Biology subjects

Klein, C.

Publications and source records attributed to Klein, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Chronic Antigen Stimulation in Solid Tumors Induces T Cell Exhaustion and Limits Efficacy of T Cell Bispecific Therapies

T cell bispecific antibodies (TCBs) have demonstrated promising results in patients with solid tumors. However, the underlying immunological and molecular mechanisms influencing these clinical outcomes require in depth evaluation. T cell exhaustion, a state induced by prolonged antigen exposure, is known to undermine T cell-based immunotherapies, though its specific impact on TCB efficacy remains unclear. In this study, we assessed the effectiveness of TCBs on tumor-specific T cells, focusing on their functional status. Utilizing a fully immunocompetent mouse model with a solid tumor expressing an immunogenic antigen, we showed that tumor-specific T cells acquire an exhausted phenotype and fail to expand under TCB treatment. By employing both mouse and human tumor-specific T cells in vitro, our study established that chronically stimulated tumor-specific T cells show impaired response to TCB treatment. The comparison of TCB efficacy in T cell-inflamed tumors with immunogenic antigens versus non-inflamed tumors with low antigen presence in mice revealed TCB success in solid tumors is more reliant on T cell functional fitness than on their abundance before treatment. The data also indicate that solid tumors with elevated levels of both, intratumoral regulatory T cells, and T cells expressing co-inhibitory receptors, show diminished responses to TCB therapy, aligning with similar observations described in hematological cancers. These findings highlight the critical role of T cell exhaustion due to chronic antigen exposure and illustrate that exhausted tumor-specific T cells are likely not the driver population redirected by TCBs for tumor elimination. Our research highlights the importance of maintaining T cell fitness and preventing T cell exhaustion to improve TCB therapy outcomes. This may help better identify patient populations with solid tumors that could benefit from TCB treatments most in clinical settings.

cancer biology↗

bioRxiv2024

Exploring the Abeta Plaque Microenvironment in Alzheimer's Disease Mice by Multimodal Lipid-Protein-Histology Imaging on a Benchtop Mass Spectrometer

Amyloid-{beta} (A{beta}) plaque deposits in the brain are a hallmark of Alzheimers disease (AD) neuropathology. Plaques consist of complex mixtures of peptides like A{beta}1-42, characteristic lipids such as gangliosides, and they are targeted by reactive microglia and astrocytes. In pharmaceutical research and development it is therefore a formidable challenge to contextualize different biomolecular classes and cell types of the A{beta} plaque microenvironment in a coherent experimental workflow on a single tissue section and on a benchtop imaging reader. Here, we developed such a workflow that combines lipid MALDI mass spectrometry imaging using a vacuum-stable matrix with histopathology stains and with MALDI HiPLEX-immunohistochemistry of plaques and multiple protein markers on a bench-top imaging mass spectrometer. The three data layers consisting of lipid, protein markers, and histology can be co-registered and evaluated together. Multimodal data analysis suggests extensive co-localization of A{beta} plaques with the peptide precursor protein, with a defined subset of lipids and with reactive glia cells on a single brain section of APP/PS1 AD model mice. Plaque-associated lipids like ganglioside GM2 and phosphatidylinositol PI38:4 isoforms were readily identified using the tandem MS-capabilities of the mass spectrometer. Taken together, our data suggests that complex pathology involving multiple lipids, proteins and cell types can be interrogated by this spatial multiomics workflow on an easy-to-use benchtop mass spectrometer.

neuroscience↗

bioRxiv2024

IL15/IL15Rα complex induces an anti-tumor immune response following radiation therapy only in the absence of Tregs and fails to induce expansion of progenitor TCF1+ CD8 T cells

BackgroundThis work seeks to understand whether IL15-incorporating treatments improve response to radiotherapy and uncover mechanistic rationale for overcoming resistance to IL15 agonism using novel therapeutic combinations. Experimental DesignOrthotopic tumor models of PDAC were used to determine response to treatment. IL15-/- and Rag1-/- mouse models were employed to determine dependence on IL15 and CTLs, respectively. Flow cytometry was used to assess immune cell frequency and activation state. Phospho-proteomic analyses were used to characterize intracellular signaling pathways. ResultsWe show that the combination of radiation therapy (RT) and an IL15/IL15Ra fusion complex (denoted IL15c) fails to confer anti-tumor efficacy; however, a CD8-driven anti-tumor immune response is elicited with the concurrent administration of an aCD25 Treg-depleting antibody. Using IL15-/- and Rag1-/- mice, we demonstrate that response to RT + IL15c + aCD25 is dependent on both IL15 and CTLs. Furthermore, despite an equivalent survival benefit following treatment with RT + IL15c + aCD25 and combination RT + PD1-IL2v, a novel immunocytokine with PD-1 and IL2R{beta}{gamma} binding domains, CTL immunophenotyping and phospho-proteomic analysis of intracellular metabolites showed significant upregulation of activation and functionality in CD8 T cells treated with RT + PD1-IL2v. Finally, we show the immunostimulatory response to RT + PD1-IL2v is significantly diminished with a concurrent lack of TCF+ CD8 T cell generation in the absence of functional IL15 signaling. ConclusionsOur results are illustrative of a mechanism wherein unimpeded effector T cell activation through IL2R{beta} signaling and Treg inhibition are necessary in mediating an anti-tumor immune response.

cancer biology↗

bioRxiv2024

Divergent response to radio-immunotherapy is defined by intrinsic features of the tumor microenvironment

BackgroundTreatment with immunotherapy can elicit varying responses across cancer types, and the mechanistic underpinnings that contribute to response vs. progression remain poorly understood. However, to date there are few preclinical models that accurately represent these disparate disease scenarios. MethodsUsing combinatorial radio-immunotherapy consisting of PD-1 blockade, IL2R{beta}{gamma} biased signaling, and OX40 agonism we were able to generate preclinical tumor models with conflicting responses, where head and neck squamous cell carcinoma (HNSCC) models responds and pancreatic ductal adenocarcinoma (PDAC) progresses. ResultsBy modeling these disparate states, we find that regulatory T cells (Tregs) are expanded in PDAC tumors undergoing treatment, constraining tumor reactive CD8 T cell activity. Consequently, the depletion of Tregs restores the therapeutic efficacy of our treatment and abrogates the disparity between models. Moreover, we show that through heterotopic implantations that the site of tumor development defines the response to therapy, as implantation of HNSCC tumors into the pancreas resulted in comparable levels of tumor progression. ConclusionsThis work highlights complexity of combining immunotherapies within the tumor microenvironment and further defines the immune and non-immune components of the tumor microenvironment as an intrinsic feature of immune suppression. What is already known on this topicO_LIIn Head and neck squamous cell carcinomas (HNSCC) and pancreatic ductal adenocarcinoma (PDAC), targeting PD-1 and IL2R{beta}{gamma} simultaneously (PD1-IL2v) has been shown to be effective when combined with radiation therapy (RT), yet complete response is still limited. The T cell co-stimulatory receptor OX40 (TNFRSF4) has pleiotropic effects, promoting T cell survival, expansion, and memory differentiation in conventional effector T cells, while subsequently limiting regulatory T cell (Treg) suppression by constraining induction and expression of Foxp3. Expression of OX40 is highly upregulated after treatment with PD1-IL2v, and we postulated that combining OX40 agonism with PD1-IL2v and RT would provide additional benefit. C_LI What this study addsO_LIUsing orthotopic models of HNSCC and PDAC, we found that the addition of OX40 agonism unexpectedly drives tumor progression in PDAC, but not HNSCC. Intriguingly, this effect dependent on the tumor microenvironment as the effect is reversed by swapping the location of tumor implantation. This progression was also abrogated by the depletion of regulatory T cells (Tregs), a known mediator of resistance in these models. C_LI How this study might affect research, practice or policyO_LIOur data demonstrate that unexpected and deleterious effects can stem from combining multiple immunotherapies. These findings hold particular translational relevance as the use of combination immunotherapies is increasingly common on trial. C_LI

immunology↗

bioRxiv2024

Dynamic alterations of dural and bone marrow B cells in an animal model of progressive multiple sclerosis

In multiple sclerosis (MS), the leptomeninges (LM) are populated with immune cell aggregates that correlate with disease progression. The impact of LM inflammation on the adjacent dura is largely unknown. Using a mouse model of MS that induces brain LM inflammation and age-dependent disease progression, we found that encephalitogenic T cells and B220high B cells accumulate substantially in the brain LM and parenchyma of both young and aged mice, while the adjacent dura remains relatively inert. We also observed a population of anti-CD20 resistant B220low B cells in the dura and bone marrow that virtually disappear at disease onset and accumulate in the brain of young mice concomitant with disease remission. In contrast, aged mice show a paucity of brain-resident B220low B cells at the expense of class-switched B220high B cells concomitant with severe, chronic disease. In summary, dynamic changes in brain, LM and dural B cells are associated with age-dependent disease severity in an animal model of progressive MS. Short SummaryFlorescu et al. investigate the temporal accumulation of immune cells within distinct meningeal compartments in an animal model of progressive MS and uncover a population of anti-CD20 resistant dural B cells that remain in the brain parenchyma at disease remission.

immunology↗

bioRxiv2024

Large transient assemblies of Apaf1 constitute the apoptosome in cells

Upon cell death signals, the apoptotic protease-activating factor Apaf1 and cytochrome c interact to form the apoptosome complex. The apoptosome is crucial for mitochondrial apoptosis, as it activates caspases that dismantle the cell. However, the assembly mechanism and appearance of the apoptosome in vivo remain unclear. We show that upon onset of apoptosis, Apaf1 molecules accumulate into multiple foci per cell. Disassembly of the foci is linked to survival of the cell. Structurally, Apaf1 foci resemble organelle-sized, cloud-like assemblies. Foci form upon specific molecular interactions with cytochrome c and depending on procaspase-9. We propose that Apaf1 foci correspond to the apoptosome in cells. Transientness and ultrastructure of Apaf1 foci suggest that the dynamic spatiotemporal organisation of apoptosome components regulates progression of apoptosis.

cell biology↗

bioRxiv2024

Multiomics analyses decipher intricate changes in the cellular and metabolic landscape of steatotic livers upon dietary restriction and sleeve gastrectomy

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic, progressive liver disease that encompasses a spectrum of steatosis, steatohepatitis (or MASH), and fibrosis. Evidence suggests that dietary restriction (DR) and sleeve gastrectomy (SG) can lead to remission of hepatic steatosis and inflammation through weight loss, but it is unclear whether these procedures induce distinct metabolic or immunological changes in MASLD livers. This study aims to elucidate the intricate hepatic changes following DR, SG or sham surgery in rats fed a high-fat diet as a model of obesity-related MASLD, in comparison to a clinical cohort of patients undergoing SG. Single-cell and single-nuclei transcriptome analysis, spatial metabolomics, and immunohistochemistry revealed the liver landscape, while circulating biomarkers were measured in serum samples. Artificial intelligence (AI)-assisted image analysis characterized the spatial distribution of hepatocytes, myeloid cells and lymphocytes. In patients and experimental MASLD rats, SG improved BMI, circulating liver injury biomarkers and triglyceride levels. Both DR and SG attenuated liver steatosis and fibrosis in rats. Metabolism-related genes (Ppara, Cyp2e1 and Cyp7a1) were upregulated in hepatocytes upon DR and SG, while SG broadly upregulated lipid metabolism on cholangiocytes, monocytes, macrophages, and neutrophils. Furthermore, SG promoted myeloid cell accumulation in the liver not only ameliorating inflammation but activating liver repair processes. Regions with potent myeloid infiltration were marked with enhanced metabolic capacities upon SG. Additionally, a disruption of periportal hepatocyte functions was observed upon DR. In conclusion, this study indicates a dynamic cellular crosstalk in steatotic livers undergoing SG. Notably, PPAR- and gut-liver axis-related processes, and metabolically-active myeloid cell infiltration indicate intervention-related mechanisms supporting SG for the treatment of MASLD.

bioinformatics↗

bioRxiv2023

EmbedGEM: A framework to evaluate the utility of embeddings for genetic discovery

Machine learning (ML)-derived embeddings are a compressed representation of high content data modalities. Embeddings can capture detailed information about disease states and have been qualitatively shown to be useful in genetic discovery. Despite their promise, embeddings have a major limitation: it is unclear if genetic variants associated with embeddings are relevant to the disease or trait of interest. In this work we describe EmbedGEM (Embedding Genetic Evaluation Methods), a framework to systematically evaluate the utility of embeddings in genetic discovery. EmbedGEM focuses on comparing embeddings along two axes: heritability and disease relevance. As measures of heritability, we consider the number of genome-wide significant associations and the mean{chi} 2 statistic at significant loci. For disease relevance, we compute polygenic risk scores for each embedding principal component, then evaluate their association with high-confidence disease or trait labels in a held-out evaluation patient set. While our development of EmbedGEM is motivated by embeddings, the approach is generally applicable to multivariate traits, and can readily be extended to accommodate additional metrics along the evaluation axes. We demonstrate EmbedGEMs utility by evaluating embeddings and multivariate traits in two separate datasets: i) a synthetic dataset simulated to demonstrate the ability of the framework to correctly rank traits based on their heritability and disease relevance, and ii) a real data from the UK Biobank including metabolic and liver-related traits. Importantly, we show that greater disease relevance does not automatically follow from greater heritability.

bioinformatics↗

bioRxiv2023

The R1441C-LRRK2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner

Considering age is the greatest risk factor for many neurodegenerative diseases, aging, in particular aging of the immune system, is the most underappreciated and understudied contributing factor in the neurodegeneration field. Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinsons disease (PD). The leucine-rich repeat kinase 2 (LRRK2) protein has been implicated in peripheral immune signaling, however, the effects of an aging immune system on LRRK2 function have been neglected to be considered. We demonstrate here that the R1441C mutation induces a hyper-responsive phenotype in macrophages from young female mice, characterized by increased effector functions, including stimulation-dependent antigen presentation, cytokine release, phagocytosis, and lysosomal function. This is followed by age-acquired immune cell exhaustion in a Lrrk2-kinase-dependent manner. Immune-exhausted macrophages exhibit suppressed antigen presentation and hypophagocytosis, which is also demonstrated in myeloid cells from R1441C and Y1699C-PD patients. Our novel findings that LRRK2 mutations confer immunological advantage at a young age but may predispose the carrier to age-acquired immune cell exhaustion have significant implications for LRRK2 biology and therapeutic development. Indeed, LRRK2 has become an appealing target in PD, but our findings suggest that more research is required to understand the cell-type specific consequences and optimal timing of LRRK2-targeting therapeutics. One Sentence SummaryThe R1441C-LRRK2 mutation causes an age-acquired immune cell exhaustion in macrophages in a sex-dependent manner

immunology↗

bioRxiv2023

Low exposure to lithium does not induce nephrogenic diabetes insipidus but microcystic dilations of collecting ducts in a long-term rat model

Lithium induces nephrogenic diabetes insipidus (NDI) and microcystic chronic kidney disease (CKD). As clinical studies suggested that NDI is dose-dependent and CKD time-dependent, we investigated the effects of low exposure to lithium in a long-term rat model. Sprague-Dawley rats were randomly fed during six months with normal diet (controls), addition of lithium to diet, or addition of lithium and amiloride to diet, allowing reaching low steady-state plasma lithium concentrations (0.25{+/-}0.06 and 0.43{+/-}0.16 mmol/L, respectively). Exposure to low plasma lithium concentrations did not induce NDI but microcystic dilations of kidney tubules, identified as collecting ducts (CDs) using immunofluorescent staining. Both hypertrophy, characterized by an increase in the ratio of nuclei per tubular area, and microcystic dilations were observed. Principal cell-to-intercalated cell ratio was higher in dilated than in hypertrophied tubules. There was no correlation between aquaporin-2 mRNA levels and cellular remodelling of CDs. Amiloride/lithium co-administration did not allow significant consistent morphometric and cellular composition changes compared to lithium administration. To conclude, rat low exposure to lithium did not induce overt NDI but microcystic dilations of CDs, which include a marked alteration in cell composition of hypertrophied and dilated CDs, suggesting two distinct underlying pathophysiological mechanisms.

pharmacology and toxicology↗

bioRxiv2023

Endosome dysfunction leads to gain-of-function TLR7 and human lupus

Hyperactive Toll-like receptor (TLR) 7 signaling has long been appreciated as a driver of autoimmune disease by breaking tolerance to self-nucleic acids in mouse models1-5. Recently, mutations in TLR7 or its associated regulator UNC93B16, 7, were identified as monogenic causes of human lupus; the unifying feature of these mutations being TLR7 gain-of-function. TLR7 is an intracellular transmembrane receptor, sensing RNA breakdown products within late endosomes8, 9. Hence, its function depends on intricate transport mechanisms and membrane interactions within the endomembrane network. Whether perturbations of any of these endosome-related processes can give rise to TLR7 gain-of-function and facilitate self-reactivity has not been investigated. Here, we show that a dysregulated endosomal compartment leads to unrestricted TLR7 signaling and human lupus. The late endosomal BLOC-1-related protein complex (BORC) together with the small Arf1-like GTPase Arl8b controls TLR7 protein levels, and a direct interaction between Arl8b and Unc93b1 is required to regulate TLR7 turnover. We identified an amino acid insertion in UNC93B1 in a patient with childhood-onset lupus, which reduces the interaction with the BORC-Arl8b complex and leads to endosomal TLR7 accumulation. Therefore, a failure to control the proper progression of TLR7 through its endocytic life cycle is sufficient to break immunological tolerance to nucleic acids in humans. Our results highlight the importance of an intact endomembrane system to prevent autoimmune disease. As the cellular mechanisms restricting TLR7 signaling can be manifold, identifying and stratifying lupus patients based on a TLR7-driven pathogenesis could be a viable strategy towards a targeted therapy.

immunology↗

bioRxiv2023

Dogs and cats are less susceptible to the omicron variant of concern of SARS-CoV-2 - a field study

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of unprecedented extent. Beside humans, a number of animal species can be infected, however, in some species differing susceptibilities were observed depending on the virus variant. Here, we serologically investigated cats and dogs living in households with human COVID-19 patients. The study was conducted during the transition period from delta as the dominating variant of concern (VOC) to omicron (BA.1/BA.2) to investigate the frequency of virus transmission of both VOCs from infected owners to their pets. The animal sera were tested by surrogate virus neutralization tests (sVNT) using either the original receptor-binding domain (RBD), enabling the detection of antibodies against the delta variant, or an omicron-specific RBD. Of the 290 canine samples, 20 tested positive by sVNT, but there were marked differences between the sampling time, and, related thereto, the virus variants, the dogs had contact to. While in November 2021 infected owners led to 50% seropositive dogs (18/36), only 0.8% (2/254) of animals with household contacts to SARS-CoV-2 between December 2021 and April 2022 tested positive. In all cases, the positive reaction was recorded against the original RBD. For cats, a similar pattern was seen, as in November 2021 38.1% (16/42) tested positive and between December 2021 and March 2022 only 5.0% (10/199). The markedly reduced ratio of seropositive animals during the period of omicron circulation suggests a considerably lower susceptibility of dogs and cats to this VOC. To examine the effect of BA.2, BA.4 and BA.5 omicron subvariants, sera taken in the second and third quarter of 2022 from randomly selected cats were investigated. 2.3% (11/372) tested seropositive and all of them showed a stronger reaction against the original RBD, further supporting the assumption of a lower susceptibility of companion animals to the omicron VOC.

microbiology↗

bioRxiv2023

Multi-omics analysis identifies LBX1 and NHLH1 as central regulators of human midbrain dopaminergic neuron differentiation

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinsons disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) iPSC reporter line, we have generated time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicted novel central regulators of mDAN differentiation and super-enhancers were used to prioritize key TFs. We find LBX1, NHLH1 and NR2F1/2 to be necessary for mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. NHLH1 is necessary for the induction of neuronal miR-124, while LBX1 regulates cholesterol biosynthesis, possibly through mTOR signaling. Consistently, rapamycin treatment led to an inhibition of mDAN differentiation. Thus, our work reveals novel regulators of human mDAN differentiation.

genomics↗

bioRxiv2022

An allelic series rare variant association test for candidate gene discovery

Allelic series are of candidate therapeutic interest due to the existence of a dose-response relationship between the functionality of a gene and the degree or severity of a phenotype. We define an allelic series as a gene in which increasingly deleterious mutations lead to increasingly large phenotypic effects, and develop a gene-based rare variant association test specifically targeted for the identification of allelic series. Building on the well-known burden and sequence kernel association (SKAT) tests, we specify a variety of association models, covering different genetic architectures, and integrate these into a COding-variant Allelic Series Test (COAST). Through extensive simulations, we confirm that COAST maintains the type I error and improves power when the pattern of coding-variant effect sizes increases monotonically with mutational severity. We applied COAST to identify allelic series for 4 circulating lipid traits and 5 cell count traits among 145,735 subjects with available whole exome sequencing data from the UK Biobank. Compared with optimal SKAT (SKAT-O), COAST identified 29% more Bonferroni significant associations with circulating lipid traits, on average, and 82% more with cell count traits. All of the gene-trait associations identified by COAST have corroborating evidence either from rare-variant associations in the full cohort (Genebass, N = 400K), or from common variant associations in the GWAS catalog. In addition to detecting many gene-trait associations present in Genebass using only a fraction (36.9%) of the sample, COAST detects associations, such as ANGPTL4 with triglycerides, that are absent from Genebass but which have clear common variant support.

genetics↗

bioRxiv2022

Humanized CAR T cells targeting p95HER2

Redirection of T lymphocytes against tumor-associated or tumor-specific antigens, via bispecifc T cells engagers (BiTEs) or chimeric antigen receptors (CARs), is a successful therapeutic strategy against certain hematologic malignancies. In contrast, so far it has failed against solid tumors. Given the scarcity of tumor-specific antigens, the vast majority of BiTEs and CAR Ts developed to date have been directed against tumor-associated antigens. These are expressed in some normal tissues and, as a consequence, frequent and serious side effects caused by on-target off-tumor activity have limited the use of BiTEs and CARs in the clinic. P95HER2 is a fragment of the tyrosine kinase receptor HER2 expressed in more than 30% of HER2-amplified tumors. It has been previoulsy shown that p95HER2 is a tumor-specific antigen. Here we present the generation of CARs targeting p95HER2. p95HER2 CAR T cells show remarkable activity against p95HER2-expressing cells in vitro and in vivo. Further, they are also effective against lung and brain metastasis. These tumor-specific CAR T cells could be used in the near future to deliver additional anti-tumor therapies in a safe manner.

cancer biology↗

bioRxiv2021

The global rarity of intact coastal regions

Management of the land-sea interface is considered essential for global conservation and sustainability objectives, as coastal regions maintain natural processes that support biodiversity and the livelihood of billions of people. However, assessments of coastal regions have focused on either strictly the terrestrial or marine realm, and as a consequence, we still have a poor understanding of the overall state of Earths coastal regions. Here, by integrating the terrestrial human footprint and marine cumulative human impact maps, we provide a global assessment of the anthropogenic pressures affecting coastal areas. Just 15.5% of coastal areas globally can be considered having low anthropogenic pressure, mostly found in Canada, Russia, and Greenland. Conversely, 47.9% of coastal regions are heavily impacted by humanity with most countries (84.1%) having >50% of their coastal regions degraded. Nearly half (43.3%) of protected areas across coastal regions are exposed to high human pressures. In order to meet global sustainability objectives, we identify those nations that must undertake greater actions to preserve and restore coastal regions so as to ensure global sustainable development objectives can be met.

ecology↗

bioRxiv2021

Human immunocompetent Organs-on-Chips platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies

AbstractTraditional drug safety assessment often fails to predict complications in humans, especially when the drug targets the immune system. Here, we show the unprecedented capability of two Organs-on-Chips to evaluate the safety profile of T-cell bispecific antibodies (TCBs) targeting tumor antigens. Although promising for cancer immunotherapy, TCBs are associated with an on-target, off-tumor risk due to low levels of expression of tumor antigens in healthy tissues. We leveraged in vivo target expression and toxicity data of TCBs targeting folate receptor 1 (FOLR1) or carcinoembryonic antigen (CEA) to design and validate human immunocompetent Organs-on-Chips safety platforms. We discovered that the Lung-Chip and Intestine-Chip could reproduce and predict target-dependent TCB safety liabilities, based on sensitivity to key determinants thereof, such as target expression and antibody affinity. These novel tools broaden the research options available for mechanistic understandings of engineered therapeutic antibodies and assessing safety in tissues susceptible to adverse events.

cancer biology↗

bioRxiv2021

Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

The level and the biological significance of mitochondrial DNA (mtDNA) methylation in human cells is a controversial topic. Using long-read third-generation sequencing technology, mtDNA methylation can be detected directly from the sequencing data, which overcomes previously suggested biases, introduced by bisulfite treatment-dependent methods. We investigated mtDNA from whole blood-derived DNA and established a workflow to detect CpG methylation with Nanopolish. In order to obtain native mtDNA, we adjusted a whole-genome sequencing protocol and performed ligation library preparation and Nanopore sequencing. To validate the workflow, 897bp of methylated and unmethylated synthetic DNA samples at different dilution ratios were sequenced and CpG methylation was detected. Interestingly, we observed that reads with higher methylation in the synthetic DNA did not pass Guppy calling, possibly affecting conclusions about DNA methylation in Nanopore sequencing. We detected in all blood-derived samples overall low-level methylation across the mitochondrial genome, with exceptions at certain CpG sites. Our results suggest that Nanopore sequencing is capable of detecting low-level mtDNA methylation. However, further refinement of the bioinformatical pipelines including Guppy failed reads are recommended.

genetics↗