Biology subjects

Hoth, M.

Publications and source records attributed to Hoth, M..

14 recordsLinked to original sources

bioRxiv2026

R-CHOP for non-Hodgkin lymphoma: Its effects on cancer cells, NK cell viability and cytotoxicity against B-lymphoma cell lines

Combining the anti-CD20 antibody rituximab with the chemotherapeutic drugs vincristine, doxorubicin, and cyclophosphamide plus the corticosteroid prednisone (R-CHOP) has been the standard first line-line therapy for many non-Hodgkin lymphomas including diffuse large B-cell lymphoma (DLBCL) for more than 20 years. Natural killer (NK) cells are considered major mediators of rituximab-induced antibody-dependent cellular cytotoxicity (ADCC). While the anti-tumor effects of vincristine, doxorubicin, and cyclophosphamide are well-documented, their impact on NK cell viability and effector function remains poorly characterized. We evaluated the single-agent and combinatorial efficacy of vincristine, doxorubicin, and the cyclophosphamide surrogate mafosfamide in four DLBCL cell lines (TMD8, WSU-DLCL2, U-2932, and RI-1) relative to their impact on NK cell survival and effector function. In single-agent experiments, vincristine eradicated all B-cell lines in a dose-dependent manner at clinically relevant concentrations, whereas doxorubicin and mafosfamide required higher doses to achieve comparable efficacy. Although all three agents reduced NK cell viability over time at clinically relevant concentrations, impaired NK cell cytotoxicity was observed only at supra-clinical doses. Combinatorial experiments revealed antagonistic interactions between vincristine and doxorubicin, without evidence of significant synergistic effects. Incorporating tumor and NK cell survival into a mathematical model support a measurable trade-off between tumor cell reduction and immune cell viability. To assess the translational relevance of our in vitro findings, we analyzed a cohort of 32 patients with B-cell non-Hodgkin lymphoma. We confirmed NK cell depletion during R-CHOP and R-mini-CHOP therapy, however NK cell loss was significantly less pronounced in patients receiving dose-reduced R-mini-CHOP. Notably, the intrinsic cytotoxicity of residual NK cells was preserved under both treatment regimens, with a trend toward enhanced cytotoxicity during R-mini-CHOP treatment. These findings suggest that R-CHOP predominantly affects NK cell abundance rather than function and support the translational relevance of the in vitro results.

cancer biology↗

bioRxiv2025

The mechanism how Pretubulysin-induced microtubule disassembly improves T cell search efficiency

To clean tissue from tumorigenic and infected cells, cytotoxic T lymphocytes (CTLs) must navigate confined environments in vivo, locate the infected cells and eliminate them. Impaired CTL migration towards the tumor can limit the efficacy of immunotherapy. Microtubules (MTs) have emerged as promising targets, because destabilizing MTs enhances T-cell migration and subsequent killing, yet the underlying mechanisms are poorly understood. Here, we use pretubulysin, a potent MT depolymerizer, to uncover how MT dynamics regulate CTL motility. Complete MT disassembly markedly increased CTL infiltration and migration in 3D matrices. To investigate how altered migration affects target elimination, we employed a persistent random-walk model parameterized solely with experimental motility data. The model shows that the increase in speed and persistence induced by pretubulysin explains enhanced search efficiency increasing the encounter rate of CTLs with target cells. The simulations also predict how these gains in search efficiency scale with tissue thickness and CTL density. Mechanistically, MT depolymerization in activated CTLs triggers localized actomyosin accumulation at the uropod. This enhances rear contraction forces and promotes faster, more persistent migration and efficient search. Our findings clarify how MT dynamics influence CTL ability to eliminate targets in 3D environments and highlights the potential of MT-targeting agents such as pretubulysin to optimize T cell-based immunotherapies.

cell biology↗

bioRxiv2024

Mitochondrial reactive oxygen species cause arrhythmias in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and caused by genetic variants that often increase sarcomeric Ca2+ sensitivity. While Ca2+ sensitization explains diastolic dysfunction, the genesis of ventricular arrhythmias is unresolved. Here, we show that HCM mutations or pharmacological interventions that increase myofilament Ca2+ sensitivity generate bioenergetic mismatch and oxidative stress during {beta}-adrenergic stimulation which provide a trigger and a substrate for arrhythmias. For any given sarcomere shortening that produces work and consumes ATP, less Ca2+ stimulates the Krebs cycle to maintain mitochondrial NADH. This reverses the mitochondrial transhydrogenase to regenerate NADH from NADPH, supporting ATP production at the cost of NADPH-dependent antioxidative capacity. The ensuing overflow of reactive oxygen species (ROS) from mitochondria and glutathione oxidation induce spontaneous Ca2+ release from the sarcoplasmic reticulum and Ca2+ waves, well-defined triggers of arrhythmias. Furthermore, transhydrogenase-dependent ROS formation slows electrical conduction during {beta}-adrenergic stimulation in vivo, providing a substrate for arrhythmias. Chronic treatment with a mitochondrially-targeted ROS scavenger abolishes the arrhythmic burden during {beta}-adrenergic stimulation in HCM mice in vivo, while inducing mitochondrial ROS with a redox cycler is sufficient to induce arrhythmias in wild-type animals. These findings may lead to new strategies to prevent sudden cardiac death in patients with HCM.

physiology↗

bioRxiv2024

Pancreatic islets undergo functional and morphological adaptation during development of Barth Syndrome

Barth syndrome is a multisystem genetic disorder caused by mutation in TAFAZZIN, a gene that encodes a phospholipid:lysophospholipid transacylase important for cardiolipin remodeling. Barth Syndrome patients suffer from a number of symptoms including early heart failure, fatigue, and systemic metabolic alterations, including hypoglycemia. The endocrine pancreas is central to glucose homeostasis, however, the impact of defective cardiolipin remodeling on pancreatic islet function and the consequences for systemic metabolism is unclear. Surprisingly, in a mouse model with global TAFAZZIN knockdown, we observed improved glucose tolerance compared to wildtype littermates. We show that pancreatic islet metabolism and secretory function are robustly maintained through various compensatory mechanisms including increased glucose uptake and increased mitochondrial volume. Transcriptomics analyses revealed increased expression of genes encoding proteins involved in N-acetylglucosamine synthesis and protein O-linked N-acetylglucosaminylation. These pathways might provide a molecular mechanism for coupling metabolic changes to mitochondrial volume regulation.

physiology↗

bioRxiv2024

T cell polarization and NFAT translocation are stiffness-dependent and are differentially regulated by Piezo1 and Orai1

Effective T cell responses against tumor cells require diverse effector functions including polarization towards tumor cells to form immunological synapses and nuclear factor of activated T-cells (NFAT)-dependent gene transcription. While the role of tumor cell softening has been associated with malignancy, stemness, and metastasis, potentially contributing to immune evasion, its impact on cellular processes in T cells is not well understood. Here, we show that both T cell polarization and NFAT nuclear translocation are modulated by target stiffness in a Ca2+ dependent manner. Using both anti-CD3 antibody-functionalized substrates with varying stiffness as surrogates for target cells or softened tumor cells, we found that both, reorientation of microtubule organizing center (MTOC) towards the tumor cells, a hallmark for T cell polarization, and NFAT translocation were impaired on softer hydrogels or following contact with softer cancer cells. The amplitudes of intracellular Ca2+ signals were dependent on stiffness, and removal of extracellular Ca2+ inhibited stiffness-dependent T cell responsiveness. While stiffness-dependent Ca2+ signaling was crucial for both, T cell polarization and NFAT translocation, Ca2+ influx through Piezo1, a mechanosensitive ion channel, mediated stiffness-dependent MTOC reorientation but not NFAT translocation. In contrast, Ca2+ influx through store-operated Orai channels mediated NFAT translocation but not MTOC reorientation. Our results demonstrate that tumor cell stiffness directly influences T cell functionality through distinct Ca2+ influx pathways, revealing cell softening as an essential mechanism employed by malignant cells to evade immune surveillance.

immunology↗

bioRxiv2023

PIEZO1-mediated mechanosensing governs NK cell killing efficiency in 3D

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK cell killing efficiency in 3D is impaired against softened tumor cells, while it is enhanced against stiffened tumor cells. Notably, the durations required for NK cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK cell functions.

immunology↗

bioRxiv2021

A noval lightsheet scattering microscopy for long-term visualization of cell-ECM interaction

Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.

biophysics↗

bioRxiv2021

Metabolic reprogramming of cytotoxic T lymphocytes by high glucose enhances pancreatic beta cell apoptosis via TRAIL

Cytotoxic T lymphocytes (CTLs) are involved in development of diabetes. However, the impact of excessive glucose on CTL-mediated antigen-independent killing remains elusive. Here, we report that TNF-related apoptosis inducing ligand (TRAIL) is substantially up- regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. The PI3K- Akt-NF{kappa}B axis and non-mitochondrial reactive oxygen species are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Metformin and Vitamin D synergistically reduce HG-enhanced expression of TRAIL in CTLs and coherently protect 1.4E7 cells from TRAIL-mediated apoptosis. Notably, in patients with diabetes, correlation between Vitamin D concentrations in plasma and glucose levels is linked to HG-enhanced TRAIL expression on CTLs. Microarray data reveal that OXCT2, an important enzyme in ketone body catabolism, is a promising target in response to vitamin D. Our work not only reveals a novel mechanism of CTL involvement in progression of diabetes, but also establishes CTLs as a target for combined metformin and vitamin D therapy to protect pancreatic beta cells of diabetic patients.

immunology↗

bioRxiv2021

Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells

Solid tumors are often characterized by condensed extracellular matrix (ECM). The impact of dense ECM on cytotoxic T lymphocytes (CTL) function is not fully understood. Here, we report that CTL-mediated cytotoxicity is substantially impaired in dense collagen matrices. Although the intrinsic killing machinery including expression of cytotoxic proteins and degranulation was intact, CTL motility was substantially compromised in dense collagen. We found that for 3D CTL migration, persistence and velocity was regulated by collagen stiffness and the porosity, respectively. Interestingly, 3D CTL velocity is strongly correlated with their nuclear deformability/flexibility during migration, which is regulated by the microtubule network. Moreover, CTL migration was completely abolished by inhibition of actin polymerization and or myosin IIA. Remarkably, disruption of the microtubule-networks significantly improves the impaired migration, search efficiency, and cytotoxicity of CTLs in dense collagen. Our work suggests the microtubule network as a promising target to rescue impaired CTL killing capacity in solid tumor related scenarios.

immunology↗

bioRxiv2021

Cytotoxic efficiency of human CD8+ T cell memory subtypes

Immunological memory is an important concept to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are relatively well defined, single TEM and TCM cell cytotoxicity has not been quantified. Here, we analyze human CD8+ subtype distribution following SEA stimulation and quantify the expression of death mediators, including perforin, granzyme B, FasL and TRAIL. We find higher perforin, granzyme B and FasL expression in TEM and compared to TCM. To quantify single TEM and TCM cytotoxicity, we develop a FRET-based fluorescent assay with NALM6 target cells stably transfected with a GFP-RFP FRET construct based on a caspase-cleavage sequence (apoptosis sensor Casper-GR). Applying this assay, TEM or TCM induced target cell apoptosis or necrosis can be quantified. We find that single TEM are much more effective than TCM in killing their targets mainly by apoptosis and secondary necrosis. The reason for this is the higher perforin expression and on a more efficient lytic immunological synapse during TEM-target contact compared to TCM-target contact. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanism should be helpful to optimize future subset-based immune therapies.

immunology↗

bioRxiv2020

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumour cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+-influx through store operated Ca2+ channels, formed by STIM-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. Here, we isolated total RNA from natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, this strategy led to the identification of KCNN4, RCAN3, CCR5 and BCL2 as potential candidates to regulate the efficiency of Ca2+-dependent target cell killing.

immunology↗

bioRxiv2020

Combined CTL and NK cell cytotoxicity against cancer cells

CTL and NK cells recognize and eliminate cancer cells. However, immune evasion, down regulation of immune function by the tumor microenvironment, or resistance of cancer cells are a major problem. While CTL and NK cells are both important to eliminate cancer, most studies address them individually. In a new experimental human model, we analysed combined primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, MART-1-specific CTL or NK cells eliminated SK-Mel-5 cells within 24 hours indicating that SK-Mel-5 cells are initially not resistant. However, at lower effector-to-target ratios, which resemble conditions of the immune contexture in human cancer, a significant number of SK-Mel-5 cells survived. Whereas CTL pre-exposure induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, NK cell pre-exposure induced resistance in surviving SK-Mel-5 cells to NK cells but not to MART-1 specific CTL. In contrast, there was even a slight enhancement of CTL cytotoxicity against SK-Mel-5 cells following NK cell pre-exposure. In all other combinations, resistance to subsequent cytotoxicity was higher, if melanoma cells were pre-exposed to larger numbers of CTL or NK cells. Increases in human leukocyte antigen class I expression correlated with resistance to NK cells, while reduction in MART-1 antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. This study quantifies the interdependence of CTL and NK cell cytotoxicity and may guide strategies for efficient CTL-NK cell anti-melanoma therapies. Key points summaryO_LICytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. CTL and NK work in parallel, but most studies address them individually. C_LIO_LIIn a new human experimental model, antigen-specific CTL and NK cell cytotoxicity interdependence against melanoma is shown. C_LIO_LIWhereas high numbers of antigen-specific CTL and NK cells eliminate all melanoma cells, lower, more physiological numbers induce resistance, in case secondary CTL or NK cell exposure follow initial CTL cell exposure or if secondary NK cell exposure follows initial NK cell exposure; only if secondary CTL exposure follows initial NK cell exposure no resistance of melanoma but even a slight enhancement of cytotoxicity was observed. C_LIO_LIAlterations in HLA-I expression correlated with resistance to NK cells, while reduction in antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous antigen. C_LIO_LIThe results should help to better understand and optimize immune therapies against cancer. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/150672v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@19fef9eorg.highwire.dtl.DTLVardef@12cae81org.highwire.dtl.DTLVardef@749eeforg.highwire.dtl.DTLVardef@6e7cf1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Optoregulated force application to cellular receptors using molecular motors

Mechanotransduction events in physiological environments are difficult to investigate, in part due to the lack of experimental tools to apply forces to mechanosensitive receptors remotely. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), here we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the polymer chains, which will in turn effectively "pulls" on engaged cell membrane receptors (integrins, cadherins...) within the illuminated area. Applied forces have the adequate magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in forcedependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate, for the first time, a functionality which at the moment cannot be achieved by any other means.

cell biology↗

bioRxiv2020

Migration of Cytotoxic T Lymphocytes in 3D Collagen Matrices

CD8+ cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main cytotoxic killer cells of the human body to eliminate pathogen-infected or tumorigenic cells (= target cells). To find their targets they have to navigate and migrate through a complex biological microenvironments, a key component of which is the extracellular matrix (ECM). The mechanisms underlying killer cells navigation are not well understood. To mimic an ECM we use a matrix formed by different collagen concentrations, and analyze migration trajectories of primary human CTLs. Different migration patterns are observed and can be grouped into three motility types: slow, fast and mixed. The dynamics are well described by a two-state persistent random walk model which allows cells to switch between slow motion with low persistence, and fast motion with high persistence. We hypothesize that the slow motility mode describes CTLs creating channels through the collagen matrix by deforming and tearing apart collagen fibers, and that the fast motility mode describes CTLs moving within these channels. Experimental evidence supporting this scenario is presented by visualizing migrating T cells following each other on exactly the same track and showing cells moving quickly in channel-like cavities within the surrounding collagen matrix. Consequently, the efficiency of the stochastic search process of CTLs in the ECM should strongly be influenced by a dynamically changing channel network produced by the killer cells themselves.

biophysics↗