Biology subjects

Kopf, M.

Publications and source records attributed to Kopf, M..

9 recordsLinked to original sources

bioRxiv2024

Investigating Polyreactivity of CD4+ T Cells to the Intestinal Microbiota

The symbiotic relationship between host and microbiota plays a pivotal role in training and development of the hosts innate and adaptive immune systems. Antigen-specific recognition of microbiota by T cells enforces tolerance at homeostasis. Conversely, dysbiosis--characterized by alterations in microbiota diversity and abundance--leads to imbalanced T cell responses and triggering of inflammatory and autoimmune diseases. Despite their significance, the identities of immunogenic microbial antigens are still largely enigmatic. Here, we leveraged an in-house developed antigen screening platform, the MCR system 1, to delineate CD4+ T cell reactivity against Akkermansia muciniphila (AKK) and Bacteroides thetaiotaomicron (BT), --two prominent members of the gut microbiota. T-cell hybridomas reactive to AKK and BT bacteria showed polyreactivity to select microbiota-derived peptides in MCR co-cultures. We discovered 13 novel antigenic epitopes from AKK and 14 from BT. Steady-state T cells recognized these epitopes in an MHC-restricted fashion. Ex vivo stimulation of peptide-specific T cells revealed induction of type 1 and type 17 immune responses, albeit with non-overlapping specificities, contrary to MCR system results. Our findings further demonstrated that most identified epitopes are broadly conserved within the given phylum and originate from both membrane and intracellular proteins. Our work showcases the potential of the MCR system for identifying immunogenic microbial epitopes, providing a valuable resource. Additionally, it indicates the existence of mucosal T cells with a tropism toward broadly conserved bacterial epitopes. Overall, our study forms the basis for decoding antigen specificity in immune system-bacterial interactions, with applications in understanding both microbiome and pathogenic bacterial immunity.

immunology↗

bioRxiv2024

Treatment of a genetic liver disease in mice through transient prime editor expression

Prime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept for in vivo prime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed an in vivo prime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at the Dnmt1 locus using AAV doses of 2.5x1013 vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2 mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5x1013 vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 {micro}mol/l to below the therapeutic threshold of 600 {micro}mol/l. Our study demonstrates the feasibility of in vivo gene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.

bioengineering↗

bioRxiv2023

CD4+ T cell-derived IL21 regulates stem cell fate in acute myeloid leukemia by activation of p38-MAPK signaling

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in acute myeloid leukemia (AML) patients. We identified CD4+ T cell-derived interleukin (IL) 21 as an important negative regulator of self-renewal of murine and human LSCs, but not hematopoietic stem cells. IL21/IL21R signaling favored asymmetric cell division and differentiation in LSCs through accumulation of reactive oxygen species (ROS) and activation of p38-MAPK signaling, resulting in reduced LSCs number and significantly prolonged survival in murine AML models. In human AML, serum IL21 at diagnosis was identified as an independent positive prognostic biomarker for outcome and correlated with better survival and higher complete remission rate in patients that underwent high-dose chemotherapy. IL21 inhibited primary AML LSCs function in vitro by activating ROS and p38-MAPK signaling and this effect was enhanced by cytarabine treatment. Consequently, promoting IL21/IL21R signaling on LSCs may be a novel approach to decrease stemness and increase differentiation in AML.

cancer biology↗

bioRxiv2022

Apolipoprotein E controls Dectin-1-dependent development of monocyte-derived alveolar macrophages upon pulmonary β-glucan-induced inflammatory adaptation

The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms which lead to the functional and developmental adaptation of lung-associated macrophages remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal {beta}-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonal macrophage adaptation. Employing single-cell transcriptomics, high dimensional imaging and flow cytometric characterization paired to in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of Dectin-1 - Card9 signaling-dependent monocyte-derived macrophages (MoAM). MoAMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic and produced large amounts of interleukin 6 upon restimulation. Myeloid cell specific ApoE ablation inhibited monocyte to MoAM differentiation dependent on M-CSF secretion, promoting MoAM cell death thus impeding MoAM maintenance. In vivo, {beta}-glucan-elicited MoAMs limited the bacterial burden of Legionella pneumophilia post infection and ameliorated fibrosis severity in a murine fibrosis model. Collectively these data identify MoAMs that are generated upon environmental cues and ApoE as an important determinant for lung immune resilience.

immunology↗

bioRxiv2021

KappaBle fluorescent reporter mice enable dynamic and low-background single-cell detection of NF-kB activity in vivo

Nuclear factor-{kappa}B (NF-{kappa}B) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-{kappa}B activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-{kappa}B reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-{kappa}B, the latter have the potential to allow the analysis of NF-{kappa}B activity at single cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knockin NF-{kappa}B reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-{kappa}B activity at the single-cell level of various cell types during inflammatory and infectious diseases.

immunology↗

bioRxiv2021

Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing

Pancreatic ductal adenocarcinoma (PDA) is an inherently immune cell deprived tumor, characterized by desmoplastic stroma and suppressive immune cells. Here we systematically dissected PDA intrinsic mechanisms of immune evasion by in vitro and in vivo CRISPR screening, and identified Rnf31 and Vps4b as essential factors required for escaping CD8+ T cell-killing. Using murine PDA cells and human PDA organoids, we demonstrate that Rnf31 protects from TNF-mediated caspase 8 cleavage and subsequent apoptosis induction. For Vps4b we found that inactivation impairs autophagy, resulting in increased accumulation of CD8+ T cell-derived granzyme B and subsequent tumor cell lysis. Orthotopic transplantation of Rnf31- or Vps4b deficient pancreatic tumors, moreover, revealed increased CD8+ T cell infiltration and effector function, and markedly reduced tumor growth in mice. Our work uncovers vulnerabilities in PDA that might be exploited to render these tumors more susceptible to the immune system.

cancer biology↗

bioRxiv2021

Bona fide alveolar macrophage generation from long-term in vitro culture of fetal precursors provides a novel platform for gene therapy and functional studies

Tissue-resident macrophage-based immune therapies have been proposed for various diseases. However, generation of sufficient numbers that possess tissue-specific functions remains a major handicap. Here, we show that fetal liver monocytes (FLiMo) cultured with GM-CSF (also known as CSF2) rapidly differentiate into a long-lived, homogeneous alveolar macrophage (AM)-like population in vitro. CSF2-cultured FLiMo remain the capacity to develop into bona fide AM upon transfer into Csf2ra-/- neonates and prevent development of alveolar proteinosis and efferocytosis of apoptotic cells for at least 1 year in vivo. Compared to transplantation of AM-like cells derived from bone marrow macrophages (BMM), CSF2-cFliMo more efficiently engraft empty AM niches in the lung and protect mice from respiratory viral infection. Harnessing the potential of this approach for gene therapy, we restored a disrupted Csf2ra gene in FLiMo and their capacity to develop into AM in vivo. Together, we provide a novel platform for generation of immature AM-like precursors amenable for genetic manipulation, which will be useful to study to dissect AM development and function and pulmonary transplantation therapy.

immunology↗

bioRxiv2021

High resolution profiling of MHC-II peptide presentation capacity, by Mammalian Epitope Display, reveals SARS-CoV-2 targets for CD4 T cells and mechanisms of immune-escape

Understanding the mechanisms of immune evasion is critical for formulating an effective response to global threats like SARS-CoV2. We have fully decoded the immune synapses for multiple TCRs from acute patients, including cognate peptides and the presenting HLA alleles. Furthermore, using a newly developed mammalian epitope display platform (MEDi), we determined that several mutations present in multiple viral isolates currently expanding across the globe, resulted in reduced presentation by multiple HLA class II alleles, while some increased presentation, suggesting immune evasion based on shifting MHC-II peptide presentation landscapes. In support, we found that one of the mutations present in B1.1.7 viral strain could cause escape from CD4 T cell recognition in this way. Given the importance of understanding such mechanisms more broadly, we used MEDi to generate a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of multiple common HLA class II molecules. Unlike other strategies, our approach is sensitive and scalable, providing an unbiased and affordable high-resolution map of peptide presentation capacity for any MHC-II allele. Such information is essential to provide insight into T cell immunity across distinct HLA haplotypes across geographic and ethnic populations. This knowledge is critical for the development of effective T cell therapeutics not just against COVID-19, but any disease.

immunology↗

bioRxiv2018

A macrophage-pericyte axis directs tissue restoration via Amphiregulin-induced TGFβ activation

The Epidermal Growth Factor Receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis following injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functions by releasing bio-active TGFb from latent complexes via integrin-v activation. Using acute injury models in two different tissues, we found that by inducing TGFb activation on mesenchymal stromal cells (aka pericytes), Amphiregulin induced their differentiation into myo-fibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration following acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.

immunology↗