Biology subjects

Thelen, M.

Publications and source records attributed to Thelen, M..

11 recordsLinked to original sources

bioRxiv2026

Memory T Cells in MHC-Deficient Humanized Mice

Major histocompatibility complexes (MHC) govern antigen presentation and T cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC TCR interactions. Humanized NOD scid IL2null (NSG) mice engrafted with human CD34+ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T cell maturation in vivo. CD34+ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single cell TCR sequencing revealed marked differences in T cell differentiation across models. Busulfan conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naive, NKT, and regulatory T cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft versus host disease and represent a refined enabling platform for human T cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.

immunology↗

bioRxiv2026

GRK-dependent ACKR3 endocytosis and chemokine scavenging is independent of receptor phosphorylation and beta-arrestin

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

cell biology↗

bioRxiv2025

Establishment and Characterization of a CCND1-Rearranged Non-Mantle Cell Lymphoma Cell Line and Patient-Derived Xenograft Model

The pathobiology of aggressive B-cell lymphomas with CCND1 rearrangements, distinct from Mantle Cell Lymphoma (MCL), presents a significant clinical challenge. These lymphomas are often difficult to diagnose and demonstrate resistance to standard immunochemotherapy, underscoring the urgent need for a deeper understanding of their underlying biology to develop more effective treatments. A major impediment to progress has been the lack of robust preclinical models that accurately reflect the complex genomics and clinical behavior of this disease. Here we directly address this critical gap by reporting the establishment and in-depth characterization of the first patient-derived cell line and a corresponding systemic patient-derived xenograft (PDX) model of a CCND1-rearranged, non-MCL lymphoma with a rapidly fatal clinical course. Through a comprehensive multi-omics approach, we demonstrate that these novel in vitro and in vivo models faithfully recapitulate the primary tumors unique immunophenotype, its intricate genetic and transcriptional landscape, and its intrinsic resistance to conventional therapeutic agents.

cancer biology↗

bioRxiv2024

ACKR5/GPR182 is a scavenger receptor for the atypical chemokine CXCL17, GPR15L and various endogenous peptides

GPR182/ACKR5, the most recently deorphanized chemokine receptor, is mainly expressed on endothelial cells and was proposed to act as a scavenger regulating the availability of a large set of chemokines. In this study, we first established the exact profiles and ranking of the chemokines binding to the human and mouse GPR182. We confirmed the high promiscuity of GPR182 towards XC, CC and CXC chemokines and a clear difference in the chemokine repertoires of the human and mouse orthologues. We next demonstrated that, beyond classical chemokines, GPR182 exhibits potent binding to the chemoattractant protein GPR15L/C10orf99, the atypical chemokine CXCL17 and various endogenous peptides, mainly from the opioid, apelin, and PACAP families. We also showed that these newly identified ligands engage GPR182 through varied binding modes. While GPR15L, just like classical chemokines, predominantly engages GPR182 via its N terminus, conversely to the C terminus-dependent binding to its cognate receptor GPR15, CXCL17 exhibits a more complex interaction, relying on both the N and C terminus. The binding mode of the newly identified peptide ligands also differ from the interactions with their cognate receptors. Our findings establish the first scavenger receptor for CXCL17 and GPR15L and advance the understanding of GPR182 ligand interactions, suggesting a regulatory role beyond chemokines.

pharmacology and toxicology↗

bioRxiv2024

T-cell development and activation in humanized mice lacking mouse major histocompatibility complexes

Humanized mice transplanted with CD34+ hematopoietic progenitor cells (HPCs) are used to study human immune responses in vivo. However, the mismatch between the mouse major histocompatibility complexes (MHCs) and the human leukocyte antigens (HLAs) is not optimal for T-cell development and can trigger xenograft reactivity. We evaluated human T-cell development in NOD.Scid.Gamma mice lacking expression of MHC class I and II (NSG-DKO). Human leukocyte engraftment was detectable at 8 weeks post-transplantation. Human CD4+ and CD8+ T-cells were detectable in blood, thymus, bone marrow and spleen of humanized NSG-DKO mice for up to 20 weeks post-transplantation. Further, we evaluated the effects of lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN- and the human cytomegalovirus gB antigen. LV delivery promoted development and activation of human central memory, {beta} and {gamma}{delta} T-cells with amplifications of the T-cell repertoire. LV administration unleashed multiple reactome pathways such as type-I interferon responses, cell cycle and metabolic processes. In summary, development of human T-cells in humanized mice does not rely on mouse MHCs and can be boosted systemically via LV administration.

immunology↗

bioRxiv2022

ADeS: a deep learning based Apoptosis Detection System for live cell imaging.

Intravital microscopy has revolutionized live cell imaging by allowing the study of spatial-temporal cell dynamics in living animals. However, the complexity of the data generated by this technology has limited the development of effective computational tools to identify and quantify cell processes. Amongst them, apoptosis is a crucial form of regulated cell death involved in tissue homeostasis and host defense. Live-cell imaging enabled the study of apoptosis at the cellular level, enhancing our understanding of its spatial-temporal regulation. However, at present, no computational method can deliver robust detection of apoptosis in microscopy time-lapses. To overcome this limitation, we developed ADeS, a deep learning-based apoptosis detection system that employs the principle of activity recognition. We trained ADeS on extensive datasets containing more than 10,000 apoptotic instances collected both in vitro and in vivo, achieving a classification accuracy above 98% and outperforming state-of-the-art solutions. ADeS is the first method capable of detecting the location and duration of multiple apoptotic events in full microscopy time-lapses, surpassing human performance in the same task. We demonstrated the effectiveness and robustness of ADeS across various imaging modalities, cell types, and staining techniques. Finally, we employed ADeS to quantify cell survival in vitro and tissue damage in vivo, demonstrating its potential application in toxicity assays, treatment evaluation, and inflammatory dynamics. Our findings suggest that ADeS is a valuable tool for the accurate detection and quantification of apoptosis in live-cell imaging and, in particular, intravital microscopy data, providing insights into the complex spatial-temporal regulation of this process.

bioinformatics↗

bioRxiv2022

Dynamic Phaeodactylum tricornutum Exometabolites Shape Surrounding Bacterial Communities

The roles of exometabolites in mediating algal-bacterial interactions and regulating microbial community composition are not well understood. Here, we identified specific exometabolites from the model diatom Phaeodactylum tricornutum affecting abundance of specific bacterial taxa in isolation and in a community setting. We examined the response of a P. tricornutum-adapted enrichment community and found that both algal exudates and algal presence drove similar changes in community composition compared to controls. Using LC-MS/MS, we identified 50 metabolites produced by axenic P. tricornutum and found that different exometabolites accumulated during different algal growth phases. Profiling growth of 12 bacterial isolates representative of the enrichment community uncovered two algal exometabolites (out of 12 tested) which supported growth of a subset of isolates as a primary carbon source. We compared enrichment community response with and without the addition of two contrasting metabolites: 4-hydroxybenzoic acid, which supported isolate growth, and lumichrome, which did not. Exogenous metabolite additions did promote increased abundances of taxa that were able to utilize the metabolite in the isolate study, but also revealed the importance of factors relating to algal presence in regulating community composition. Collectively, this work demonstrates the influence of specific algal exometabolites in driving microbial community composition.

microbiology↗

bioRxiv2022

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence SummaryNaturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.

immunology↗

bioRxiv2022

A pipeline to track unlabeled cells in wide migration chambers using pseudofluorescence

Cell migration is a pivotal biological process, whose dysregulation is found in many diseases including inflammation and cancer. Advances in microscopy technologies allow now to study cell migration in vitro, within microenvironments that resemble in vivo conditions. However, when cells are observed within large 3D migration chambers at low magnification and for extended periods of time, data analysis becomes difficult. Indeed, cell detection and tracking are hampered due to the large pixel size, the possible low signal-to-noise ratio and distortions in the cell shape due to changes in the z-axis position. Although fluorescent staining can be used to facilitate cell detection, it may alter cell behavior and suffer from fluorescence loss over time (photobleaching). Here we describe the application of an image analysis pipeline based on deep learning to convert the transmitted light signal from unlabeled lymphoma cells to pseudofluorescence. Such pipeline confers a significant improvement in tracking accuracy while not suffering from photobleaching. This is reflected in the possibility of tracking cells for three-fold longer periods of time.

cell biology↗

bioRxiv2021

Small peptides modulate the immune function of the ion channel-like protein ACD6 in Arabidopsis thaliana

ACCELERATED CELL DEATH 6 (ACD6) mediates a trade-off between growth and defense in Arabidopsis thaliana. However, the precise biochemical mechanism by which ACD6 and related proteins in plants act remains enigmatic. Here, we identified two loci, MODULATOR OF HYPERACTIVE ACD6 1 (MHA1) and its paralog MHA1-LIKE (MHA1L), that code for [~]7 kDa proteins that differentially interact with specific ACD6 variants. MHA1L enhances accumulation of an ACD6 complex, thereby increasing activity of the ACD6 standard allele for regulating plant growth and defenses. ACD6 is a multipass transmembrane protein with intracellular ankyrin repeats that are structurally similar to those found in mammalian ion channels. Several lines of evidence link increased ACD6 activity to enhanced calcium influx, likely mediated by ACD6 itself and with MHA1L as a direct regulator of ACD6.

plant biology↗

bioRxiv2019

Semi-supervised machine learning facilitates cell colocalization and tracking in intravital microscopy

2-photon intravital microscopy (2P-IVM) is a key technique to investigate cell migration and cell-to-cell interactions in organs and tissues of living organisms. Focusing on immunology, 2P-IVM allowed recording videos of leukocytes during the immune response, highlighting unprecedented mechanisms of the immune system. However, the automatic analysis of the acquired videos remains challenging and poorly reproducible. In fact, both manual curation of results and tuning of bioimaging software parameters among different experiments, are required. One of the most difficult tasks for a user is transferring to a computer the knowledge on what a cell is and how it should appear with respect to the background, other objects, or other cell types. This is possibly due to the low specificity of acquisition channels which may include multiple cell populations and the presence of similar objects in the background. In this work, we propose a method based on semi-supervised machine learning to facilitate colocalization. In line with recently proposed approaches for pixel classification, the method requires the user to draw some lines on the cells of interest and some line on the other objects/background. These lines embed knowledge, not only on which pixel belongs to a class or which pixel belongs to another class but also on how pixels in the same object are connected. Hence, the proposed method exploits the information from the lines to create an additional imaging channel that is specific for the cells fo interest. The usage of this method increased tracking accuracy on a dataset of challenging 2P-IVM videos of leukocytes. Additionally, it allowed processing multiple samples of the same experiment keeping the same mathematical model.

systems biology↗