Biology subjects

Dopazo, A.

Publications and source records attributed to Dopazo, A..

13 recordsLinked to original sources

bioRxiv2026

Extracellular Vesicles from Senescent Tumor Cells Are Necessary and Sufficient to Drive Paracrine Senescence

Cellular senescence exerts powerful non-cell autonomous effects through the senescencelzlassociated secretory phenotype (SASP). This SASP comprises soluble factors and extracellular vesicles (EVs). Although soluble SASP components can induce senescence in neigbouring cells, the specific contribution of EVs to paracrine senescence is poorly defined. Here, we show that EVs released by senescent tumor cells are necessary and sufficient to propagate senescence. Conditioned media from bleomycinlzlinduced senescent A549 cells triggered a permanent growth arrest with morphological changes and upregulation of senescence markers in recipient tumor cells. Pharmacological inhibition of EV biogenesis using GW4869 or genetic downregulation of the EV secretion mediator RAB27A markedly attenuates paracrine senescence without affecting soluble SASP factor secretion or the senescent state of producer cells. Proteomic characterization reveals that senescent EVs exhibit a distinct molecular signature enriched for extracellular components and processes related to wound healing and hemostasis. Importantly, purified senescent EVs, devoid of soluble SASP factors, fully recapitulated paracrine senescence induction. These findings identify senescent EVs as key autonomous SASP effectors and highlight vesicular pathways as potential therapeutic targets in cancer and therapylzlinduced senescence.

cancer biology↗

bioRxiv2025

HDL-associated phosphatidylserine blunts myeloid activation and protects from atherosclerosis

Background and aimsLipids play a critical role in atherosclerosis. Low-density lipoprotein (LDL)-cholesterol and certain lipid classes like sphingomyelins are associated with inflammation and poor cardiovascular outcomes. Phosphatidylserine (PS), on the other hand, is a negatively charged anti-inflammatory phospholipid class involved in efferocytosis. In this study, we sought to investigate its anti-atherosclerotic properties through a combination of complementary human lipidomics analyses, in vitro assays and in vivo experiments in Apoe-/- mice. MethodsHuman lipidomics studies were performed on the 300OB cohort comprising 300 obese and overweight individuals at risk of cardiovascular disease. In vitro assays were carried out using human monocytes and macrophages, and in vivo experiments included histopathological, immunophenotyping and single-cell transcriptomic analyses. ResultsIn humans, we identified PS as an anti-inflammatory and atheroprotective biomarker. Hence, we developed a high-density lipoprotein (HDL)-like formulation enriched in PS to exploit its properties in a targeted fashion in mice. In vitro, this formulation potently inhibited inflammatory cytokine production on human myeloid cells. Our in-depth in vivo experiments provided evidence of the formulations potent plaque-stabilizing and anti-inflammatory actions. These effects were mediated by a shift in the monocyte/macrophage compartment toward homeostatic/repairing phenotypes. ConclusionsCollectively, our results demonstrate that HDL-associated PS potently suppresses inflammation and atheroprogression, and holds promise as a viable approach to improve immunomodulatory therapies.

immunology↗

bioRxiv2025

A high-resolution, unbiased analysis of the cellular immune response to Epstein-Barr virus

More than 95% of humans are infected with Epstein-Barr virus (EBV), yet although EBV infection has been associated with inflammatory and autoimmune diseases, lymphoproliferative disorders, and several types of cancer, for the vast majority of infected people the infection is asymptomatic as EBV replication is controlled by the immune system. Immunity against this virus has been studied since the discovery of EBV in the 1960s, and although important insights have been made, no unbiased, global studies of immune responses to EBV in healthy seropositive subjects have been reported. Here we describe a novel protocol to study the cellular immune response to EBV, detecting lymphocytes that respond to EBV via analyses of proliferation or induced expression of activation markers and cytokines. Using this system we sequenced, for the first time at a single-cell level, the transcriptome of all cells capable of responding to EBV in healthy individuals and in patients with inborn errors of immunity (IEI) associated with susceptibility to EBV infection. Lymphocyte cytotoxicity appears to be crucial for the proper control of EBV-infection, while a proportionate T-regulatory cell response likely helps to avoid excessive immunity and immune pathology. We also show that {gamma}{delta} T cells expressing the TCR V{delta}1 chain use various activating natural killer (NK) cell receptors to recognise and kill EBV-infected lymphoblastoid cell lines (LCLs) and thus could be a promising candidate for allogeneic cell therapy for EBV-associated lymphoproliferative disorders in patients with either primary or secondary immunodeficiencies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/681317v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@116ad85org.highwire.dtl.DTLVardef@1da33d7org.highwire.dtl.DTLVardef@81a2a6org.highwire.dtl.DTLVardef@dd571d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

CIRCADIAN MODULATION OF NEUTROPHIL FUNCTION DETERMINES COLLATERAL PERFUSION AND OUTCOME AFTER ISCHEMIC STROKE

Stroke is a leading cause of mortality and disability, driven by complex and time-dependent mechanisms that aggravate ischemic damage. Among them, collateral perfusion determines the initial size of the ischemic core, the rate of its expansion, and the extent of the penumbra both at stroke onset and over time. Insufficiency of collaterals may occur due to genetic factors or other determinants, such as aging or cardiovascular risk factors, which reduce the number of collaterals or the diameter of those that remain. But aspects of less structural nature could also affect the effectiveness of these pathways by decreasing their patency. We hereby show that diurnal fluctuations in infarct volume in ischemic stroke mouse models are neutrophil phenotype-dependent, since differences in infarct volumes were abolished by depleting neutrophils or blocking their circadian clock, and linked to the collateral circulation: during the inactive phase of mice (daytime), collateral perfusion in the ipsilesional hemisphere was reduced, coinciding with an increase in intravascular neutrophil accumulation, suggestive of microvascular stalling. Single-cell transcriptomics, ex vivo functional assays and in vivo pharmacological and genetic strategies confirmed enhanced neutrophil extracellular traps (NETs) formation at this time. Importantly, in a cohort of human stroke patients, we identified diurnal oscillations in neutrophil and NET-related biomarkers, peaking during the human inactive phase (evening/night), and similarly associated with reduced collateral flow and poorer clinical outcomes. These findings underscore the critical role of neutrophils, their circadian dynamics and NET release in driving collateral insufficiency and ischemic brain damage, suggesting novel personalized therapeutic strategies based on circadian rhythms for the treatment of stroke.

immunology↗

bioRxiv2024

Deficient Memory, Long-Term Potentiation and Hippocampal Synaptic Plasticity in Galectin-4-KO Mice.

BACKGROUNDBrain function is influenced by the gut through the microbiota-gut-brain axis. Non-physiological microbiota-depletion or induced gut infection in animal models, have been instrumental to link intestinal alterations to cognitive and mood dysfunctions. However, the effects of specific, controlled, physiologically relevant shifts in commensal microbiota composition on brain function remain poorly understood. METHODSMice deficient in galectin-4 (Lgals4-KO) were used in this study. Gut microbiota was analysed by 16S-rRNA sequencing. Cognitive and mood status were evaluated with specific behavioral tests. Long-term potentiation (LTP) was tested ex vivo and in vivo by electrophysiological methods and in vitro by immunofluorescence and western blot. RNA-sequencing was used for transcriptomic analyses. Golgi-Cox staining and transmission electron microscopy were used for quantitative and morphological assessments of dendritic spines and synapses. RESULTSLgals4-KO mice present an altered intestinal commensal microbiota in the absence of pathogens, deficient memory formation, and impaired hippocampal LTP in vivo and ex vivo. Furthermore, Lgals4-KO neurons show a reduced activation of AMPA receptors and of CaMKII upon chemically induced LTP in vitro. These mice also display significantly lower dendritic spine density and shorter spine length in hippocampal dendrites, as well as an increased area of the postsynaptic densities CONCLUSIONSOur results define a new role for galectin-4 in the modulation of commensal bacteria. We also show that the absence of galectin-4 induces changes in gut microbial composition, along with synaptic alterations and memory impairment, supporting our hypothesis that variations in endogenous microbiota may cause or contribute to relevant neurological pathologies.

neuroscience↗

bioRxiv2024

Role of Lamin A/C on dendritic cell function in antiviral immunity

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFN{gamma}-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFN{gamma}-producing -CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naive CD4 T cells. Lamin A/C deletion induces alterations in NF{kappa}B nuclear localization, thereby influencing NF{kappa}B-dependent transcription. Furthermore, lamin A/C ablation modifies the epigenetic signature of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and elucidates the molecular mechanisms through which lamin A/C modulates DC function via epigenetic and transcriptional regulation.

immunology↗

bioRxiv2024

Evolutionary convergence of sensory circuits in the pallium of amniotes

The amniote pallium contains sensory circuits structurally and functionally equivalent, yet their evolutionary relationship remains unresolved. Our study employs birthdating analysis, single-cell RNA and spatial transcriptomics, and mathematical modeling to compare the development and evolution of known pallial circuits across birds (chick), lizards (gecko) and mammals (mouse). We reveal that neurons within these circuits stations are generated at varying developmental times and brain regions across species, and found an early developmental divergence in the transcriptomic progression of glutamatergic neurons. Together, we show divergent developmental and evolutionary trajectories in the pallial cell types of sauropsids and mammals. Our research highlights significant differences in circuit construction rules among species and pallial regions. Interestingly, despite these developmental distinctions, the sensory circuits in birds and mammals appear functionally similar, which suggest the convergence of high-order sensory processing across amniote lineages.

developmental biology↗

bioRxiv2023

BirthSeq, a new method to isolate and analyze dated cells from any tissue in vertebrates

Embryonic development is a complex and dynamic process that unfolds over time and involves the production of increasing numbers of cells, as well as the diversification of different cell types. The impact of developmental time on the formation of the central nervous system is well-documented, with evidence showing that time plays a critical role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via FACS, and analysis using high-throughput techniques. We demonstrate the effectiveness of BirthSeq for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in brain development across three vertebrate species (mouse, chick, and gecko). Overall, BirthSeq provides a versatile tool for studying any tissue in any vertebrate organism, helping to fill the necessity in developmental biology research by targeting cells and their temporal cues. SUMMARY STATEMENTBirthSeq allows the isolation and investigation of alive cells according to their birthdate, in any kind of tissue and vertebrate species.

developmental biology↗

bioRxiv2023

NGFR regulates germinal center B-cell activation and negative selection

The expression of the Nerve growth factor receptor (NGFR) has been described in follicular dendritic cells (FDCs), the major lymphoid stromal cell (LSC) compartment regulating B-cell activation within germinal centers (GCs). However, the role of NGFR in humoral response is not well defined. In this work, we have studied the effect of Ngfr KO in LNs organization and function. Ngfr KO led to spontaneous GC formation and expansion of GC B-cell compartment which were related to Ngfr depletion in non-hematopoietic radioresistant compartment. In agreement, Ngfr KO mice showed alterations in LSC with an increased frequency of FDCs harboring an activated phenotype characterized by the overexpression of CD21/35, MAdCAM-1, and VCAM-1. Moreover, Ngfr KO mice showed GC ectopic location, loss of polarization, impaired high-affinity antibody production, and increased circulating autoantibodies. In addition, Ngfr KO/Bcl2 Tg mice displayed increased levels of autoantibodies, higher incidence of autoimmunity, and decreased overall survival. Our work shows that NGFR maintains GC structure and functionality, being involved in the regulation of antibody production and immune tolerance.

immunology↗

bioRxiv2023

Long-term cytotoxic NK cells with broad anti-tumour capacity proliferate selectively, without exhaustion, after BCG priming and extremely low doses of cytokines

Background: Natural killer (NK) cell-based immunotherapies, currently under investigation, appear to be safe, efficient treatments in patients with haematological tumours. Nevertheless, the short-lived nature of these cells combined with the need to infuse large number of cells for efficient tumour elimination represent important challenges for the development of NK cell-based therapies. Although NK cell anti-tumour activity is regulated by cytokines, constant stimulation together with the immunosuppressive tumour environment can result in NK cell exhaustion. Therefore, improved approaches to produce highly cytotoxic and longer-lived NK cells are of considerable clinical interest. Methods: Peripheral blood mononuclear cells (PBMC) are primed in vitro with a pulse of either Bacillus Calmette-Guerin (BCG) vaccine or a cell wall extract of M. bovis, followed by weekly stimulations with low doses of IL12, 15 and 21. The phenotype and anti-tumour fitness of the activated NK cell culture were examined using scRNA-seq, flow cytometry and functional assays, including degranulation, specific cytotoxicity and IFN{gamma} release. Results: we describe a novel strategy for the generation of long-lived activated NK cells capable of killing a broad range of solid tumours. A unique subset of cytotoxic NK cells (CD56high CD16+ NKG2A+) specifically proliferated in vitro, and was further expanded without functional exhaustion under minimal survival cytokine combinations. Mycobacterial cell-wall fractions also activated NK cells that recognised tumours efficiently, and proliferated well, and this approach has the advantage that no live bacteria are present in the cultures. Conclusions: We propose that BCG-priming to expand anti-tumour NK cells, without cell sorting, could be a scalable and economical basis for the development of safe and universal cellular immunotherapies against solid tumours. Key messagesAdoptive therapy with sorted NK cells grown in IL12, 15, 18 are being tested in clinical trials, but are only efficient for haematological tumours. In addition, their survival in vivo is limited. Here, we define culture conditions that drive the selective proliferation of long-lived natural killer (NK) cells, without the need of cell sorting, in minimal doses of cytokines, after priming with BCG or mycobacteria components. BCG-primed NK cells grow and maintain effective cytotoxic function against a variety of solid tumours in vitro, without exhaustion for at least 28 days of culture. This new approach provides the basis for the generation of innate adoptive cell therapy tools.

immunology↗

bioRxiv2023

Incongruence between transcriptional and vascular pathophysiological cell states

The Notch pathway is a major regulator of transcriptional specification and vascular biology. Previous studies have suggested that targeting the ligand Dll4 or the Notch-receptors results in similar molecular and angiogenesis outcomes. Here, we analyzed single and compound genetic mutants for all Notch signaling members and found very significant differences in the way ligands and receptors regulate vascular homeostasis. Loss of Notch receptors, leads to minor vascular pathology featuring hypermitogenic MAPK-driven cell-cycle arrest and senescence. In contrast, loss of Dll4 triggers a strong Myc-driven switch towards cell proliferation and sprouting and major organ pathology. Targeting of Myc completely suppressed the proliferative and tip-cell angiogenic states induced by Dll4 loss-of-function, however, this did not avoid vascular pathology. Only VEGF blockade prevented the pathology induced by Dll4 loss, but without fully suppressing its transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states and adult vascular phenotypes and related pathophysiology.

physiology↗

bioRxiv2022

Antiviral State of CD1c+ cDC contributes to Increased Maturation and Activation of Cytotoxic Natural Killer cells in Sjogrens Syndrome

ObjectivePrimary Sjogrens syndrome (pSS) is an inflammatory autoimmune disorder characterized by damage of exocrine glands and linked to IFN responses and the induction of autoreactive adaptive immune cells. However, the role of innate immune cells in pSS pathology remains understudied. MethodsWe studied differential phenotypical characteristics of different NK cell, conventional dendritic cell (cDC) and monocyte subsets in the blood and salivary glands from pSS individuals. Transcriptional patterns of circulating cDC and Mo from pSS and healthy controls were also compared. Finally, in vivo alterations in these cell populations in the salivary gland were investigated in a mouse model. ResultsHere, we identified CD16+ CD56hi NK cells enriched in pSS patients which associates with higher natural cytotoxic function and increased proportions of circulating CD64+ CD1c+ cDC exhibiting antiviral transcriptional IFN signatures. CD64hi cDC and NK cell were detected infiltrated into the salivary glands from pSS patients and a murine SS model. CD1c+ cDC from patients with pSS expressed high levels of ligands for activating NK receptors and increased ability to activate NK cells ex vivo. Finally, the antiviral RIG-I and DDX60 sensors regulated the expression of NK cell receptor ligands on CD1c+ cDC. ConclusionsTherefore, the interplay of CD1c+ cDCs and NK cells could contribute to pSS pathology.

immunology↗

bioRxiv2021

Single cell clonal analysis identifies an AID-dependent pathway of plasma cell differentiation

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody secreting plasma cells. Activation Induced Deaminase (AID) initiates antibody diversification in GCs by somatic hypermutation and class switch recombination. Here we have addressed the role of AID in the terminal differentiation of GC B cells by combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells. We identified 8 transcriptional clusters that include dark zone and light zone GC subsets, plasmablasts/plasma cells (PB), 4 subsets of MBCs and a novel prePB subset, which shares the strongest clonal relationships with PBs. Mice lacking AID have various alterations in the size and expression profiles of these transcriptional clusters. We find that AID deficiency leads to a reduced proportion of prePB cells and severely impairs transitions between the prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

immunology↗