Cell Compartment Research Articles

Exhaustive stress causes a rapid immunological response in the humoral and cellular haemolymph compartments of the pale octopus (Octopus pallidus)

Cephalopods are economically and ecologically important species across the world, yet information linking physiological stress and associated immunological responses is limited in the current literature. Here, the effects of exhaustive exercise in a holobenthic octopus species, Octopus pallidus, were examined by evaluating immunological parameters. In whole haemolymph, the pH and refractive index were measured. To assess the cellular function of the haemolymph, the total count, cell vitality and phagocytosis capacity of the haemocytes were also measured. To assess enzymatic function, activities of the phenoloxidase system and lysozyme were quantified in the plasma and cellular components of the haemolymph. Overall, exhaustive exercise led to rapid changes in the haemolymph with a significant decrease in the pH and phagocytosis capacity though the number of cells and cell vitality were not affected. Exercise also triggered the increase of activated phenoloxidase (PO-like) activity and the decrease of the inactive zymogen prophenoloxidase (ProPO-like), total PO-like and lysozyme activity in plasma and an increase in total PO-like activity in the hemocyte compartment. These responses indicated that a realistic energetic demand had substantial, rapid impact on immune function. These results also provide an important baseline to understand the immune physiology of cephalopods that will further efforts to identify the mechanisms underlying the impacts of stressors.

Abstract A031: Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy

Abstract Preclinical and clinical studies have used antibodies to deplete CD4-expressing cells to promote anti-tumor immunity. Specifically, a monoclonal anti-CD4 antibody (anti-CD4) has been employed to remove FOXP3+ CD4+ regulatory T cells (Tregs), which are known to impede anti-tumor immunity. Our previous studies demonstrated that administering anti-CD4 during B16 melanoma tumor growth in mice leads to the generation of tumor Ag-specific CD8 T cells that disseminate and form memory following surgical tumor excision. However, the importance of co-depleting other CD4-expressing cell compartments for anti-CD4 treatment efficacy are unknown. We utilized flow cytometry and single-cell RNA sequencing (scRNAseq) to evaluate the primary response of tumor Ag-specific CD8 T cells (pmel) in B16 melanoma tumor bearing mice treated with anti-CD4. Responses were compared to those induced by anti-PD1 and anti-CTLA4 dual immune checkpoint blockade (ICB). To determine the importance of homeostatic space in priming, homeostatic expansion and priming were decoupled, and neutralizing antibodies were administered. Treg-depleted (Foxp3-DTR) mice were used for comparison, and Batf3-/- mice and IL-2R blocking antibodies were used to determine requirements for CD8 T cell priming. Finally, scRNAseq was conducted on CD4 T cells from human melanoma tumors previously treated with ICB to characterize transcriptional properties of intratumoral CD4 T cells. Compared to ICB, anti-CD4 treatment elicited a more robust, disseminated, and persistent primary tumor Ag-specific CD8 T cell response. ScRNAseq revealed that anti-CD4 treatment induced more stem-like (TCF1+) memory precursor cells, whereas ICB induced more differentiated effector (Gzmb+) cells. Removal of homeostatic space or blockade of IL-7R and IL-15 during priming did not diminish pmel responses in anti-CD4 treated mice, indicating that homeostatic space was not required. Importantly, pmel cells were shown to produce their own IL-2, and pmel priming was significantly lower after IL-2 receptor blockade, suggesting dependence on autocrine IL-2 signals. Using DT-treated FOXP3-DTR mice, we found that targeted Treg depletion was insufficient for the pmel priming that disseminated and formed memory, indicating that total CD4 T cell depletion was required for this systemic response. Depletion of total CD4 T cells uniquely increased cDC1 proportions in tumor-draining LNs as well as their expression of CD86. However, treatment of tumor-bearing RAG-/- mice with anti-CD4 confirmed that depletion of CD4-expressing APC compartments does not contribute to anti-CD4 treatment efficacy. ScRNAseq of CD4 T cells from human melanoma tumors showed dominance of Tr1-like cells and other subsets without transcriptional evidence of helper differentiation, despite prior ICB. In mice, combination of anti-CD4 with dual ICB restored the systemic response of tumor-specific CD8 T cells. This study underscores the importance of eliminating conventional CD4 T cells in tumor-bearing hosts to elicit systemic primary and memory CD8 T cell responses. Citation Format: Christo P. C. Dragnev, Delaney E. Ramirez, Tyler G. Searles, Nathaniel Spicer, TIffany Chen, J. Louise Lines, Aaron R. Hawkes, Wilson L. Davis III, Asmaa Mohamed, Keisuke Shirai, Joesph D. Phillips, Pamela C. Rosato, Yina H. Huang, Mary Jo Turk. Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A031.

Lysine succinylation precisely controls normal erythropoiesis.

Lysine succinylation (Ksu) has recently emerged as a protein modification that regulates diverse functions in various biological processes. However, the systemically and precise role of lysine succinylation in erythropoiesis remains to be fully elucidated. In this study, we noted a prominent increase of succinyl-CoA and lysine succinylation during human erythroid differentiation. To explore the functional significance of succinylation, we inhibited succinylation by either knock downing key succinyltransferases or overexpressing desuccinylases. Succinylation inhibition led to suppressed cell proliferation, increased apoptosis, and disrupted erythroid differentiation. In vivo overexpression of the desuccinylases SIRT5 delayed erythroid differentiation. Furthermore, integrative proteome and succinylome analysis identifies 939 succinylated proteins with 3,562 Ksu sites, distributed across various cellular compartments and involved in multiple cellular processes. Significantly, inconsistencies between protein expression levels and succinylation levels were observed, indicating that the succinylation of certain proteins may function independently of expression. Mechanistically, we implicated KAT2A-mediated succinylation of histone H3 K79, leading to chromatin remodeling and subsequently erythropoiesis regulation. Specially, we identified CYCS as a key regulator of erythropoiesis, which depends on its succinylation sites K28/K40. Taken together, our comprehensive investigation of the succinylation landscape during erythropoiesis provides valuable insights into its regulatory role and offer potential implications for erythroid-related diseases.

Evolutionary origins of the lysosome-related organelle sorting machinery reveal ancient homology in post-endosome trafficking pathways

The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution.

Evolutionary Grid Optimization and Deep Learning for Improved In Vitro Cellular Spheroid Localization

The recently developed high-throughput system for cell spheroid generation (SpheroWell) is a promising technology for cost- and time-efficient in vitro analysis of, for example, chondrogenic differentiation. It is a compartmental growth surface where spheroids develop from a cell monolayer by self-assembling and aggregation. In order to automatize the analysis of spheroids, we aimed to develop imaging software and improve the localization of cell compartments and fully formed spheroids. Our workflow provides automated detection and localization of spheroids in different formation stages within Petri dishes based on images created with a low-budget camera imaging setup. This automated detection enables a fast and inexpensive analysis workflow by processing a stack of images within a short period of time, which is essential for the extraction of early readout parameters. Our workflow combines image processing algorithms and deep learning-based image localization/segmentation methods like Mask R-CNN and Unet++. These methods are refined by an evolution strategy for automated grid detection, which is able to improve the overall segmentation and classification quality. Besides the already pre-trained neural networks and predefined image processing parameters, our evolution-based post-processing provides the required adaptability for our workflow to deliver a consistent and reproducible quality. This is especially important due to the use of a low-budget imaging setup with various light conditions. The to-be-detected objects of the three different stages show improved results using our evolutionary post-processing for monolayer and starting aggregation with Dice coefficients of 0.7301 and 0.8562, respectively, compared with the raw scores of 0.2879 and 0.8187. The Dice coefficient of the fully formed spheroids in both cases is 0.8829. With our algorithm, we provide automated analyses of cell spheroid by self-assembling in SpheroWell dishes, even if the images are created using a low-budget camera setup.

Single-cell transcriptomics reveal potent extrafollicular B cell response linked with granzyme K+ CD8 T cell activation in lupus kidney

ObjectivesB and T cells constitute the majority of infiltrating lymphocytes in the kidney and represent the local perpetrators in lupus nephritis (LN), but the underlying pathogenic mechanisms are not well...

The heat shock response of plants: new insights into modes of perception and signaling and how hormones contribute.

Plants have evolved specific temperature preferences, and shifts above this range cause heat stress with detrimental effects such as physiological disruptions, metabolic imbalances, and growth arrest. To reduce damage, plants utilize the heat shock response (HSR), signaling cascades that activate the heat shock factors (HSFs), transcription factors that control the heat stress-responsive transcriptome for activation of protective measures. While the core HSR is well-studied, we still know relatively little about heat stress perception and signal integration or cross-talk with other pathways. In the last few years, however, significant progress has been made in this area, which is summarized here. It has emerged that the plant hormones brassinosteroids (BRs) and abscisic acid (ABA) contribute to heat stress tolerance by impacting HSF modes of activity. Also, we began to understand that heat stress is sensed in different cellular compartments and that events in the nucleus, such as nuclear condensate formation via liquid-liquid phase separation, play a key role. In the future, it will be important to explore how these multilayered perception and signaling modes are utilized to understand how environmental context and developmental stage determine the outcome of heat stress effects on plant growth and development.

Spatial proteomics identifies JAKi as treatment for a lethal skin disease.

Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction triggered by common medications and is an emerging public health issue1-3. Patients with TEN undergo severe and sudden epidermal detachment caused by keratinocyte cell death. Although molecular mechanisms that drive keratinocyte cell death have been proposed, the main drivers remain unknown, and there is no effective therapy for TEN4-6. Here, to systematically map molecular changes that are associated with TEN and identify potential druggable targets, we utilized deep visual proteomics, which provides single-cell-based, cell-type-resolution proteomics7,8. We analysed formalin-fixed, paraffin-embedded archived skin tissue biopsies of three types of cutaneous drug reactions with varying severity and quantified more than 5,000 proteins in keratinocytes and skin-infiltrating immune cells. This revealed a marked enrichment of type I and type II interferon signatures in the immune cell and keratinocyte compartment of patients with TEN, as well as phosphorylated STAT1 activation. Targeted inhibition with the pan-JAK inhibitor tofacitinib in vitro reduced keratinocyte-directed cytotoxicity. In vivo oral administration of tofacitinib, baricitinib or the JAK1-specific inhibitors abrocitinib or upadacitinib ameliorated clinical and histological disease severity in two distinct mouse models of TEN. Crucially, treatment with JAK inhibitors (JAKi) was safe and associated with rapid cutaneous re-epithelialization and recovery in seven patients with TEN. This study uncovers the JAK/STAT and interferon signalling pathways as key pathogenic drivers of TEN and demonstrates the potential of targeted JAKi as a curative therapy.

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins.

Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins

AbstractSulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Improving the Utility of a Cancer Gene Mutation Panel for Multiple Myeloma Patients

Abstract Multiple myeloma is a plasma cell malignancy that accounts for 1-2% of all cancer diagnoses and 10-15% of hematopoietic neoplasms. Clonal rearrangement of the IG locus suggests a monoclonal model for pathogenesis. The presence of cytogenetic abnormalities in >90% of cases suggests transformation by chromosomal instability and rearrangement. Classification and consensus guidelines by The International Myeloma Working Group (IMWG) and others reflect this by categorizing myeloma based on ploidy, translocations, and chromosomal abnormalities. Interestingly, a portion of Emory’s test volume for cancer gene mutation profiling by second generation short-read sequencing is for multiple myeloma cases. This suggests our 75-gene myeloid neoplasm panel (MMP75) is being utilized for medical purposes that are not met by cytogenetic and microarray characterizations performed during the diagnostic workup of multiple myeloma. Inspired by this observation, this study investigates providers motivations for ordering MMP75 and assesses the adequacy of our current implementation for their stated purposes. Anecdotes from ordering physicians indicate interest in MMP75 results when informing prognosis, selecting treatments, and evaluating for treatment associated secondary neoplasms. A more formal survey is underway that will statistically capture how the results of MMP75 testing were ultimately used: to inform diagnosis, determine prognosis, guide treatment, explain progression, identify/rule-out secondary neoplasia, or no effect on management. We also explore the variants called by MMP75 when performed using bone marrow from patients who received a cytogenetic workup for multiple myeloma at some point. A retrospective analysis of the 5-year interval between Jan 2019 and Jan 2024 identified 156 cases meeting this criterion. The number of pathogenic variants per case ranged between 1 and 8, with mutations detected across 42 genes. The genes most frequently identified with pathogenic mutations were TP53 (22.4%), DNMT3A (19.2%), TET2 (14.7%), and ASXL1 (12.2%). All four have been associated with adverse outcomes in patients with multiple myeloma. Activating events in therapeutic targets included mutations in the RAS/RAF pathway (16.7%), JAK/STAT signaling (2.6%), and the kinase KIT (0.6%). While these findings support the use of MMP75 for detection of relevant mutations, the genetic variants identified are not unique to multiple myeloma. Mutations detected in DNMT3A, TET2, and ASXL1 are common in clonal hematopoiesis of indeterminant potential (CHIP) and myeloid neoplasms. Hotspot mutations in TP53 and activating events in the RAS/RAF pathway also occur in various hematologic malignancies. This test therefore detects events that occur in both the plasma cell compartment and other cells of origin. This encumbers distinguishing variants that inform on a patient’s multiple myeloma from events that suggest a secondary neoplasm. Future directions include determining whether the effect of plasma cell enrichment on a mutations variant allele frequency (VAF) in repeat testing can help distinguish true multiple myeloma variants from events in other cells.

Hybrid Protocells Based on Coacervate-Templated Fatty Acid Vesicles Combine Improved Membrane Stability with Functional Interior Protocytoplasm.

Prebiotically-plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self-assembly and coacervate droplets formed by liquid-liquid phase separation. Both types of structures form spontaneously and can be related to cellular compartmentalization motifs in today's living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation and membranes providing superior boundaries. Herein, protocell models constructed by spontaneous encapsulation of coacervate droplets by mixed fatty acid/phospholipid and by purely fatty acid membranes are described. Coacervate-supported membranes form over a range of coacervate and lipid compositions, with membrane properties impacted by charge-charge interactions between coacervates and membranes. Vesicles formed by coacervate-templated membrane assembly exhibit profoundly different permeability than traditional fatty acid or blended fatty acid/phospholipid membranes without a coacervate interior, particularly in the presence of magnesium ions (Mg2+). While fatty acid and blended membrane vesicles are disrupted by the addition of Mg2+, the corresponding coacervate-supported membranes remain intact and impermeable to externally-added solutes. With the more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, can be performed inside the protocell model due to the simple diffusion of FDA and then following with the coacervate-mediated abiotic hydrolysis to fluorescein.

Breaking the barriers: Overcoming cancer resistance by targeting the NLRP3 inflammasome.

Inflammation has a pivotal role in the initiation and progression of various cancers, contributing to crucial processes such as metastasis, angiogenesis, cell proliferation and invasion. Moreover, the release of cytokines mediated by inflammation within the tumour microenvironment (TME) has a crucial role in orchestrating these events. The activation of inflammatory caspases, facilitated by the recruitment of caspase-1, is initiated by the activation of pattern recognition receptors on the immune cell membrane. This activation results in the production of proinflammatory cytokines, including IL-1β and IL-18, and participates in diverse biological processes with significant implications. The NOD-Like Receptor Protein 3 (NLRP3) inflammasome holds a central role in innate immunity and regulates inflammation through releasing IL-1β and IL-18. Moreover, it interacts with various cellular compartments. Recently, the mechanisms underlying NLRP3 inflammasome activation have garnered considerable attention. Disruption in NLRP3 inflammasome activation has been associated with a spectrum of inflammatory diseases, encompassing diabetes, enteritis, neurodegenerative diseases, obesity and tumours. The NLRP3 impact on tumorigenesis varies across different cancer types, with contrasting roles observed. For example, colorectal cancer associated with colitis can be suppressed by NLRP3, whereas gastric and skin cancers may be promoted by its activity. This review provides comprehensive insights into the structure, biological characteristics and mechanisms of the NLRP3 inflammasome, with a specific focus on the relationship between NLRP3 and tumour-related immune responses, and TME. Furthermore, the review explores potential strategies for targeting cancers via NLRP3 inflammasome modulation. This encompasses innovative approaches, including NLRP3-based nanoparticles, gene-targeted therapy and immune checkpoint inhibitors.

Transcriptomics combined with physiological analysis provided new insights into the enhancing Dendrobium nobile resilience to Cu stress through Ce modulation

In the face of escalating environmental challenges, understanding the mechanisms through which plants can resist heavy metal stress is paramount. This study delves into the efficacy of cerium (Ce), a rare earth element, in mitigating copper (Cu) toxicity in Dendrobium nobile Lindl., an orchid plant with significant medicinal and ornamental value. Through a comprehensive experimental approach that encompasses physiological, biochemical, and molecular analyses, we demonstrate that Ce application notably enhances D. nobile's tolerance to Cu stress. The investigation revealed that Ce supplementation significantly ameliorated the adverse effects of Cu on plant growth, evidenced by a notable decrease in Cu accumulation within the plant tissues. At the molecular level, transcriptome sequencing uncovered the upregulation of genes involved in antioxidant defense mechanisms, heavy metal chelation, and stress response signaling pathways. Key findings include the enhanced activities of antioxidant enzymes such as superoxide dismutase, peroxidase, and catalase, alongside an increase in the levels of antioxidants and phytochelatins, which collectively contribute to the detoxification of Cu. Furthermore, Ce was found to influence the subcellular distribution of Cu, promoting its sequestration in less harmful cellular compartments. Our study provides novel insights into the role of Ce in modulating plant responses to heavy metal stress, highlighting its potential as a mitigative agent against Cu toxicity. The elucidation of specific gene regulations and pathway activations offers a foundation for future research aimed at enhancing plant resilience to environmental stresses through biotechnological interventions.

Molecular and microbiological methods for the identification of nonreplicating Mycobacterium tuberculosis.

Chronic tuberculosis (TB) disease, which requires months-long chemotherapy with multiple antibiotics, is defined by diverse pathological manifestations and bacterial phenotypes. Targeting drug-tolerant bacteria in the host is critical to achieving a faster and durable cure for TB. In order to facilitate this field of research, we need to consider the physiology of persistent MTB during infection, which is often associated with the nonreplicating (NR) state. However, the traditional approach to quantifying bacterial burden through colony enumeration alone only informs on the abundance of live bacilli at the time of sampling, and provides an incomplete picture of the replicative state of the pathogen and the extent to which bacterial replication is balanced by ongoing cell death. Modern approaches to profiling bacterial replication status provide a better understanding of inter- and intra-population dynamics under different culture conditions and in distinct host microenvironments. While some methods use molecular markers of DNA replication and cell division, other approaches take advantage of advances in the field of microfluidics and live-cell microscopy. Considerable effort has been made over the past few decades to develop preclinical in vivo models of TB infection and some are recognized for more closely recapitulating clinical disease pathology than others. Unique lesion compartments presenting different environmental conditions produce significant heterogeneity between Mycobacterium tuberculosis populations within the host. While cellular lesion compartments appear to be more permissive of ongoing bacterial replication, caseous foci are associated with the maintenance of M. tuberculosis in a state of static equilibrium. The accurate identification of nonreplicators and where they hide within the host have significant implications for the way novel chemotherapeutic agents and regimens are designed for persistent infections.

CTLA4 blockade abrogates KEAP1/STK11-related resistance to PD-(L)1 inhibitors.

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients willbenefit from dual ICB1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial3. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy-a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8+ cytotoxic T cells, but relative sparing of CD4+ effector subsets. Dual ICB potently engaged CD4+ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that-together with CD4+ and CD8+ T cells-contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

Rapid and effective protocol to measure glutathione peroxidase activity

Abstract Background Intracellular hydroperoxides are harmful reactive oxygen species that can cause damage. Cells depend on antioxidant enzymes such as glutathione peroxidase (GPx) to prevent their accumulation. GPx is present in all cell compartments, including the cytosol, mitochondria, and peroxisomes. This article will present a rapid and reliable method for measuring GPx activity, which has been rigorously tested and proven dependable. Results The enzyme samples are incubated in a phosphate buffer containing specific concentrations of glutathione (GSH) and peroxide and then incubated for a suitable time to react before Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) is added. This method, designed for ease of use, is effective and straightforward. When GSH reacts with DTNB, mixed disulfides (GS-S-DTNB) are formed, where the thiol group replaces one of the nitrobenzoic acid moieties in DTNB. The release of the nitrobenzoic acid group as nitro-2-thiobenzoate anion (TNB−) creates a yellow anion that can be measured spectrophotometrically at a wavelength of 412 nm. The intensity of the yellow color is directly proportional to the unreacted glutathione concentration. GPx activity was linked to decreased absorbance of the TNB− anion, providing a sensitive and relatively specific method for detecting thiol groups in various biological samples. Conclusion This study utilized a Box–Behnken design (BBD) to apply to response surface modeling (RSM) to determine the best conditions for measuring GPx activity. The efficiency of the new protocol was validated by comparing GPx activity in matched samples using the Bland–Altman plot analysis and reference methods. The new protocol was just as accurate as the reference one, with a correlation coefficient of 0.9991. Graphical abstract

High-dimensional Immune Profiles and Machine Learning May Predict Acute Myeloid Leukemia Relapse Early following Transplant.

Identification of early immune signatures associated with acute myeloid leukemia (AML) relapse following hematopoietic stem cell transplant (HSCT) is critical for patient outcomes. We analyzed PBMCs from 58 patients with AML undergoing HSCT, focusing on T cell subsets and functional profiles. High-dimensional flow cytometry coupled with Uniform Manifold Approximation and Projection dimensionality reduction and PhenoGraph clustering revealed distinct changes in CD4+ and CD8+ T cell populations in 16 patients who relapsed within 1 y of HSCT. We observed increased IL-2, IL-10, and IL-17-producing CD4+ T cells, alongside decreased CD8+ T cell function early in relapsing patients. Notably, relapsing patients exhibited increased TCF-1intermediate cells, which lacked granzyme B or IFN-γ production in the CD4+ T cell compartment. We then developed a supervised machine learning algorithm that predicted AML relapse with 90% accuracy within 30 d after HSCT using high-throughput assays. The algorithm leverages condensed immune phenotypic data, alongside the ADASYN algorithm, for data balancing and 100 rounds of XGBoost supervised learning. This approach holds potential for detecting relapse-associated immune signatures months before clinical manifestation. Our findings demonstrate a distinct immunological signature potentially capable of predicting AML relapse as early as 30 d after HSCT.

Jag1 represses Notch activation in lateral supporting cells and inhibits an outer hair cell fate in the medial cochlea.

Notch signaling patterns the cochlear organ of Corti, and patients with the JAG1/NOTCH2-related genetic disorder Alagille syndrome can thus experience hearing loss. We investigated the function of Jag1 in cochlear patterning and signaling using Jag1Ndr/Ndr mice, a model of Alagille syndrome. Jag1Ndr/Ndr mice exhibited expected vestibular and auditory deficits, a dose-dependent increase in ectopic inner hair cells, and a reduction in outer hair cells. Single cell RNA sequencing of the organ of Corti demonstrated a global dysregulation of genes associated with inner ear development and deafness. Analysis of individual cell types further revealed that Jag1 represses Notch activation in lateral supporting cells and demonstrated a function for Jag1 in gene regulation and development of outer hair cells. Surprisingly, ectopic "outer hair cell-like" cells were present in the medial compartment and pillar cell region of Jag1Ndr/Ndr cochleae, yet they exhibited location-dependent expression of the inner hair cell fate-determinant Tbx2, suggesting Jag1 is required for Tbx2 to drive inner hair cell commitment. This study thus identifies new roles for Jag1 in supporting cells, and in outer hair cell specification and positioning.

A Novel Chimeric Oncolytic Virus Mediates a Multifaceted Cellular Immune Response in a Syngeneic B16 Melanoma Model

Background/Objectives: Oncolytic virotherapy is a promising approach in cancer immunotherapy. We have previously described a recombinant hybrid oncolytic virus (OV), VSV-NDV, which has a favorable safety profile and therapeutic immunogenicity, leading to direct oncolysis, abscopal effects, and prolonged survival in syngeneic in vivo tumor models. While OVs are known to mediate systemic anti-tumor immune responses, the detailed characterization of local and systemic immune responses to fusogenic oncolytic virotherapy remains unexplored. Methods and Results: We analyzed immune cell compartments in the spleen, blood, tumor-draining lymph nodes (TDLNs), and tumors over the course of VSV-NDV therapy in a bilateral syngeneic melanoma mouse model. Our results revealed significant local infiltration and activation of T lymphocytes in tumors and globally in the blood and spleen. Notably, in vivo CD8+ T cell depletion led to complete abrogation of the tumor response, highlighting the crucial role of T cells in promoting the therapeutic effects of oncolytic VSV-NDV. In vitro co-culture experiments enabled the interrogation of human immune cell responses to VSV-NDV-mediated oncolysis. Human peripheral blood mononuclear cells (PBMCs) were efficiently stimulated by exposure to VSV-NDV-infected cancer cells, which recapitulates the in vivo murine findings. Conclusions: Taken together, these data characterize a broad anti-tumor immune cell response to oncolytic VSV-NDV therapy and suggest that CD8+ T cells play a decisive role in therapeutic outcome, which supports the further development of this chimeric vector as a multimechanistic immunotherapy for solid cancers.