Biology subjects

Hu, L.

Publications and source records attributed to Hu, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Cell atlas of the developing human meninges reveals a dura origin of meningioma

The vertebrate central nervous system is enveloped by the meninges, consisting of the pia, arachnoid, and dura layers. The arachnoid is hypothesised to give rise to the most common primary intracranial tumours, meningiomas. However, molecular evidence supporting this hypothesis is lacking. There are no effective medical therapies to treat meningiomas that are resistant to local interventions, encumbered by our limited understanding of their cellular origin. To address this limitation in our understanding of meningioma biology, we generated a comprehensive reference single cell and spatial transcriptomic atlas of human fetal meninges at post-conceptional weeks 5-13. We found that the meningeal layers develop concurrently, and identified an inner CDH1-positive dura cell layer expressing tight junction genes consistent with barrier function. We show that transcriptionally, meningioma cells resemble dura-lineage cells, and that common meningioma driver genes were expressed preferentially in the dura lineage. Our findings suggest that meningiomas originate from dura lineage cells. HIGHLIGHTSO_LIscRNA-seq and spatial transcriptomics reveals architecture of human fetal meninges development C_LIO_LIMeningeal layers are formed concurrently, by a gradual refinement of cell states C_LIO_LIA CDH1+ inner dura sublayer expresses tight junction genes like the arachnoid barrier C_LIO_LIMeningioma tumours likely originate from inner dura lineage cells, not arachnoid C_LI

developmental biology↗

bioRxiv2025

A Universal RT-qPCR Method for DNA Aptamer Quantification

Aptamers are widely used in various applications; however, their quantification methods remain underdeveloped. In this study, we established a universal RT-qPCR-based method for DNA aptamer quantification. By incorporating reverse transcription, primers and probes could be added to aptamers, enabling their detection via qPCR (Figure 1). We developed a software tool for primer and probe design and optimized the reverse transcription process. Two aptamers, CD9-aptamer and CD63-aptamer, were selected as model systems representing two distinct aptamer types: the CD9-aptamer contains an intrinsic primer-binding site within its sequence, whereas the CD63-aptamer does not. Using this method, the limit of detection (LOD) for CD9-aptamer reached 10-14 M, while the LOD for CD63-aptamer was 10-12 M. Compared to existing quantification methods, this approach significantly improves accuracy and cost-effectiveness. Additionally, the method supports SYBR Green, TaqMan, and one-step RT-qPCR assays, broadening its applicability and enhancing precision for various aptamer-based applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/662296v1_fig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@44ec4corg.highwire.dtl.DTLVardef@f59184org.highwire.dtl.DTLVardef@2480fdorg.highwire.dtl.DTLVardef@906845_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Schematic illustration of RT-qPCR-Based Aptamer Quantification. C_FIG

molecular biology↗

bioRxiv2025

Development of a replication-defective MPXV platform for fundamental and therapeutic research

The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying monkeypox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a novel strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineered replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5L)--genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. Our work demonstrates the utility of replication-defective MPXV particles as a reliable platform for high-throughput antiviral discovery, offering significant advantages for both fundamental virology studies and therapeutic development against orthopoxviruses.

microbiology↗

bioRxiv2025

Population-specific brain charts reveal Chinese-Western differences in neurodevelopmental trajectories

Human brain charts provide unprecedented opportunities for decoding neurodevelopmental milestones and establishing clinical benchmarks for precision brain medicine 1-7. However, current lifespan brain charts are primarily derived from European and North American cohorts, with Asian populations severely underrepresented. Here, we present the first population-specific brain charts for China, developed through the Chinese Lifespan Brain Mapping Consortium (Phase I) using neuroimaging data from 43,037 participants (aged 0-100 years) across 384 sites nationwide. We establish the lifespan normative trajectories for 296 structural brain phenotypes, encompassing global, subcortical, and cortical measures. Cross-population comparisons with Western brain charts (based on data from 56,339 participants aged 0-100 years) reveal distinct neurodevelopmental patterns in the Chinese population, including prolonged cortical and subcortical maturation, accelerated cerebellar growth, and earlier development of sensorimotor regions relative to paralimbic regions. Crucially, these Chinese-specific charts outperform Western-derived models in predicting healthy brain phenotypes and detecting pathological deviations in Chinese clinical cohorts. These findings highlight the urgent need for diverse, population-representative brain charts to advance equitable precision neuroscience and improve clinical validity across populations.

neuroscience↗

bioRxiv2025

Tuning the interaction of a ParA-type ATPase with its partner separates bacterial organelle positioning from partitioning

The maintenance of carboxysome distribution (Mcd) system comprises the proteins McdA and McdB, which spatially organize carboxysomes to promote efficient carbon fixation and ensure their equal inheritance during cell division. McdA, a member of the ParA/MinD family of ATPases, forms dynamic gradients on the nucleoid that position McdB-bound carboxysomes. McdB belongs to a widespread but poorly characterized class of ParA/MinD partner proteins, and the molecular basis of its interaction with McdA remains unclear. Here, we demonstrate that the N-terminal 20 residues of H. neapolitanus McdB are both necessary and sufficient for interaction with McdA. Within this region, we identify three lysine residues whose individual substitution modulates McdA binding and leads to distinct carboxysome organization phenotypes. Notably, lysine 7 (K7) is critical for McdA interaction: substitutions at this site result in the formation of a single carboxysome aggregate positioned at mid-nucleoid. This phenotype contrasts with that of an McdB deletion, in which carboxysome aggregates lose their nucleoid association and become sequestered at the cell poles. These findings suggest that weakened McdA-McdB interactions are sufficient to maintain carboxysome aggregates on the nucleoid but inadequate for partitioning individual carboxysomes across it. We propose that, within the ParA/MinD family of ATPases, cargo positioning and partitioning are mechanistically separable: weak interactions with the cognate partner can mediate positioning, whereas effective partitioning requires stronger interactions capable of overcoming cargo self-association forces.

microbiology↗

bioRxiv2025

UniCell: Towards a Unified Solution for Cell Annotation, Nomenclature Harmonization, Atlas Construction in Single-Cell Transcriptomics

Standardizing cell type annotations across single-cell RNA-seq datasets remains a major challenge due to inconsistencies in nomenclature, variation in annotation granularity, and the presence of rare or previously unseen populations. We present UniCell, a hierarchical annotation framework that combines Cell Ontology structure with transcriptomic data for scalable, interpretable, and ontology-aware cell identity inference. UniCell leverages a multi-task architecture that jointly optimizes local and global classifiers, yielding coherent predictions across multiple levels of the ontology-defined hierarchy. When benchmarked across 20 human and mouse datasets, UniCell consistently outperformed state-of-the-art tools, including CellTypist, scANVI, OnClass, and SingleR, in annotation performance, and sensitivity to low-abundance populations. In disease settings, UniCell effectively identified previously unseen cell types through confidence-guided novelty detection. Applied to 45 human and 23 mouse tissue atlases, UniCell enabled cross-dataset and cross-species harmonization by embedding cells into a unified latent space aligned with Cell Ontology structure. Moreover, when used to supervise single-cell foundation models, UniCell substantially improved downstream annotation accuracy, rare cell detection, and hierarchical consistency. Together, these results establish UniCell as a generalizable framework that supports high-resolution annotation, nomenclature standardization, and atlas-level integration, providing a scalable and biologically grounded solution for single-cell transcriptomic analysis across diverse biological systems.

bioinformatics↗

bioRxiv2025

Breakthrough infection elicits synergistic broadly neutralizing antibodies targeting non-dominant epitopes on RBD and NTD in an individual of inactivated SARS-CoV-2 vaccine

Background/ObjectivesIdentification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. MethodsThrough single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were respectively measured with ELISA and pseudovirus-based neutralization assay. Their epitopes were mapped by competition ELISA and site- directed mutation. ResultsAmong a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13, and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20, and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. ConclusionsThese four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection.

immunology↗

bioRxiv2025

Novel Strategy against AML by Rescuing Loss of Phase Separation of Pathogenic NPM1c

Nucleophosmin (NPM1) is a nucleolar protein that participates in many biological processes through phase separation. In acute myeloid leukemia (AML), nearly one-third of cases harbor NPM1c mutations. Previous studies mainly focused on the contribution of the additional NES in NPM1c mislocalization and AML development; however, the mechanisms underlying the relationship between aberrant localization and phase separation of NPM1c remain poorly understood. In this study, we demonstrate that loss of phase separation (LoPS) with RNA is associated with NPM1c mislocalization, which uncovers a new strategy for therapeutic targeting of NPM1c. Leveraging high-throughput screening, we identify a potent small molecule capable of rescuing NPM1c LoPS in vitro and effectively restoring the nucleolar localization of NPM1c within cells. Remarkably, this compound demonstrates anti-leukemia activity in both AML cell lines and patient-derived blasts. Overall, these findings indicate a promising avenue for developing new AML therapies against NPM1c by reinstating its phase separation capabilities. One-Sentence SummaryRescuing the lost phase separation capacity of NPM1c unveils a promising AML therapy.

cancer biology↗

bioRxiv2025

Genome-wide analysis reveals the genetic basis of key agronomic traits and modern wheat breeding in Henan Province

Despite the continuous improvement of wheat cultivars in Henan Province, the largest Chinese wheat-producing area, there have been no reports of studies on their breeding advances in the past decades. This study aims to elucidate their genetic basis and breeding advances. A total of 351 landraces and 265 cultivars in Henan Province were employed in selective sweep studies and genome-wide association studies. All the wheat accessions were planted in five locations in Henan Province in 2016-2017, 2017-2018 and 2018-2019. Candidate genes were identified through differential expression, GO annotation, gene homology, and haplotype analyses. Breeding advances were summarized based on four breeding periods (1950s, 1960s-1970s, 1980s-1990s, and post-2000s) of all 265 cultivars. The results demonstrated that 263 known genes with selection signals were found to be associated with important traits. Around the detected quantitative trait loci, 51 genes had been previously reported, while 252 genes were identified as candidate genes. Of the 303 genes and loci identified, 80 genes and 50 loci contributed to the improvement of 12 traits like plant height, and 12 key candidate genes was verified by qRT-PCR, while superior haplotypes of 24 genes explained cultivar climate adaptation. The superior haplotypes of 164 genes are available for future breeding. Transmission of superior haplotypes in four varietal pedigrees revealed pyramiding of superior haplotypes in elite cultivars. The analysis of post-2000 cultivars suggests a broader range of germplasm resources should be considered in parental selection than only recent accessions. This study provides a genetic basis and guide for wheat improvement.

genetics↗

bioRxiv2025

Synergistic neuroprotective and cognitive-enhancing effects of Walnut Peptide and Theanine in human brain organoid and mouse stress models

Stress is a prevalent mental health concern emerging predominantly in late adolescence or early adulthood. Since 2007, the Food and Drug Administration (FDA) has not approved any novel anxiolytic pharmaceuticals, fueling interest in nutritional supplements as alternative therapies for stress management. Building on prior zebrafish research, this study investigates the synergistic effects of Theanine (Th) and Walnut Peptide (WP) on stress mitigation and cognitive enhancement. Utilizing the human brain organoid stress (BO-stress) model, WP+ Th were observed to reduce stress and regulate expression of neurotransmitters, including Gamma-Aminobutyric Acid (GABA), serotonin (5-HT), dopamine (DA), and acetylcholine (Ach), as well as brain-derived neurotrophic factor (BDNF) and serotonin transporter (SERT). Subsequent in vivo study using C57BL/6J mouse-stress model demonstrated that the treatment (Th 85 mg/mL + WP 200 mg/mL), or administrated with vehicles, significantly improved their performance in stress and cognitive assessments, partially normalized neurotransmitter imbalances by modulating SERT and BDNF expression. These findings highlighted the potential of using WP + Th, particularly when delivered with vehicles (eg: powder/yogurt/milk), as a novel combined therapeutic approach for stress management and cognitive enhancement. We performed a correlation analysis between BO-stress model and mouse-stress model, revealing a alignement in the SERT levels.In addition, SERT was highly correlated with other markers in the mouse hippocampus and may represent a key target for modulating the balance between stress and cognition. Significance StatementThis study established an innovative human brain organoid-stress model and, in conjunction with mouse-stress model, elucidated the synergistic actions of Theanine and Walnut Peptide in mitigating stress and augmenting cognitive function. Their beneficial effects were mediated through the regulation of SERT and BDNF, presenting a promising non-pharmacological avenue for mental health care. In addition, species differences between humans and mice were also examined through correlation analysis of brain organoids and mouse models. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/640260v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1a81886org.highwire.dtl.DTLVardef@e2281dorg.highwire.dtl.DTLVardef@1b7b167org.highwire.dtl.DTLVardef@dff9aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Neural variability reliably and selectively encodes pain discriminability

Neural activity varies dramatically across time. While such variability has been associated with cognition, its relationship with pain remains largely unexplored. Here, we systematically investigated the relationship between neural variability and pain, particularly pain discriminability, in five large electroencephalography (EEG) datasets (total N = 489), collected from healthy individuals (Datasets 1-4) and patients with postherpetic neuralgia (PHN; Dataset 5) who had received painful or nonpainful sensory stimuli. We found robust correlations between neural variability and interindividual pain discriminability. These correlations were (1) replicable in multiple datasets, (2) pain selective, as no significant correlations were observed in nonpain modalities, and (3) clinically relevant, as they were partly disrupted in patients with PHN. Importantly, variability and amplitude of EEG signals were mutually independent and had distinct temporal and oscillatory profiles in encoding pain discriminability. These findings demonstrate that neural variability is a replicable and selective indicator of pain discriminability above and beyond amplitude, thereby enhancing the understanding of neural encoding of pain discriminability and underscoring the value of neural variability in pain studies.

neuroscience↗

bioRxiv2025

RegFormer: A Single-Cell Foundation Model Powered by Gene Regulatory Hierarchies

Single-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of cellular diversity, but current computational models often fail to incorporate regulatory priors, handle data sparsity, or efficiently process long gene sequences. Here, we present RegFormer, a foundation model that integrates gene regulatory networks (GRNs) with Mamba-based state-space modeling, overcoming the scalability and context-length limitations of Transformer architectures. RegFormer encodes each gene through dual embeddings, a value embedding for quantitative expression and a token embedding for regulatory identity, organized within a GRN-guided gene order to capture both expression dynamics and hierarchical regulation. Pretrained on 26 million human single cells spanning 45 tissues and diverse biological contexts, RegFormer achieves superior scalability and biological fidelity. Across comprehensive benchmarks, it consistently outperforms state-of-the-art single-cell foundation models (scGPT, Geneformer, scFoundation, and scBERT), delivering higher clustering accuracy, improved batch integration, and more precise cell type annotation. RegFormer also reconstructs biologically coherent GRNs, accurately models transcriptional responses to genetic perturbations, and enhances drug response prediction across cancer cell lines. By combining regulatory priors with efficient long-sequence Mamba modeling, RegFormer establishes a biologically grounded and scalable framework for single-cell representation learning, enabling deeper mechanistic insight into gene regulation and cellular state transitions.

bioinformatics↗

bioRxiv2025

ViFIT-assisted Histopathology: From H&E Style Standardization to Virtual Fiber Image Transformation

Deep learning-based virtual fiber staining provides a promising complement to routine H&E pathology. However, the reliance on predefined staining style inputs and manual intervention limits the clinical applicability of existing methods. To address these challenges, we introduce ViFIT-assisted histopathology, a two-stage diagnostic approach that integrates our proposed unsupervised deep learning-based virtual fiber transformation model (ViFIT). This approach enables the conversion of H&E-stained images with diverse styles into pathologist-preferred H&E images, while simultaneously generating content-consistent virtual fiber images containing label-free collagen fibers and stained reticular and elastic fibers. ViFIT-assisted histopathology reveals tumor-associated fibers and provides quantitative metrics across multiple intraoperative and postoperative cases. Experimental results demonstrate that ViFIT significantly outperforms state-of-the-art unsupervised methods in both style standardization and virtual staining, across various downstream tasks and cancer types. By eliminating the need for staining variation and manual annotation, ViFIT-assisted histopathology streamlines histopathology workflows, making it well-suited for multi-center consultations and differential diagnosis.

pathology↗

bioRxiv2025

Volatile-suppressed peptide signaling enhances volatile responses in plant-plant interactions

Plant volatiles shape plant-plant interactions by acting as defense regulators and response factors. While plant volatile biosynthesis is well understood, how their emission is regulated remains largely elusive. Here, we show that small peptide signaling regulates induced volatile release in maize. Following herbivore attack, green leaf volatiles such as (Z)-3-hexenyl acetate (HAC) are released and induce terpene and indole emissions from neighboring plants. This process is accompanied by reduced expression of the ZmCLE1E9 gene and the ZmBAM1A, ZmBAM1B and ZmBAM3C receptor genes in HAC-exposed plants. Exogenous ZmCLE1E9 peptide inhibits HAC-triggered volatile release by limiting stomatal aperture. This inhibition disappears in the Zmbam1a/Zmbam1b/Zmbam3c triple mutant. Molecular docking supports ZmCLE1E9 and ZmBAMs as ligand-receptor pairs. Furthermore, Zmcle1e9 and Zmbams triple mutants show increased volatile emissions upon HAC exposure. In summary, we show that upon HAC perception, maize plants enhance their capacity to release terpenes and indole via the suppression of CLE1E9 signaling. This behavior allows maize plants to rapidly deploy volatile cues in response to stress volatiles and thus shape the infochemical dynamics of multitrophic environments.

plant biology↗

bioRxiv2025

Label-free Detection and Characterization of Metabolism in Fresh and Cryopreserved Macrophages

Cryopreservation is a widely used technique to preserve biological samples for extended periods of time at low temperatures. Even though it is known to have significant effects on cell viability, its effect on their metabolism remains unexplored. Studying how cryopreservation influences the metabolism of cells is important to guarantee the reliability of samples transported between sites for analysis. Optical metabolic imaging allows for the study of cellular metabolism in a label-free manner by using the autofluorescence properties of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), two metabolic coenzymes. The goal of this research is to study the metabolic changes in macrophages after cryopreservation and compare these results with freshly isolated macrophage samples to evaluate if the metabolic data is retained after cryopreservation. The metabolism of macrophages was analyzed with fluorescence lifetime imaging microscopy (FLIM) using a multiphoton microscope. Monocytes were isolated from human whole blood and separated into two groups. In Group 1, freshly isolated monocytes were differentiated into macrophages using macrophage-colony stimulating factor (M-CSF) over 8 days. In Group 2, isolated monocytes were cryopreserved after separation from whole blood and then thawed and stimulated with M-CSF for 8 days. Single-cell analysis showed there are significant changes in FLIM parameters between the groups suggesting that the metabolic data of macrophages is altered after a cryopreservation and cell thawing cycle. Our research bridges the current gap by studying the metabolic changes of cells after cryopreservation using a non-invasive and label-free imaging technique.

bioengineering↗

bioRxiv2025

Optical metabolic imaging identifies metabolic shifts and mitochondria heterogeneity in POLG mutator macrophages

The polymerase gamma (POLG) gene mutation is associated with mitochondria and metabolism disorders, resulting in heterogeneous responses to immunological activation and posing challenges for mitochondrial disease therapy. Optical metabolic imaging captures the autofluorescent signal of two coenzymes, NADH and FAD, and offers a label-free approach to detect cellular metabolic phenotypes, track mitochondria morphology, and quantify metabolic heterogeneity. In this study, fluorescence lifetime imaging (FLIM) of NAD(P)H and FAD revealed that POLG mutator macrophages exhibit a decreased NAD(P)H lifetime, and optical redox ratio compared to the wild-type macrophages, indicating an increased dependence on glycolysis. FLIM revealed that both wild-type and POLG mutator macrophages switch to a decreased NAD(P)H{tau} 1, and{tau} m after immune stimulation by Lipopolysaccharides (LPS). Furthermore, a bimodality index of subpopulation analysis identified heterogenous populations of POLG mutator macrophage responses under immune challenge by LPS. Moreover, to quantify the mitochondria variations in POLG mutator macrophages, a customized thresholding image processing pipeline was developed to segment mitochondria regions within each cell from the NADH image, allowing for the feature analysis of mitochondria clusters. Consequently, the wild-type macrophages exhibited a higher percentage of mitochondria-containing pixels and longer lengths of connected mitochondria, as compared with POLG mutated macrophages. Altogether, these results illustrate the potential of optical metabolic imaging for non-invasive detection and quantification of cellular metabolism, metabolic heterogeneity within cell populations, and intra-cellular mitochondria morphology differences in POLG mutator macrophages. Optical metabolic imaging will be valuable for studying POLG-mutation diseases and evaluating efficacy of potential therapies.

cell biology↗

bioRxiv2025

Activity dependent Clustering of Neuronal L-Type Calcium Channels by CaMKII

Neuronal depolarization activates L-type voltage-gated Ca{superscript 2} channels (LTCCs), increasing local Ca{superscript 2} concentrations to initiate excitation-transcription (E-T) coupling. We show that depolarization enhances clustering of CaV1.2 and CaV1.3 LTCCs in cultured hippocampal neurons, coinciding with increased nuclear CREB phosphorylation. LTCC clustering and LTCC-dependent CREB phosphorylation are selectively disrupted by 1,6-hexanediol, implicating biomolecular condensation. Activated CaMKII holoenzymes assemble complexes containing multiple CaV1.2 and/or CaV1.3 1 subunits. Complex assembly is facilitated by co-expression of CaMKII-binding {beta}2a subunits and Shank3 and selectively disrupted by 1,6-hexanediol. In HEK293 cells, pharmacological LTCC activation enhances clustering only when wild-type CaMKII is co-expressed. A CaMKII mutant that cannot bind LTCC N-terminal domains fails to support LTCC subunit complex formation in vitro and LTCC clustering in HEK293 cells. In neurons, the knockdown of CaMKII expression disrupts depolarization-induced (co-)clustering of CaV1.2 and CaV1.3. Together, these findings indicate that CaMKII-dependent clustering of plasma membrane LTCCs via biomolecular condensation is essential for initiating long-range signaling to activate gene expression following neuronal depolarization.

neuroscience↗

bioRxiv2024

Phosphotyrosine Signal Profiling of Clinical CAR-T Reveals Tonic Signaling Associated with Therapeutic Efficacy

Chimeric Antigen Receptor T (CAR-T) therapies have revolutionized the treatment of cancers such as relapsed and refractory B cell malignancies. However, the precise therapeutic mechanism of CAR-T action remain to be elucidated. In this study, we systematically analyzed CAR signaling via phosphotyrosine (pTyr) proteomics in CAR-T cells from both clinical patients and healthy donors. We found that CAR-T products from clinical patients displayed heightened tyrosine phosphorylation, particularly in the JAK-STAT, MAPK and TCR signaling cascades. We also identified the significantly regulated pTyr sites in primary CAR-T cells under tonic signaling or upon stimulation by antigen-presenting CD19-K562 cells. Although both CD28{zeta} and 4-1BB{zeta} CAR-T cells exhibited comparable pTyr changes, CD28{zeta} CAR-T cells displayed a more pronounced activation in the TCR signaling pathway. Additionally, comparative analysis between clinical and primary CAR-T cells suggested that CAR-T products were subject to heightened tonic signaling, which may be related to therapeutic relapse. Our findings reveal the state of clinical CAR-T products and refine the CAR-T signal transduction network, providing comprehensive insights and informed guidance for CAR-T therapies.

immunology↗