Browse Articles

Cross-gated feature interaction network for fetal abdominal circumference image segmentation

m6A RNA-binding Protein as a Novel Autoantigen in Rheumatoid Arthritis 2250120

Abstract Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. While several autoantigens have been implicated in RA, the role of m6A RNA modifications in autoimmunity remains unclear. Here, we identify the m6A RNA-binding protein as a novel autoantigen in RA. Methods Immunoprecipitation followed by Western blot revealed a distinct 63-kDa protein in RA patient serum but not in healthy controls. Both a monoclonal antibody (BR2) against the m6A RNA-binding protein and a commercial antibody showed strong immunostaining in RA synovium, particularly in fibroblast-like synoviocytes (FLS). Confocal microscopy confirmed colocalization of BR2 with the commercial antibody. Results The protein was also detected in RA synovial fluid by ELISA. Functionally, siRNA knockdown of the m6A RNA-binding protein, as well as treatment with BR2, reduced expression of proinflammatory mediators including TNF-α, IL-6, MMP-1, and MMP-3. Conclusion These results suggest that the m6A RNA-binding protein contributes to RA pathogenesis and may serve as both a biomarker and a potential therapeutic target. Funding Source (NSTC113-2622-B-039-002) Topic Categories Basic Autoimmunity (BA)

Dissecting Zika Virus pathogenesis through maternal-fetal immune interactions and sex-based susceptibility 2260838

Abstract Introduction Despite extensive research on ZIKV vertical transmission and its threat to pregnant women and their babies, the mechanisms by which maternal inflammation may contribute to developmental impairments in the offspring remain unclear. This is crucial given growing evidence that maternal immune activation can have long-term effects on the progeny. Considering this, we aimed to demonstrate that ZIKV-induced maternal inflammation is important for its pathogenesis in the offspring. Methods Wild-type (WT) females, resistant to ZIKV, were mated with hSTAT2 knock-in (KI) males, generating offspring expressing hSTAT2 in fetal-derived tissues but not in the mother, creating ZIKV-susceptible fetuses within a resistant maternal environment. Conversely, hSTAT2 KI females (susceptible to ZIKV) were mated with WT males, generating both hSTAT2 KI and WT within a susceptible maternal environment. Pregnant females were infected at embryonic day 10.5. Results In hSTAT2 KI placentas, viral load was comparable between susceptible and resistant dams. However, placentas from susceptible dams showed higher inflammatory markers and increased CD11c+MHC-II+ and CD8+ T cells. A pronounced sex difference in female offspring revealed higher viral load, greater pro-inflammatory cytokine expression, and elevated NK1.1+ and CD8+ T lymphocytes in the placenta. This increased female susceptibility may be linked to higher AXL and lower type III interferon receptor expression in the placenta. After birth, offspring from susceptible dams showed increased vocalization, with females most affected. By four weeks, female offspring exhibited a higher frequency of effector CD8+, CD4+ T cells, and NK cells in the spleen, indicating a sex-specific immune impact of prenatal ZIKV exposure. Conclusion Maternal inflammation and sex-based susceptibility are relevant factors in ZIKV pathogenesis, emphasizing the need for long-term monitoring of prenatally exposed children beyond Congenital Zika Syndrome. Funding Source n/a Topic Categories Viral Immunology (VIR)

Bee Venom Peptide Melittin Targets Mitochondrial Vulnerabilities in Melanoma via MT-CO1 Regulation 2266872

Abstract Introduction Melanoma cells undergo metabolic reprogramming that suppresses mitochondrial oxidative phosphorylation and enhances glycolytic dependence. Transcriptomic analysis shows strong downregulation of the mitochondrial gene MT-CO1, which encodes cytochrome c oxidase subunit I, in the A375 melanoma cell line. Melittin, a peptide from bee venom, has demonstrated potent lytic and pro-apoptotic effects in multiple cancer models through mitochondrial disruption. This project aims to determine whether melittin treatment alters MT-CO1 expression, induces mitochondrial stress, and reduces melanoma cell viability. Methods A375 melanoma cells will be treated with melittin at graded concentrations and time points. MT-CO1 mRNA and protein levels will be quantified by qPCR and Western blotting. Mitochondrial function will be assessed through ATP production and reactive oxygen species (ROS) assays. Computational modeling will predict melittin—membrane interactions and potential apoptotic pathways activated by mitochondrial stress. Results Preliminary analysis predicts that melittin will increase ROS accumulation, impair mitochondrial function, and partially restore MT-CO1 expression relative to untreated controls. Reduced cell viability is expected in a dose-dependent manner, reflecting enhanced sensitivity to mitochondrial disruption. Conclusion Targeting MT-CO1, a downregulated mitochondrial gene in melanoma, with melittin may exploit metabolic vulnerabilities in tumor cells. Melittin-induced mitochondrial stress could promote apoptosis and limit melanoma cell growth, offering a potential therapeutic avenue that integrates natural cytolytic peptides with metabolic targeting strategies. Funding Source n/a Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

TLR11/12-mediated regulation of optimal myelopoiesis during Toxoplasma gondii infection 2309722

Abstract Introduction While host resistance to systemic Type 1 infections depends on the egress of newly generated Ly6Chi inflammatory monocytes from bone marrow, this process remains poorly understood. Methods In this study, we performed a kinetic analysis of myeloid progenitors in bone marrow isolated from Toxoplasma gondii-infected wild-type mice alongside mice lacking major toxoplasma pattern recognition receptors TLR11 and TLR12, which display acute mortality and impaired monocyte recruitment to infected tissues. Results As early as day 4 post-infection, both wild-type and TLR11/12-/- mice exhibit significant loss of multipotent myeloid precursors (CMP, GMP, and MDP), indicating that these quantitative differences alone cannot explain distinct survival outcomes. However, the population of committed monocyte progenitors (cMoP), which also diminished initially, was subsequently recovered only in wild-type and not TLR11/12-/- mice. Moreover, intracellular staining revealed that cMoP in bone marrow from wild-type mice, concomitant with IFN-γ-induced Ly6A/E expression, also upregulated the transcription factors PU.1 and IRF4, known to positively regulate myelopoiesis. In contrast, the diminishing population of cMoP in bone marrow from TLR11/12-/- animals failed to upregulate PU.1 and IRF4 despite displaying an increased level of Ly6A/E. Conclusion Together, these findings indicate that the signaling cascade initiated by TLR11/12 activation plays a dual protective role during T. gondii infection: promoting innate inflammation and sustaining bone marrow myelopoiesis. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Functional Characterization of ZFC3H1 and DYNLT3 in A375 Melanoma Cells Using CRISPR Knockout 2266763

Abstract Introduction Melanoma progression is driven by transcriptional and cytoskeletal regulators that influence cell proliferation, migration, and immune evasion. ZFC3H1, a zinc finger RNA-binding protein involved in nuclear RNA decay, and DYNLT3, a dynein light-chain component implicated in intracellular transport and signaling, are both aberrantly expressed in melanoma according to public transcriptomic datasets. This study investigates the functional roles of ZFC3H1 and DYNLT3 in A375 melanoma cells to determine whether their dysregulation contributes to tumor growth or survival. Methods Differential expression analysis of RNA-seq data from melanoma and normal skin samples identified ZFC3H1 and DYNLT3 as upregulated genes. CRISPR-Cas9 knockouts were generated in A375 cells to assess loss-of-function effects. RNA extraction and cDNA synthesis confirmed knockout efficiency by qPCR. Cell viability and proliferation were evaluated using trypan blue exclusion and standard colorimetric assays. Ongoing assays are examining changes in morphology and expression of stress-response genes following knockout. Results Preliminary results confirmed robust expression of ZFC3H1 and DYNLT3 in wild-type A375 cells and successful CRISPR knockout in target lines. Early viability assays suggest reduced proliferation in DYNLT3-deficient cells, while ZFC3H1 knockout shows altered morphology consistent with cytoskeletal disruption. Additional analyses are underway to quantify transcriptional and phenotypic effects relative to control cells. Conclusion Loss-of-function analysis of ZFC3H1 and DYNLT3 in A375 melanoma cells provides new insight into their potential roles in tumor cell growth and regulation. These findings may help define RNA-processing and transport mechanisms that support melanoma progression and identify novel targets for therapeutic intervention. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

BRCA1 Expression and Its Role in Glioblastoma Growth and Survival 2258351

Abstract Introduction Glioblastoma multiforme (GBM) is one of the most aggressive brain cancers, characterized by rapid proliferation and poor clinical outcomes. The BRCA1 gene, classically associated with hereditary breast and ovarian cancers, has emerging relevance in brain tumors where its overexpression may enhance tumor cell survival. This study investigates the expression and functional role of BRCA1 in glioblastoma to determine whether its dysregulation contributes to tumor progression. Methods Public RNA-seq datasets were analyzed to compare BRCA1 expression in glioblastoma versus normal brain tissue. RNA was extracted from GBM cell lines, and qPCR was performed to validate expression patterns. A BRCA1 knockout was designed using CRISPR-Cas9 technology, and ongoing assays are assessing changes in proliferation and cell viability between wild-type and knockout lines. Results Computational analysis confirmed that BRCA1 expression is significantly elevated in glioblastoma relative to normal controls. Preliminary qPCR data validated this upregulation in cultured GBM cells. BRCA1 knockout studies are underway to evaluate how gene loss influences proliferation and survival, providing functional insight into its potential role in tumor maintenance. Conclusion Increased BRCA1 expression in glioblastoma supports its potential involvement in promoting tumor cell survival. Continued knockout experiments will clarify whether BRCA1 contributes to glioblastoma growth through mechanisms of enhanced proliferation or stress tolerance. These findings may inform future therapeutic strategies targeting BRCA1-associated pathways in brain cancer. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)

High-dimensional flow cytometry analysis reveals temporal dynamics of lung macrophages during Mycobacterium tuberculosis infection 2261143

Abstract Introduction Mycobacterium tuberculosis (Mtb) spends most of its lifespan within macrophages. During early infection, alveolar macrophages (AMs) facilitate rapid Mtb growth, whereas recruited monocyte-derived interstitial macrophages (IMs) restrict replication. However, macrophage subsets harboring Mtb in the presence of protective T cell—mediated adaptive immunity at the later stage of infection remain poorly defined. Many studies have relied on scRNA-seq to characterize macrophages without detailed phenotypic or functional analysis. Methods Our study aims to establish consensus on identifying lung macrophage subsets and analyze their dynamics and permissiveness across infection stages using high-dimensional flow cytometry and lineage tracing with scRNA-seq datasets. Results At 2 weeks post infection, CD11bhi AMs highly expressed CD63, CD38, and iNOS, consistent with a pro-inflammatory AM signature. Lineage tracing further confirmed the presence of monocyte-derived AMs. Four IM subsets were identified, with CCR2loCD11chiSca-1+ IMs being the predominant subset infected with Mtb. At 4 weeks post infection, six subsets of IMs were identified, and only three of them harbored Mtb. Notably, all three Mtb-infected IM subsets expressed high levels of CD11c, CD38, and iNOS. Conclusion These data provide a high-resolution analysis on heterogeneous lung macrophages and highlight shifts of their phenotypes during infection. It lays the foundation for future studies on how these macrophage alterations affect bacterial control. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

“Red Blood Cells are critical determinants of survival in radiation-induced hematologic injury” 2260138

Abstract Introduction Whole-body ionizing radiation induces acute hematopoietic injury that critically limits survival. While radiation-induced leukocyte loss has been extensively characterized, the role of red blood cell (RBC) depletion in determining mortality remains poorly understood. Because RBCs are vital cells of blood, studying RBCs following radiation can uncover key determinants of survival and therapeutic targets in hematopoietic injury. Methods Wild-type C57BL/6 mice (Male and Female, 8—10 weeks of age) were exposed to 5 Gy total body irradiation (TBI), and peripheral blood parameters were monitored longitudinally on day 0 to day 60 post-radiation. White blood cell (WBC), red blood cell (RBC), and hemoglobin levels were quantified at each time point using complete blood counts (CBCs). RBC morphology was examined by Wright—Giemsa staining to assess structural abnormalities. To evaluate the functional impact of RBC loss, an additional cohort of mice received lethal 6 Gy TBI and were transfused with RBCs on days 2 and 5 post-irradiation, while another group of mice received PBS as a control. Survival was monitored for all groups. Results WBC counts declined rapidly, showing >90% reduction by day 1, with recovery beginning after day 20. In contrast, RBC counts and hemoglobin levels exhibited a delayed but progressive decline, reaching their lowest levels around day 15. Radiation also caused distinct RBC morphological abnormalities, including poikilocytes, echinocytes, and schistocytes. The start of mortality in irradiated mice closely coincided with the loss of RBCs and hemoglobin. Remarkably, transfusion of RBCs on days 2 and 5 post-irradiation significantly improved survival, 40% lethality in transfused mice vs. 100% in controls; P = 0.0022. Conclusion These findings identify that RBC depletion is a major driver of radiation-induced mortality. Protection or restoration of RBCs strikingly improves survival, highlighting RBC maintenance as a critical therapeutic target in hematopoietic radiation injury. Funding Source n/a Topic Categories Hematopoiesis and Immune System Development (HEM)

Topology‐Orchestrated Multi‐physical Energy Control in Turtle Shell‐Inspired Metamaterials

ABSTRACT Achieving metamaterials that are simultaneously mechanically, acoustically, and thermally functional remains a central challenge in architected material design. In this research, we report a turtle shell bioinspired multifunctional lattice metamaterial (BMLM) that overcomes this limitation through topology‐driven hybrid‐coupling of distinct dissipation and transport pathways within a single hierarchical framework. The integration of arcuate plate‐strut geometries with engineered microporosity enables coordinated mechanical energy absorption, acoustic attenuation, and convective thermal transport. Experimentally validated, the proposed architecture achieves an exceptional specific compressive energy absorption of 55.7 kJ/kg alongside broadband acoustic performance, exhibiting an average sound absorption coefficient of 0.954 and near‐unity absorption (>0.9) sustained continuously from 2150 to 4250 Hz. Notably, the structure maintains acoustic stability under severe deformation; even at 40% compressive strain, absorption remains above 0.9 across a 2.1 kHz bandwidth, demonstrating deformation‐resilient functionality. Moreover, the interconnected open‐cell topology facilitates efficient airflow and convective thermal dissipation through its high surface‐area‐to‐volume ratio, comparing superior to standard plate‐fin heat sinks. Collectively, this work establishes a topology‐driven strategy for designing bio‐inspired metamaterials with robust, cross‐domain performance.

Interfacial Confinement‐Programmed Hydrogen Spillover on Ag/CoNiS Boosts Nitrate‐to‐Ammonia Electrosynthesis

ABSTRACT Electrochemical nitrate reduction (NO 3 RR) under ambient conditions offers a sustainable route for ammonia (NH 3 ) synthesis; however, its efficiency is restricted by the kinetic mismatch between water dissociation and nitrate hydrogenation. Here, we design Ag/CoNiS heterostructures in which Ag loading density programs interfacial confinement to regulate hydrogen spillover from CoNiS water‐activation domains to Ag‐associated nitrate/nitrogen oxide (NO x ) intermediates, thereby coupling * H generation, relay, and deep nitrate hydrogenation. The optimized Ag M /CoNiS achieves an NH 3 yield of 22.31 mg h −1  cm −2 with 99.13% Faradaic efficiency. In situ Raman, distribution of relaxation times (DRT) analysis, hydrogen/deuterium (H/D) isotope experiments, and tert‐butanol (TBA) perturbation tests reveal that the confined Ag–CoNiS interface regulates interfacial water and establishes a balanced * H supply–consumption regime, thereby suppressing competing hydrogen evolution. Density functional theory (DFT) calculations further show that Ag facilitates nitrate deoxygenation, whereas excessive Ag coverage weakens Co/Ni‐centered water activation, explaining the volcano‐type activity trend. Coupling NO 3 RR with the sulfide oxidation reaction (SOR) further enables a low‐voltage NO 3 RR||SOR electrolyzer, requiring only 0.70 V at 50 mA cm −2 for energy‐saving co‐production of ammonia and sulfur.

Hyperparameter-optimized deep learning for next-day pH forecasting in distributed water quality monitoring systems

Exploiting Virus-like Particles (VLP)-Peptide Platforms to Target SA-85 Trans-Sialidase for Chagas Disease Immunotherapy 2238706

Abstract Introduction Chagas disease is a potentially life-threatening illness caused by Trypanosoma cruzi. An estimated 6-7 million people are infected worldwide, and over 70 million are at risk of infection. There has been an increase in reports of Chagas disease in non-endemic regions such as Europe and the United States. There are only two approved medications for treatment, which are only effective in the acute stage with overwhelming side effects. No vaccine is approved for human use. T.cruzi lacks the ability to synthesise Sialic acid and scavenges it from its host glycoproteins via enzymes called trans-sialidases (TS). Trans-sialidase SA-85 is one of the most promising targets for vaccine development, as it is conserved across different strains and forms of the parasite Methods SSA-85-derived immunogenic peptides were predicted using immunoinformatic tools, and three peptides (TS-1, TS-2, TS-3) were synthesised and displayed on Q-Beta Bacteriophage Virus-like particles. Mice of C57BL/6 background were immunized with the peptides supplemented with AddaS03 adjuvant. Three doses of the peptides were administered two weeks apart between each immunization. Thereafter, serum samples and spleen homogenate samples were utilized to evaluate the immune response against the peptides. Results Anti-SA85 Immunoglobulin G (IgG) antibodies were assayed via indirect ELISA, showing a strong immune response with the peptides compared to the control group (p < 0.0001). Furthermore, cytokines (TNF-α, IFN-γ and Granzyme B) profile using spleen homogenates and Griess reaction showed significantly elevated levels in our peptide groups in comparison to the controls. TS-2 values for IFN-γ up to 1890pg/ml and TNF-α at 271pg/ml. This shows a Th-1 biased T-cell activation. Conclusion While these results serve as a preliminary proof of concept trial for SA-85 peptide vaccine, evidence suggests they can elicit a strong immune response and are viable candidates for further preclinical research into developing a vaccine for Chagas disease. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

IFNλ4 cell surface accumulation establishes localized innate immune environment and influences cancer risk 2310095

Abstract Introduction Interferon lambda 4 (IFNλ4) is a type III interferon encoded by IFNL4 at the 19q13.2 region. Up to 50% of people carry the rs368234815-ΔG allele in IFNL4 exon 1 and can produce functional IFNλ4 protein. IFNλ4 production (the ΔG allele) is associated with impaired HCV clearance and increased liver and prostate cancer risk. In contrast, results from the Ovarian Cancer Association Consortium and FinnGen suggest that the ΔG allele strongly protects against mucinous ovarian cancers and cysts. Here, we aimed to characterize IFNλ4 effects related to these protective associations. Methods Differential expression analyses after IFNλ4 induction in primary human hepatocytes or HepG2 (hepatocellular carcinoma) identified pathways likely relevant to mucin production, ovarian cysts, and cancer. To determine how IFNλ4 might drive cyst or tumor formation, we measured IFNλ4 overexpression effects on apoptosis in HepG2 and HEY-T30 (ovarian carcinoma) and observed IFNλ4 intercellular localization and cell surface interactions using confocal microscopy and western blot. Results Differential expression analyses revealed that IFNλ4 induced IRF1, a transcriptional regulator with tumor suppressor functions, as well as several mucin genes. Consistent with IRF1 upregulation, IFNλ4 overexpression induced apoptosis in vitro. Confocal and western blot analyses of cell surface interactions revealed accumulation of secreted IFNλ4 on the producer cell surfaces and transfer to adjacent cells. Conclusion IFNλ4 induction of IRF1, apoptosis, and mucin production may prevent cysts and mucinous cancer in the ovary, explaining the genetic associations. Cell surface accumulation, potentially facilitated by upregulated mucin production, may allow IFNλ4-producing cells to restrict signaling to only adjacent cells. This suggests a role for IFNλ4 in eliciting localized immune responses, thereby targeting cancerous cells or pathogens while preventing widespread inflammation that can damage sensitive microenvironments like in the ovary. Funding Source n/a Topic Categories Cytokines and Chemokines and their Receptors (CCR)

Modeling the Impact of Memory T Cell Activation on Lacrimal Gland Physiology 2267619

Abstract Introduction Recent evidence shows the dominance of memory T cells in the immune landscape of the mouse lacrimal gland (LG) but their role in idiopathic lacrimal dysfunction is unknown, and there is no current model able to selectively manipulate this subset of cells. We hypothesized that viral infection results in resident memory T cell (TRM) establishment in the LG, and that reactivation of TRM results in pro-inflammatory cytokine production and reduction in lacrimal secretions. Methods Female mice received an adoptive transfer of OT-I T cells followed by intranasal (IN) infection of vesicular stomatitis virus expressing OVA. A minimum of 30 days later, LGs were harvested for immunohistochemistry (IHC) or processed for flow cytometry. For IHC, LGs were stained for OT-Is, and for flow cytometry, LG lymphocytes were stained for TRM markers. Lacrimation was assessed using in vitro measurement of protein release from LGs in response to neural stimuli. Results Data showed that VSVova infection seeds persistent antigen-experienced CD8+ T cells and TRM populations in the murine LG. Following infection, a subset of LG CD8s were CD69+CD103- and smaller subset were CD69+CD103+. Also, a subset of CD8s were OT-Is, found by IHC and flow cytometry. Following in vitro reactivation, OT-Is expressed high levels of cytokines. Conclusion These data indicate that VSVova results in LG TRM populations, and that LG OT-Is can reactivate in vitro. Ongoing work is testing the impact of TRM reactivation on LG inflammation and function. Funding Source R01EY033712 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Defining Heterogeneity of CAR T cell populations to treat B16F10 melanoma 2310016

Abstract Introduction Chimeric antigen receptors (CAR) T cells recognize tumor associated antigen and have been approved to treat relapsed/refractory B cell lymphomas but not yet for solid tumors. In patients, the pre-transfer CAR product is heterogenous, containing T cells from distinct differentiation stages which can influence proliferation and persistence in vivo. These subsets may also adopt distinct effector, memory or exhausted states correlated with the antigen load and tissue environment. Understanding this heterogeneity is critical for optimizing CAR T cell therapy. Methods To investigate how CAR T cell subsets contribute individually and collectively, we are using DARLIN in-vivo lineage tracing and scRNA-seq to determine clonal relationships between the pre-transfer product and CAR T cells found at Day 7 and Day 40 after transfer. This will reveal the differentiation capacity and spatial localization of CAR T populations to provide further insights that may improve CAR-T therapy. Results We have demonstrated in a B16F10 melanoma model, TA99-CAR T cells (TRP1-specific), infiltrating the tumor are characterized by a CD44+ Ly6c+ PD-1+ population, indicating an activated and potentially exhausted phenotype. In contrast, CAR T cells localized in secondary lymphoid organs predominantly express CD62L and lack Ly6c expression, suggesting a central memory-like state with greater proliferative potential. Furthermore, when armoring CAR T cells with superkine IL-2 and IL-33, these cells demonstrated enhanced infiltration into the tumor microenvironment and adopted a terminal effector phenotype, characterized by high expression of terminal differentiation marker, KLRG1. Conclusion These data suggest that CAR T cell intrinsic and tissue-specific cues could shape CAR T cell differentiation potential. Funding Source NIH R01CA297297-01 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Regulatory maturation of cDC1 accelerates effector CD8 T cell contraction after severe influenza A virus infection 2267170

Abstract Introduction XCR1+ dendritic cells (cDC1) initiate CD8 T cell responses against Influenza A virus (IAV) infection. In the resolution phase, sustained cytokine production by CD8 T cells prolong lung damage, however, whether cDC1 regulate effector CD8 T cells is unknown. Methods cDC1 and CD8 T cells were characterized by flow cytometry. The transcriptome of cDC1 populations was analyzed by scRNA-seq. To investigate the function of mature cDC1 we co-cultured lung cDC1 and OT-I cells, performed adoptive transfers of OT-I cells, and transplanted bone marrow (BM) into fluorescent mice. Results IAV infection triggered two rounds of cDC1 maturation. The first round during the acute infection and is followed by recruitment of effector CD8 T cells. The second round occurs in the resolution phase and is followed by contraction of antigen specific CD8 T cells. The transcriptome of mature cDC1 showed that cDC1 maturation is driven by TNFa and IFNg in naïve and infected mice respectively. In the resolution phase, mature cDC1 express genes related to efferocytosis and immunoregulation. Transplant of WT BM into fluorescent mice showed that cDC1 acquire apoptotic cells only in the resolution phase. Our in vivo and in vitro experiments showed that mature cDC1 from the resolution phase reduced T-bet and IFNg production in OT-I cells while expanding RORgt OT-I cells. Conclusion The mature cDC1 that accumulate in the lungs during the resolution phase of IAV infection regulate effector CD8 T cell responses. Funding Source R01 AI153413 Topic Categories Viral Immunology (VIR)

Characterizing MTRNR2L1 as a Novel Pseudogene Associated with Glioblastoma Cell Survival 2258390

Abstract Introduction Glioblastoma multiforme (GBM) is an aggressive and treatment-resistant brain tumor with limited understanding of the regulatory mechanisms that promote tumor persistence. The pseudogene MTRNR2L1 has been reported to be upregulated in several cancers, but its function in glioblastoma remains unknown. This study aims to characterize MTRNR2L1 expression in GBM and determine whether it contributes to tumor cell survival and growth. Methods RNA was extracted from U87-MG glioblastoma cells, and MTRNR2L1 expression was confirmed through cDNA synthesis and quantitative PCR. A gene knockout was designed using CRISPR technology, and ongoing experiments are assessing how MTRNR2L1 loss affects cell viability and proliferation compared with wild-type controls. Results qPCR analysis confirmed MTRNR2L1 expression in U87-MG glioblastoma cells. Preliminary knockout studies suggest that loss of MTRNR2L1 may reduce glioblastoma cell proliferation and survival. Additional validation is underway to clarify its role in regulating tumor cell behavior. Conclusion MTRNR2L1 is a previously uncharacterized pseudogene expressed in glioblastoma that may influence tumor growth and cell survival. Functional characterization of this gene will help clarify its contribution to cancer-associated pseudogene networks and may reveal new targets for future glioblastoma research. Funding Source n/a Topic Categories Translational and Interventional Immunology (TI)

Goal-oriented transgenic TCR-T cell design for efficient cancer immunotherapy 2266120

Abstract Introduction Adoptive cell transfer (ACT) therapy is a promising approach for cancer treatment facing several limitations. Tumor-infiltrating lymphocyte (TILs) therapy is limited by patient condition and tumor accessibility. Peripheral lymphocyte redirection strategies by low transduction efficiency and potency. We previously showed that transgenic TCR-T cells suffer inconsistent expression of transgenic receptor and surface dilution by endogenous TCR, both limiting effective avidity for antigen. In this work we compare 3 general strategies for transgenic TCR-T cell engineering: Viral transduction, viral transduction coupled with endogenous TCR knock-out and Crispr-cas9 knock-in under endogenous TCR promoter. Thus, we identify their use cases for goal oriented therapeutic T-cells design. Methods We use pMIGII retroviral vector for viral transduction. For endogenous TCR knock-out we deliver CRISPR-CAS9-gRNA RNP targeting TRAC and TRBC loci, using NEON system. For CRISPRS-CAS9 knock-in we use the above method followed by AAV6 transduction to deliver HDR template targeting TRAC. For Cytolytic Assay we use Chromium 51 release. Results Regular viral transduction resulted in transgenic TCR-T cells with low cytolytic activity, that were rescued by CRISPR-CAS9 knock-out of endogenous TCR. Both these approaches, however, resulted in loss of cytolytic activity and receptor expression with subsequent restimulations, due to semi-random gene insertion by viral vector. CRISPR-CAS9 HDR knock-in under endogenous TCR promoter produced consistent near-physiological transgenic TCR expression level, and cytolytic activity, maintained over multiple restimulations. Conclusion Viral Transduction couples with endogenous TCR knock-out is sufficient to produce highly potent transgenic TCR -T cells for short-term use, with repeated infusions or in the context of in vivo transduction. Precision knock-in under endogenous TCR locus produces TCR-T cells more suitable for off-the-shelf reagent with long lasting cytolytic activity. Funding Source MRIE at SKCC, DOD CDMP HT9425-25-1-0902 Topic Categories Technological Innovations in Immunology (TECH)

Characterization of the Functional and Phenotypic Profile of a Naïve Conventional Innate-like Population of CD8+ T Cells in Neonatal Cord Blood 2260244

Abstract Introduction Neonates, while highly susceptible to serious infections, may harbor unique features of adaptive immunity that protect during infancy before durable memory is established. Previous work in our lab has identified a naive conventional CD8+ T cell population, which we termed fetal innate-like T cells (FITs), that is unique to cord blood (CB) and shares phenotypic features with innate-like unconventional T cells and NK cells. This FIT population is defined by co-expression of KLRG1, CD161 and CD8αβ, but more investigation is needed to identify their overlapping and distinct functionality with respect to conventional and innate CD8α+ T cells. We hypothesize that FITs share functions with innate lymphoid cells, including cytotoxic mediators and cytokines, that provide alternative paths to protection in the neonatal period. Methods To test our hypothesis, we performed high dimensional spectral flow and automated clustering to compare phenotypic and functional profiles of in vitro-stimulated FITs and non-FITs populations from neonates and adults. Results Upon stimulation, FITs showed increased expression of either TNFα or Granzyme K, and there were no detectable dual-staining cells. FITs were negative for IFNγ, IL8, and IL17A. FITs could be further distinguished from other conventional CD8+ events by their CD337 (NKp30) expression, a marker more often associated with anti-tumor activity in natural killer cells. Conclusion These findings support the presence of a unique subpopulation of conventional CD8αβ T cells that overlap with innate-like lymphoid populations, raising their potential for alternative pathways to protection or immunopathology during early life. Funding Source U24AI152176 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)