engineering for the treatment of hematologic malignancies and autoimmune diseases 2256860
Abstract Introduction Chimeric antigen receptor (CAR) T cell therapy has significantly advanced treatment of hematologic malignancies, including B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL), and multiple myeloma (MM). However, conventional autologous CAR-T therapies are hampered by logistical complexity, high costs, and lengthy manufacturing timelines, limiting accessibility–particularly for patients with aggressive disease. To address these challenges, we developed an in vivo CAR-T platform that directly engineers functional CAR T cells within the body, eliminating the need for ex vivo manipulation. Methods Here we report the development and validation of Ori-TTV, a rationally designed T cell—targeting lentiviral vector optimized for safe and efficient in vivo CAR-T generation. Key innovations of Ori-TTV include: (1) an Ori Multiple Targeting Domain (Ori-MTD) for selective T cell transduction; (2) a synthetic T cell—specific promoter for lineage-restricted CAR expression; (3) a phagocytosis-resistant modification to evade macrophage clearance; and (4) a de-targeted fusogen to enhance safety and reduce off-target effects. These features enable selective in vivo reprogramming of T cells. Results Ori-TTV encodes a dual-target CAR recognizing both CD19 and BCMA, facilitating simultaneous elimination of malignant B and plasma cells. This expands therapeutic potential across relapsed/refractory B-ALL, NHL, and MM, and B/plasma cell—driven autoimmune disorders such as systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), and systemic sclerosis (SSc). In vitro, the T cell—specific promoter drives CAR expression at levels comparable to EF-1α, supporting robust and multi-round cytotoxicity against CD19+ and BCMA+ cells. In vivo, Ori-TTV administration induced significant and durable tumor regression in humanized PBMC and CD34+ mouse models. Conclusion In summary, Ori-TTV offers a safe, scalable, cost-effective platform to broaden access to CAR-T immunotherapy. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
A bivalent multi-adjuvant Chlamydia vaccine targeting secreted and outer membrane antigens reduces genital infection and pathology in mice 2257258
Abstract Introduction Chlamydia trachomatis is the most common bacterial STI and a major cause of hydrosalpinx and infertility, making a vaccine urgently needed. We developed a vaccine by conjugating the secreted Chlamydial Protease-like Activity Factor (CPAF) to the STING agonist CL1151 (CPAF-CL1151). Although intramuscular (i.m.) immunization elicited strong Th1 responses, it did not prevent oviduct pathology following C. muridarum infection. We hypothesized that co-delivery of CPAF-CL1151 with the Major Outer Membrane Protein (MOMP) and additional adjuvants (CpG1826 and QS-21) via combined i.m./intranasal (i.n.) routes would enhance mucosal and systemic Th1 immunity and protection. Methods Female C57BL/6 mice were immunized with CPAF-CL1151 ± QS-21 (i.m.) or CPAF-CL1151 + QS-21 ± MOMP ± CpG1826 (i.m.+i.n.). Antigen-specific T-cell responses were assessed by IFNγ/IL-17A ELISpot and flow cytometry, and serum IgG by ELISA. After challenge, cervical chlamydia burden was quantified by inclusion forming unit assay, and oviduct hydrosalpinx was assessed macroscopically. Results CPAF-CL1151 + QS-21 i.m. immunization increased IFNγTNFα CD4+ T cells 2.4-fold compared to CPAF-CL1151 alone. The combined i.m.+i.n. CPAF-CL1151 + MOMP + CpG + QS-21 regimen further doubled this population. MOMP-specific CD4+ T-cell responses were modest (0.75% of CD4+ cells) compared to CPAF (28.7%), but high anti-Chlamydia IgG titers (∼106 endpoint) were generated. The bivalent triple-adjuvant vaccine reduced cervical burden by 1.3 log10 (P < 0.0001) and shortened infection by 11 days compared to PBS controls (P = 0.0002), and decreased hydrosalpinx by ∼50% relative to CPAF-CL1151 + QS-21. Conclusion Thus, a bivalent CPAF-MOMP vaccine delivered via i.m.+i.n. routes induces robust Th1 immunity, accelerates clearance, and reduces oviduct pathology. Ongoing studies aim to define the minimal antigen, adjuvant, and route combinations required for optimal protection. Funding Source NIAID F31AI194796, NIAID U01AI182180 Topic Categories Vaccines and Immunotherapy (VAC)
BCMA-CAR Natural Killer Cells Suppress Myeloma in Bone Marrow but Allow Emergence of Extramedullary Disease 2266680
Abstract Introduction Multiple myeloma (MM) is an aggressive blood cancer arising from plasma cells. B cell maturation antigen (BCMA)-targeted chimeric antigen receptor T cell (α-BCMA-CAR-T) immunotherapies currently provide life-saving treatment for MM patients. Unfortunately, severe toxicities along with the high cost and complexity of autologous CAR-T manufacturing remain important limitations. Novel research is underway to use CAR-expressing natural killer (NK) cells as an allogeneic CAR-T alternative, but studies have yet to evaluate long-term CAR-NK efficacy against MM. Methods NK cells were isolated, expanded via feeder-cell stimulation, and engineered to express α-BCMA-CAR and IL-15 co-expression. The functional characteristics of α-BCMA-CAR-IL15-expressing NK cells were initially assessed in vitro, followed by long-term testing in a luciferase-expressing MM-xenograft mouse model to examine the persistence and therapeutic effect of α-BCMA-CAR-IL15 NK. Results α-BCMA-CAR NK cells have enhanced cytokine production and cytotoxicity against BCMA-high MM cells compared to untransduced NK cells, with IL-15 co-expression required for CAR-NK persistence. When injected into NSG mice, both α-BCMA-CAR and IL-15 expression were required for persistent restriction of MM growth. Despite near complete and sustained elimination of MM in hematopoietic tissues, long-term assessment of mice treated with α-BCMA-CAR-IL15 NK cells revealed the emergence of extramedullary disease (EMD) in the form of BCMA-positive MM plasmacytomas. Conclusion This study showcases α-BCMA-CAR-IL15 NK cell therapy as a potent anti-MM therapeutic, achieving sustained MM elimination from the bone marrow and greatly extending survival in a MM-xenograft model. However, α-BCMA-CAR-IL15 NK cells appeared ineffective at eliminating extramedullary disease. By demonstrating the strengths and weaknesses of α-BCMA-CAR-IL15 cells, our study provides a valuable pre-clinical MM model for studying and developing interventions for aggressive MM-EMD. Funding Source Canadian Institutes of Health Research Topic Categories Translational and Interventional Immunology (TI)
T cell dysfunction during Staphylococcus aureus osteomyelitis in humanized mice 2257659
Abstract Introduction Staphylococcus aureus is the leading cause of implant-associated osteomyelitis. It has a high recurrence rate, a low post-operative cure rate, and can lead to sepsis, multiorgan failure, and death. This indicates a need for future innovation in therapeutics and vaccines. With our humanized mouse model, we aimed to investigate the human T cell response during infection, as T cells play a crucial role in controlling bacterial growth during chronic infections. Using immunohistochemistry, we have found preliminary evidence of T cell dysfunction in the bone marrow niche. This has led to our hypothesis that CD4 T cells are becoming exhausted in the bone marrow due to chronic infection. Methods Female humanized NSG-SGM3 BLT mice (20-24 weeks old) underwent transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux) or sham surgery. At fourteen days post-infection, bone marrow cells were isolated and subjected to flow cytometry and single-cell RNA sequencing. Results In the bone, we observed an influx of Th1/Th17 cells through single-cell RNA sequencing. Using spectral flow cytometry, we observed increases in immune checkpoint proteins LAG-3, PD-1, and TIM-3 on CD4 T cells. Interestingly, we also discovered that TIM-3-positive CD4 T cells exhibited reduced Ki67 staining, suggesting potentially impaired functional capacity. To explore this further, we examined the effector profile (IFN-γ, IL-17A, and TNF-α) of these cells and observed diminished cytokine production in TIM-3-positive cells. Finally, we analyzed human serum from arthroplasty patients and found TIM-3 levels to be highly predictive of adverse outcomes. Conclusion These results suggest CD4 T cells may be dysfunctional and contribute to the chronicity of infection in S. aureus osteomyelitis. Ultimately, this work will provide novel mechanistic insights into bacteria-T cell interactions during S. aureus bone infections, hoping to inform better diagnostics and therapeutics. Funding Source NIAMS Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)
Effects of an Interactive Response Platform on Learning Outcomes in Medical and Pharmacy Students 2299728
Abstract Introduction Hotspot is an interactive audience response system, powered by PointSolutions technology, that allows students to answer questions by selecting areas of interest on images, diagrams, and figures, rather than choosing from standard multiple-choice options. This form of active learning is implemented to promote real-time engagement, higher-order questioning, and immediate feedback during lectures, thereby deepening understanding of complex concepts such as immunology. Methods Faculty at California Northstate University incorporated subject-related Hotspot questions into the lectures of their first-year medical and pharmacy courses across three different matriculating classes. Following these sessions, students completed an anonymous Likert scale survey administered electronically via SurveyMonkey. Survey items assessed student perceptions of engagement, learning, and ease of use on a 7-point Likert scale in this IRB-approved study. Results Students reported that Hotspot technology made it easier to pay attention during lectures (average Likert score of 5.93 out of 7, n = 102). They expressed interest in adding more Hotspot-style questions (5.70 out of 7, n = 102), and felt the questions were a good use of class time (6.18 out of 7, n = 102). Students also indicated that the interactive format challenged them and reinforced their understanding better than standard multiple-choice questions (5.67 out of 7, n = 102). Conclusion Students preferred Hotspot questions over traditional multiple-choice formats and believed they better prepared them for exams. They encouraged the integration of Hotspot technology and viewed it as convenient and highly effective. This study supports the incorporation of active learning strategies in education to enhance engagement, deepen comprehension, and improve lecture attendance. We hope our investigation encourages other educators to consider the use of unique, active-learning teaching styles to support future clinicians and scientists. Funding Source n/a Topic Categories Immunology Education and Communication (EDU)
Defining the role of Oncostatin M in lung homeostasis 2261154
Abstract Introduction As the primary site of gas exchange, the lung presents a large surface area that is continuously exposed to the external environment. To preserve lung function, cells within the alveolar niche must maintain homeostasis while defending against pathogens. Alveolar macrophages (AMs) and alveolar type II (ATII) epithelial cells collaborate through reciprocal signaling to preserve this delicate balance. Our previous work identified a novel role for macrophage-derived Oncostatin M (OSM) in restoring the epithelial barrier following viral-induced injury and revealed that OSM regulates epithelial transcriptional programs even at steady state. However, the mechanisms through which OSM maintains alveolar homeostasis are not yet fully defined. Methods We performed transcriptional profiling of ATII and AM populations isolated from wild-type and OSM-deficient mice at steady state, as well as following administration of exogenous OSM. Complementary analyses, including flow cytometry, immunofluorescence microscopy, ELISA, and cholesterol assays, were used to further characterize the alveolar niche. Results Loss of OSM altered the transcriptional states of both ATIIs and AMs. In ATIIs, genes regulating cholesterol homeostasis were downregulated, while lipid metabolism pathways were altered in AMs. Administration of exogenous OSM to the lung restored the transcriptional profiles of both cell types toward wild-type states. Conclusion These findings suggest that OSM helps maintain the steady-state alveolar niche by regulating cholesterol homeostasis through modulation of ATII cell states. Ongoing work will define how macrophages sense and respond to the alveolar environment to sustain epithelial homeostasis. Funding Source National Institute of Health (NIH) grant # R35GM150816 (RAF) and the Harvard FAS Dean’s Competitive Fund for Promising Scholarship (RAF). YLwas supported by NIH grant # TL1TR002543. Topic Categories Cytokines and Chemokines and their Receptors (CCR)
Characterizing the Roles of Dysferlin Deficiency in Function, Polarization, and Secretome of Human iPSC-Derived Macrophages 2251399
Abstract Introduction Limb-girdle muscular dystrophy R2/2B (LGMDR2/2B) is an untreatable and progressive late-onset skeletal muscle disease caused by the loss of a membrane-repair protein dysferlin. Even before disease symptom onset, LGMDR2 muscles are infiltrated by pro-inflammatory macrophages (MP), implicating immune cells in disease pathogenesis. While MPs express dysferlin, defining the cell-autonomous roles of dysferlin in MP function has been challenging in vivo due to complex multicellular interactions and altered microenvironment in LGMDR2 muscle. Methods To address this, we generated human induced pluripotent stem cell (hiPSC)-derived macrophages (iMPs) from three healthy and three LGMDR2 donors to delineate cell-autonomous roles of dysferlin in macrophage: 1) polarization, 2) transcriptional profile, 3) secretome, and 4) phagocytotic and endocytic function. Results Despite exhibiting comparable polarization under well-characterized pro- and anti-inflammatory cues, RNAseq analyses revealed downregulation of Gene Ontology terms related to cytokine secretion, phagocytosis, and receptor-mediated endocytosis in LGMDR2 iMPs. Proteomic analysis of iMP conditioned media revealed significant differences in 72 secreted proteins, including numerous chemokines, cytokines, and growth factors, suggesting an altered secretory phenotype. Functional assays found no significant differences in the phagocytosis of E. coli bioparticles or fluorescent myotube debris. However, receptor-mediated endocytosis of AcLDL was significantly lower in both M0 and M2 LGMDR2 vs. healthy iMPs. Pharmacological screens identified clathrin-dependent endocytosis as the primary pathway for AcLDL uptake in both genotypes, with altered clathrin trafficking and reduced scavenger receptor expression likely underlying LGMDR2 endocytic deficits. Conclusion Overall, dysferlin loss in iMPs results in cell-autonomously altered transcriptome, secretome, and endocytic function, which may contribute to LGMDR2 muscle pathology and disease progression. Funding Source Jain Foundation grant, NIH grant 1R01AR082979-01, National Science Foundation Graduate Research Fellowship Topic Categories Immune Mechanisms of Human Disease (HUM)
Mechanisms of CD070, an Active Compound from Chinese Herbal Medicine, in Regulating and Inhibiting PD-1 Expression 2257112
Abstract Introduction PD-1 antibodies have shown clinical success in cancer treatment, but limitations such as limited efficacy, immune-related side effects, and high costs highlight the need for alternative approaches. There is an urgent need to identify new agents that inhibit PD-1 signaling and directly reduce its expression. This study aims to identify compounds that downregulate PD-1 expression on CD3-stimulated T cells and explore their mechanisms. Methods This investigation focused on CD070; a bioactive compound extracted from Chinese herbal medicine. We used CD3-stimulated Jurkat T cells and peripheral blood mononuclear cells (PBMCs), which were isolated from three different donors, to assess the effects of CD070 on PD-1 expression by Flow cytometry and Western blotting. Seahorse extracellular acidification rate (ECAR) analysis was used to assess glycolytic activity, and 2-deoxyglucose (2-DG), a glycolysis inhibitor, was added to investigate the role of glycolysis in PD-1 regulation. Results Our results showed CD070 treatment significantly downregulated PD-1 protein expression induced by CD3-stimulated Jurkat cells. In PBMCs from all donors, CD070 also reduced PD-1 expression across the CD3+, CD4+, and CD8+ T cell populations. Notably, Seahorse ECAR analysis revealed CD070 inhibited glycolysis in T cells in a dose-dependent manner. When using 2-DG, it similarly reduced PD-1 expression in CD3-stimulated T cells, supporting the connection between glycolysis and PD-1 regulation. Moreover, we found that increasing glucose concentrations in glucose-free culture media led to increased PD-1 expression, further reinforcing the idea that glucose metabolism plays a critical role in immune checkpoint regulation. Conclusion This study shows that CD070 reduces PD-1 expression on CD3-stimulated T cells by modulating glycolysis, offering a novel metabolic approach to enhance cancer immunotherapy. Funding Source This work was partially financially supported by the National Science and Technology Council, Taiwan; and the “Chinese Medicine Research Center, China Medical University” from the Featured Areas Research Center Program within the framework of the Higher E Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Colon-Specific Signatures for Tissue Resident Memory T cells in Health and Colorectal Cancer 2258023
Abstract Introduction Tissue-resident memory T cells (TRM) are one of the predominant T cell subsets in the human intestine, yet their role in intestinal anti-tumor immunity remains poorly defined. In colorectal cancer (CRC), TRM are positively prognostic. However, their baseline features during homeostasis are not well delineated, limiting our understanding of their behavior within and in response to CRC. Here, we leverage our unique human organ donor tissue resource spanning multiple mucosal and lymphoid tissues to define colon TRM signatures across diverse host variables (age, sex, BMI, CMV serostatus, etc.) and how these programs are altered in CRC. Methods We performed CITE-seq profiling of colon and blood samples from 8 human organ donors, integrated with a multi-tissue in-house single-cell dataset (24 donors) and publicly available datasets from healthy colon, CRC, and CRC-adjacent tissue (73 donors). We conducted differential gene expression (DE) analysis comparing colon TRM to those in the small intestine (jejunum) and other mucosal tissues. DE results, in combination with single-cell hierarchical Poisson factorization, were used to define homeostatic colon TRM gene signatures that were then queried in disease states. Results We found that colon CD4 and CD8 TRM express genes associated with tissue residency, stemness and quiescence (GPR15, LEF1, BACH2, IKZF2, ZEB1, TGFBR3) at significantly higher levels than jejunum TRM. We found that expression of these gene programs was lost in tumor-infiltrating TRM in both microsatellite stable and microsatellite instable CRC subtypes. Additionally, we identified several tumor-specific TRM gene co-expression programs, including an innate-cytotoxic program in CD8+ TRM enriched in tumor-adjacent tissue, and a CD4+ TEMRA-like program enriched in CRC but also present in tumor-adjacent and healthy tissue. Conclusion These data establish a comprehensive framework for dissecting how the tissue environment and tumor context shape features of human colon TRM. Funding Source Herbert Irving Comprehensive Cancer Center Human Tissue Immunity Initiative Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
T-ALL Elicits Robust T Cell Responses Despite Low Tumor Mutational Burden in multiple tissues with distinct TMEs 2267295
Abstract Introduction T-cell acute lymphoblastic leukemia (T-ALL) is a devastating pediatric malignancy arising from immature T cells. Despite improved survival rates, treatment still relies on intensive, toxic chemotherapy that fails to cure all patients. More effective and targeted therapies are urgently needed. Most immunotherapy efforts in T-ALL have focused on engineered cells (e.g., CAR T cells), while strategies leveraging patients’ pre-existing anti-tumor T cells remain largely unexplored. This is partly due to the assumption that T-ALL’s low tumor mutational burden (TMB) limits immunogenicity. However, our data challenge this paradigm using both murine models and patient samples. Methods We used murine transplantation models in which spontaneous T-ALL from CD45.2+ LN3 or LMO2 mice were transferred into immune-competent CD45.1+ congenic hosts, allowing distinction between malignant and host T cells. OTI/II and NUR77-GFP mice were used to assess TCR-dependent activation. Results T-ALL-bearing mice showed high frequencies of TOX+PD-1+ T cells, consistent with chronic, antigen-specific activation. Ex vivo stimulation revealed that PD-1+ T cells retained function, with increased IFN-γ production. To confirm TCR specificity, we transplanted T-ALL into OT-I/II mice (OVA-specific), where exhausted phenotypes were not induced–supporting a TCR-dependent mechanism. NUR77-GFP mice further confirmed high TCR signal strength in T cells from T-ALL-bearing hosts. Finally, treatment with aPD-1 or aCD40 improved survival, with enhanced benefit in combination. Conclusion These findings provide proof-of-concept that robust, functional, leukemia-specific T cell responses are mounted against T-ALL despite low TMB. This challenges existing dogma and supports the development of immunotherapy strategies that activate endogenous T cells. Ongoing studies aim to define how divergent tumor microenvironments regulate these responses and identify targets for rational combination immunotherapy. Funding Source na Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Limited sustainability of regulatory T cell repertoire revealed by novel genetic switches 2257672
Abstract Introduction Regulatory T (Treg) cells carry a diverse T-cell antigen receptor (TCR) repertoire, playing a crucial role in suppressing inflammatory T cells reactive to self-antigens and innocuous foreign antigens. Despite their long lifespan, questions arise about how the diverse Treg repertoire is faithfully sustained during extended antigen stimulation. Methods In this study, we employ a novel genetic approach to demonstrate that Treg cells possess substantial stemness for self-renewal in native physiological conditions. Results The suppressive capacity of existing Treg cells declines over time, leading to impaired fitness and stability. Timestamp tracing further reveals constant thymic Treg development and influx into the peripheral Treg pool, which is essential for maintaining the integrity of Treg repertoire. Conclusion These findings demonstrate the limited sustainability of Treg repertoire, providing valuable guidance for Treg-based treatment of autoimmune disease. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)
A bacterial immunotherapeutic drives long-term immunity in Glioblastoma 2301770
Abstract Introduction Glioblastoma (GBM) is the most aggressive primary brain tumor, with most patients succumbing to disease within 2 years. Current standard of care is ineffective with recurrence within 6 months, partly due to weak anti-tumor immune responses. Despite successes in other cancers, immunotherapies have largely failed in GBM because of its inherently immune-excluded and immunosuppressive nature. We report a genetically attenuated Brucella melitensis strain (BmΔvjbR) that targets tumors and modulates their immune microenvironment. Prior studies in non-brain tumors showed that BmΔvjbR promotes polarization of myeloid cells to an activated state and increases the number and function of CD8+ T cells. Here, we tested the hypothesis that BmΔvjbR can induce durable anti-tumor immunity in GBM. Methods BmΔvjbR therapeutic efficacy was tested in a syngeneic orthotopic CT2A high grade glioma mouse model, transduced with luciferase. Mice received an intratumoral injection of either PBS or 1.10^8 CFU BmΔvjbR. Mice were followed for survival, and tumor growth was monitored using bioluminescence imaging. Survivors were rechallenged with the same cell line and compared to age-matched naïve controls. Results BmΔvjbR treatment reduced tumor development and achieved 50% long-term survival, compared to PBS controls. Upon rechallenge, previously cured mice cleared established tumors resulting in 100% survival (n = 3), whereas age-matched controls (n = 8) developed tumors and the majority died. Conclusion These preliminary findings indicate that BmΔvjbR can elicit durable and protective anti-tumor immunity in GBM, reducing tumor progression and improving survival upon rechallenge. Ongoing exploration holds promise for uncovering the full therapeutic potential of bacterial-based strategies for reprogramming the immunosuppressive environment in GBM and other cancers. Funding Source Siteman cancer center pilot grant Topic Categories Vaccines and Immunotherapy (VAC)
Butyrophilin-like 9 cooperates with JAM-A to drive anti-tumor immunity 2261194
Abstract Introduction Butyrophilin-like 9 (BTNL9), is downregulated across multiple cancer types, including ovarian cancer (OV), its decreased expression directly correlates with poorer patient outcomes, and it is known to bind T cells, suggesting a role in anti-tumor immunity. Methods To investigate this, in vitro, NY-ESO-1+ OV cells were transduced with BTNL9 ORF or empty plasmid and incubated overnight with antigen-specific T cells, and IFNy (ELISA) and cytotoxicity were measured. In vivo, BL/6 mice were implanted intraperitoneally with BTNL9-proficient or BTNL9-knockout OV tumors and were administered aCD8 antibodies or control IgG every three days while survival was recorded (n = 5 per condition). Additionally, we performed immunoprecipitation of BTNL9 from OV cells followed by LC-MS2 and identified BTNL9 binding partners. Results T cells incubated with cells overexpressing BTNL9 displayed significantly more IFNy and cytotoxicity (∼2 fold increase) while mice bearing BTNL9-proficient tumors survived significantly longer than those with BTNL9-deficient tumors; this advantage disappeared with CD8 T cell depletion. LC-MS2 identified the junctional adhesion molecule JAM-A as a binding partner of BTNL9. Blockade of JAM-A’s receptor, LFA-1, abrogates the stimulatory phenotype of BTNL9 in our in vitro assays. Conclusion This work serves to characterize a novel mechanism in OV by which BTNL9 enhances T cell stimulation via the JAM-A:LFA-1 interaction and provides new opportunities to potentially improve cancer immunotherapies. Funding Source NIH, NJCCR Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)
Exportin-5 Links miRNA Biogenesis to the Preservation of B Cell Receptor Surface Expression 2310271
Abstract Introduction The B cell receptor (BCR) dictates B lymphocyte identity and function, allowing for a robust adaptive immune response through antigen recognition. Strict regulation of BCR surface density dictates proper B cell signaling, immune regulation, and the prevention of malignancy, yet the factors regulating this density remain undefined. To systematically identify these regulators, we performed a genome-wide CRISPR interference (CRISPRi) screen in Ramos B cells, a human non-Hodgkin’s lymphoma cell line that undergoes constitutive somatic hypermutation (SHM) and identified Exportin-5 (XPO5) as a central regulator of BCR surface expression. Methods We transduced dCas9-KRAB Ramos cells with a genome-wide guide RNA library and isolated BCR-positive and -negative populations by flow cytometry. Hits were validated with individual guide RNAs. Mechanisms were investigated using bulk and small RNA sequencing, and BCR mutation frequencies were quantified via the TRUST4 pipeline. Results XPO5 deficient cells exhibited an accelerated loss of surface BCR with no change in transcript levels, suggesting a potential post-transcriptional regulatory mechanism. Further analysis revealed XPO5 depletion led to increased mutation rates specifically within the BCR light chain, implicating XPO5 in regulating the rate and fidelity of somatic hypermutation (SHM). Transcriptomic and small RNA sequencing revealed a global reduction in miRNA levels and enrichment of target gene sets indicative of cell cycle arrest and increased DNA damage response. Conclusion We identify XPO5 as a multi-faceted regulator of B cell identity. By maintaining the global miRNA landscape, XPO5 supports cell cycle progression and regulates the fidelity of somatic hypermutation. Loss of XPO5 uncouples these processes, driving light chain biased hypermutation and the loss of functional BCR surface expression. This highlights a novel link between miRNA nuclear export machinery and the preservation of BCR integrity. Funding Source n/a Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)