34 results on '"De Munter S"'
Search Results
2. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities
- Author
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Caeneghem, Y. (Yasmine) van, De Munter, S. (Stijn), Tieppo, P. (Paola), Goetgeluk, G. (Glenn), Weening, K. (Karin), Verstichel, G. (Greet), Bonte, S. (Sarah), Taghon, T. (Tom), Leclercq, G. (Georges), Kerre, T. (Tessa), Debets, J.E.M.A. (Reno), Vermijlen, D. (David), Abken, H. (Hinrich), Vandekerckhove, B. (Bart), Caeneghem, Y. (Yasmine) van, De Munter, S. (Stijn), Tieppo, P. (Paola), Goetgeluk, G. (Glenn), Weening, K. (Karin), Verstichel, G. (Greet), Bonte, S. (Sarah), Taghon, T. (Tom), Leclercq, G. (Georges), Kerre, T. (Tessa), Debets, J.E.M.A. (Reno), Vermijlen, D. (David), Abken, H. (Hinrich), and Vandekerckhove, B. (Bart)
- Abstract
Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord
- Published
- 2017
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3. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities
- Author
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Van Caeneghem, Y, De Munter, S, Tieppo, P, Goetgeluk, G, Weening, K, Verstichel, G, Bonte, S, Taghon, T, Leclercq, G, Kerre, T, Debets, Reno, Vermijlen, D, Abken, H, Vandekerckhove, B, Van Caeneghem, Y, De Munter, S, Tieppo, P, Goetgeluk, G, Weening, K, Verstichel, G, Bonte, S, Taghon, T, Leclercq, G, Kerre, T, Debets, Reno, Vermijlen, D, Abken, H, and Vandekerckhove, B
- Published
- 2017
4. P.1.i.056 Mass spectrometry imaging as a powerful tool for detailed molecular investigation of brain pathologies: molecular microscope for peroxisomal disorders
- Author
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Skraskova, K., primary, Khmelinskii, A., additional, Abdelmoula, W.M, additional, De Munter, S., additional, Baes, M., additional, and Heeren, R.M.A., additional
- Published
- 2015
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5. ThymoSpheres culture: A model to study human polyclonal unconventional T cells.
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Billiet L, Jansen H, Pille M, Boehme L, Sanchez Sanchez G, De Cock L, Goetgeluk G, Pascal E, De Munter S, Deseins L, Ingels J, Michiels T, De Vos R, Zolfaghari A, Vandamme N, Roels J, Kerre T, Dmitriev RI, Taghon T, Vermijlen D, and Vandekerckhove B
- Abstract
In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)
- Published
- 2024
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6. Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.
- Author
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De Munter S, Buhl JL, De Cock L, Van Parys A, Daneels W, Pascal E, Deseins L, Ingels J, Goetgeluk G, Jansen H, Billiet L, Pille M, Van Duyse J, Bonte S, Vandamme N, Van Dorpe J, Offner F, Leclercq G, Taghon T, Depla E, Tavernier J, Kerre T, Drost J, and Vandekerckhove B
- Subjects
- Humans, Animals, Mice, T-Lymphocytes immunology, T-Lymphocytes metabolism, Lymphoma, Large B-Cell, Diffuse immunology, Lymphoma, Large B-Cell, Diffuse therapy, Lymphoma, Large B-Cell, Diffuse genetics, Gene Knockout Techniques, Cell Line, Tumor, CRISPR-Cas Systems, CD27 Ligand, Immunotherapy, Adoptive methods, Xenograft Model Antitumor Assays, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen genetics
- Abstract
CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target., (©2024 American Association for Cancer Research.)
- Published
- 2024
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7. Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum.
- Author
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Ingels J, De Cock L, Stevens D, Mayer RL, Théry F, Sanchez GS, Vermijlen D, Weening K, De Smet S, Lootens N, Brusseel M, Verstraete T, Buyle J, Van Houtte E, Devreker P, Heyns K, De Munter S, Van Lint S, Goetgeluk G, Bonte S, Billiet L, Pille M, Jansen H, Pascal E, Deseins L, Vantomme L, Verdonckt M, Roelandt R, Eekhout T, Vandamme N, Leclercq G, Taghon T, Kerre T, Vanommeslaeghe F, Dhondt A, Ferdinande L, Van Dorpe J, Desender L, De Ryck F, Vermassen F, Surmont V, Impens F, Menten B, Vermaelen K, and Vandekerckhove B
- Subjects
- Humans, Male, Female, Middle Aged, Aged, Dendritic Cells immunology, Lung Neoplasms immunology, Lung Neoplasms pathology, Cancer Vaccines immunology, Carcinoma, Non-Small-Cell Lung immunology, Carcinoma, Non-Small-Cell Lung pathology, Carcinoma, Non-Small-Cell Lung therapy, Antigens, Neoplasm immunology, Cell Differentiation immunology, Vaccination, T-Lymphocytes immunology
- Abstract
Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively, these data support the feasibility, safety, and immunogenicity of this treatment in resected NSCLC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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8. HES6 knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.
- Author
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De Vos T, Oatman N, Boehme L, Putteman T, Velghe I, Van Droogenbroeck Y, De Munter S, Cesnekova M, Van Nieuwerburgh F, Vandekerckhove B, Philippe J, and Taghon T
- Abstract
Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models. Using bulk and scRNA-seq data, we show that HES6 is expressed during erythroid/megakaryocyte and pDC development, as well as in multipotent precursors and at specific stages of T- and B-cell development following preBCR and preTCR signalling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, pDCs, Band T-cells. In addition, HES6 knockdown HSPCs displayed reduced colony forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression impacts cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that impact these perturbations. Thus, our study uncovers new insights for a role of HES6 in human hematopoiesis.
- Published
- 2024
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9. The Wiskott-Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation.
- Author
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Pille M, Avila J, Sanchez GS, Goetgeluk G, De Munter S, Jansen H, Billiet L, Weening K, Xue H, Bonte S, Ingels J, De Cock L, Pascal E, Deseins L, Kerre T, Taghon T, Leclercq G, Vermijlen D, Davis B, and Vandekerckhove B
- Subjects
- Humans, Animals, Mice, Cell Lineage, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, Cell Differentiation, Wiskott-Aldrich Syndrome Protein metabolism, Wiskott-Aldrich Syndrome
- Abstract
The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro , their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)
+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pille, Avila, Sanchez, Goetgeluk, De Munter, Jansen, Billiet, Weening, Xue, Bonte, Ingels, De Cock, Pascal, Deseins, Kerre, Taghon, Leclercq, Vermijlen, Davis and Vandekerckhove.)- Published
- 2023
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10. Single-cell profiling identifies a novel human polyclonal unconventional T cell lineage.
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Billiet L, De Cock L, Sanchez Sanchez G, Mayer RL, Goetgeluk G, De Munter S, Pille M, Ingels J, Jansen H, Weening K, Pascal E, Raes K, Bonte S, Kerre T, Vandamme N, Seurinck R, Roels J, Lavaert M, Van Nieuwerburgh F, Leclercq G, Taghon T, Impens F, Menten B, Vermijlen D, and Vandekerckhove B
- Subjects
- Adult, Humans, Cell Lineage, Cell Differentiation, Thymus Gland, T-Lymphocytes metabolism, Receptors, Antigen, T-Cell, alpha-beta genetics, Receptors, Antigen, T-Cell, alpha-beta metabolism
- Abstract
In the human thymus, a CD10+ PD-1+ TCRαβ+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8β expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny., (© 2023 Billiet et al.)
- Published
- 2023
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11. Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles.
- Author
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Berdecka D, Harizaj A, Goemaere I, Punj D, Goetgeluk G, De Munter S, De Keersmaecker H, Boterberg V, Dubruel P, Vandekerckhove B, De Smedt SC, De Vos WH, and Braeckmans K
- Subjects
- Humans, Organophosphorus Compounds, Macromolecular Substances, T-Lymphocytes, Nanoparticles
- Abstract
Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)
- Published
- 2023
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12. The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency.
- Author
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Wahlen S, Matthijssens F, Van Loocke W, Taveirne S, Kiekens L, Persyn E, Van Ammel E, De Vos Z, De Munter S, Matthys P, Van Nieuwerburgh F, Taghon T, Vandekerckhove B, Van Vlierberghe P, and Leclercq G
- Subjects
- Humans, Core Binding Factor Alpha 1 Subunit metabolism, Gene Expression Regulation, Killer Cells, Natural metabolism, Transcription Factors metabolism
- Abstract
Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rβ expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations., Competing Interests: SW, FM, WV, ST, LK, EP, EV, ZD, SD, PM, FV, TT, BV, PV, GL No competing interests declared, (© 2022, Wahlen et al.)
- Published
- 2022
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13. Small-scale manufacturing of neoantigen-encoding messenger RNA for early-phase clinical trials.
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Ingels J, De Cock L, Mayer RL, Devreker P, Weening K, Heyns K, Lootens N, De Smet S, Brusseel M, De Munter S, Pille M, Billiet L, Goetgeluk G, Bonte S, Jansen H, Lint SV, Leclercq G, Taghon T, Menten B, Vermaelen K, Impens F, and Vandekerckhove B
- Subjects
- Antigens, Neoplasm genetics, Humans, Immunotherapy, Peptides, RNA, Messenger genetics, Cancer Vaccines, Lung Neoplasms, Neoplasms genetics, Neoplasms therapy
- Abstract
Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA. The authors have developed MIDRIX
NEO , a personalized mRNA-loaded dendritic cell vaccine targeting tumor neoantigens, which is currently being evaluated in a phase 1 clinical study in lung cancer patients. To facilitate this study, the authors set up a Good Manufacturing Practice (GMP)-compliant production process for the manufacture of small batches of personalized neoantigen-encoding mRNA. In this article, the authors describe the complete mRNA production process and the extensive quality assessment to which the mRNA is subjected. Validation runs have shown that the process delivers mRNA of reproducible, high quality. This process is now successfully applied for the production of neoantigen-encoding mRNA for the clinical evaluation of MIDRIXNEO . To the authors' knowledge, this is the first time that a GMP-based production process of patient-tailored neoantigen mRNA has been described., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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14. Photothermal nanofibres enable safe engineering of therapeutic cells.
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Xiong R, Hua D, Van Hoeck J, Berdecka D, Léger L, De Munter S, Fraire JC, Raes L, Harizaj A, Sauvage F, Goetgeluk G, Pille M, Aalders J, Belza J, Van Acker T, Bolea-Fernandez E, Si T, Vanhaecke F, De Vos WH, Vandekerckhove B, van Hengel J, Raemdonck K, Huang C, De Smedt SC, and Braeckmans K
- Subjects
- Animals, CRISPR-Cas Systems genetics, Cell Survival drug effects, Cell- and Tissue-Based Therapy, Humans, MCF-7 Cells, Melanoma, Experimental genetics, Melanoma, Experimental immunology, Melanoma, Experimental therapy, Mice, Nanofibers chemistry, Nanoparticles therapeutic use, Neoplasms genetics, Neoplasms immunology, Neoplasms pathology, Programmed Cell Death 1 Receptor antagonists & inhibitors, Programmed Cell Death 1 Receptor immunology, RNA, Small Interfering chemistry, RNA, Small Interfering genetics, RNA, Small Interfering immunology, Transfection, Immunotherapy, Adoptive, Nanoparticles chemistry, Neoplasms therapy, RNA, Small Interfering pharmacology
- Abstract
Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR-Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2021
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15. In vitro OP9-DL1 co-culture and subsequent maturation in the presence of IL-21 generates tumor antigen-specific T cells with a favorable less-differentiated phenotype and enhanced functionality.
- Author
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Bonte S, de Munter S, Billiet L, Goetgeluk G, Ingels J, Jansen H, Pille M, de Cock L, Weening K, Taghon T, Leclercq G, Vandekerckhove B, and Kerre T
- Subjects
- Antigens, Neoplasm, Coculture Techniques, Humans, Interleukins, Phenotype, Leukemia, Myeloid, Acute therapy, T-Lymphocytes
- Abstract
T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms' tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, T
SCM ) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro , starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable TSCM -like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)- Published
- 2021
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16. Human Thymic CD10 + PD-1 + Intraepithelial Lymphocyte Precursors Acquire Interleukin-15 Responsiveness at the CD1a - CD95 + CD28 - CCR7 - Developmental Stage.
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Billiet L, Goetgeluk G, Bonte S, De Munter S, De Cock L, Pille M, Ingels J, Jansen H, Weening K, Van Nieuwerburgh F, Kerre T, Taghon T, Leclercq G, and Vandekerckhove B
- Subjects
- Adult, Cell Proliferation, Cells, Cultured, Child, Humans, CD8-Positive T-Lymphocytes cytology, CD8-Positive T-Lymphocytes metabolism, Cell Differentiation, Interleukin-15 pharmacology, Receptors, Cell Surface metabolism, Thymocytes cytology, Thymocytes metabolism
- Abstract
Human thymic CD8αα
+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a- fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10+ PD-1+ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a- CD10+ PD-1+ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.- Published
- 2020
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17. RepoMan stimulates the chromosome-dependent pathway of microtubule assembly.
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De Munter S, Van der Hoeven G, and Bollen M
- Subjects
- Carrier Proteins genetics, Cell Cycle Proteins genetics, Centrosome metabolism, HEK293 Cells, HeLa Cells, Humans, Microtubule-Associated Proteins metabolism, Nuclear Proteins genetics, Phosphorylation, Protein Phosphatase 1 metabolism, Transfection, Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Chromosomes, Human metabolism, M Phase Cell Cycle Checkpoints genetics, Microtubules metabolism, Nuclear Proteins metabolism, Signal Transduction genetics
- Abstract
RepoMan is a chromosome-associated scaffold protein that integrates signaling of multiple kinases and phosphatases to coordinate spindle-kinetochore interactions, chromosome (de)condensation and nuclear envelope (dis)assembly during mitosis. Another key mitotic event is the assembly of a microtubule-based spindle, which involves redundant pathways emanating from the centrosomes, microtubules and chromosomes. Here we describe a novel mitotic function of RepoMan in regulating chromosome-dependent microtubule assembly. At limiting concentrations of microtubule-destabilizing agents, RepoMan-depleted cells showed enhanced chromosome clustering. This clustering was completely dependent on the partial inhibition of microtubule growth originating from the chromosome-dependent pathway. We also demonstrated that RepoMan interacts with prime regulators of the chromosome-dependent spindle assembly such as NuSAP1, NuMA, and TPX2. In addition, RepoMan was required to enable or maintain phosphorylation of NuSAP1 at CDK sites, thereby enabling activation of NuSAP1 through dissociation of inhibitory importin β/7. Our data identify RepoMan as an enhancer of microtubule assembly at chromosomes.
- Published
- 2020
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18. Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation.
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Raes L, Stremersch S, Fraire JC, Brans T, Goetgeluk G, De Munter S, Van Hoecke L, Verbeke R, Van Hoeck J, Xiong R, Saelens X, Vandekerckhove B, De Smedt S, Raemdonck K, and Braeckmans K
- Abstract
Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells. In this study, we demonstrate that gold nanoparticle-mediated vapor nanobubble (VNB) photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells. Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration, while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%. Importantly, compared to electroporation, which is the reference technology for nonviral transfection of T cells, a fivefold increase in the number of transfected viable Jurkat T cells was observed. Altogether, our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells, with promising potential for the future engineering of cells in therapeutic and fundamental research applications.
- Published
- 2020
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19. TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment.
- Author
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Depreter B, Weening KE, Vandepoele K, Essand M, De Moerloose B, Themeli M, Cloos J, Hanekamp D, Moors I, D'hont I, Denys B, Uyttebroeck A, Van Damme A, Dedeken L, Snauwaert S, Goetgeluk G, De Munter S, Kerre T, Vandekerckhove B, Lammens T, and Philippé J
- Subjects
- Adult, Child, Humans, Immunotherapy, Male, Nuclear Proteins, Receptors, Antigen, T-Cell, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute therapy
- Abstract
Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML., (Copyright© 2020 Ferrata Storti Foundation.)
- Published
- 2020
- Full Text
- View/download PDF
20. T-cells with a single tumor antigen-specific T-cell receptor can be generated in vitro from clinically relevant stem cell sources.
- Author
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Bonte S, De Munter S, Goetgeluk G, Ingels J, Pille M, Billiet L, Taghon T, Leclercq G, Vandekerckhove B, and Kerre T
- Subjects
- Hematopoietic Stem Cells, Humans, Neoplasms therapy, Antigens, Neoplasm immunology, Immunotherapy, Adoptive, Receptors, Antigen, T-Cell genetics, T-Lymphocytes
- Abstract
Chimeric antigen receptor (CAR) T-cells have shown great promise in the treatment of B-cell malignancies. For acute myeloid leukemia (AML), however, the optimal target surface antigen has yet to be discovered. Alternatively, T-cell receptor (TCR)-redirected T-cells target intracellular antigens, marking a broader territory of available target antigens. Currently, adoptive TCR T-cell therapy uses peripheral blood lymphocytes for the introduction of a transgenic TCR. However, this can cause graft-versus-host disease, due to mispairing of introduced and endogenous TCR chains. Therefore, we started from hematopoietic stem and progenitor cells (HSPC), that do not express a TCR yet, isolated from healthy donors, patients in remission after chemotherapy and AML patients at diagnosis. Using the OP9-DL1 in vitro co-culture system and agonist selection, TCR-transduced HSPC develop into mature tumor antigen-specific T-cells with only one TCR. We show here that this approach is feasible with adult HSPC from clinically relevant sources, albeit with slower maturation and lower cell yield compared to cord blood HSPC. Moreover, cryopreservation of HSPC does not have an effect on cell numbers or functionality of the generated T-cells. In conclusion, we show here that it is feasible to generate TA-specific T-cells from HSPC from adult healthy donors and patients and we believe these T-cells could be of use as a very valuable form of patient-tailored T-cell immunotherapy., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)
- Published
- 2020
- Full Text
- View/download PDF
21. Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.
- Author
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Huyghe L, Van Parys A, Cauwels A, Van Lint S, De Munter S, Bultinck J, Zabeau L, Hostens J, Goethals A, Vanderroost N, Verhee A, Uzé G, Kley N, Peelman F, Vandekerckhove B, Brouckaert P, and Tavernier J
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Mice, Transgenic, Immunotherapy, Neoplasms therapy, Tumor Necrosis Factor-alpha
- Abstract
Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T-cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8-targeted type I interferon AcTakine. Co-treatment with a CD13-targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2020
- Full Text
- View/download PDF
22. Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly.
- Author
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De Munter S, Van Parys A, Bral L, Ingels J, Goetgeluk G, Bonte S, Pille M, Billiet L, Weening K, Verhee A, Van der Heyden J, Taghon T, Leclercq G, Kerre T, Tavernier J, and Vandekerckhove B
- Subjects
- Cell Line, Genetic Vectors, Humans, Lymphocyte Activation, Polymerase Chain Reaction, Receptors, Antigen, T-Cell genetics, Single-Chain Antibodies genetics, T-Lymphocytes immunology, Immunotherapy, Adoptive, Receptors, Antigen, T-Cell immunology, Single-Chain Antibodies immunology, Single-Domain Antibodies immunology
- Abstract
Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs., Competing Interests: Jan Tavernier is affiliated with Orionis Biosciences (as a scientific advisor and/or an employee) and holds equity interests in Orionis Bioscience. Jan Tavernier received financial research support from Orionis Biosciences NV. All the other authors declare no conflict of interest.
- Published
- 2020
- Full Text
- View/download PDF
23. Developmental and Degenerative Cerebellar Pathologies in Peroxisomal β-Oxidation Deficiency.
- Author
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De Munter S and Baes M
- Subjects
- Animals, Axons metabolism, Axons pathology, Humans, Oxidation-Reduction, Purkinje Cells metabolism, Purkinje Cells pathology, Cerebellum metabolism, Cerebellum pathology, Peroxisomes metabolism
- Abstract
The integrity of the cerebellum is exquisitely dependent on peroxisomal β-oxidation metabolism. Patients with peroxisomal β-oxidation defects commonly develop malformation, leukodystrophy, and/or atrophy of the cerebellum depending on the gene defect and on the severity of the mutation. By analyzing mouse models lacking the central peroxisomal β-oxidation enzyme, multifunctional protein-2 (MFP2), either globally or in selected cell types, insights into the pathomechanisms could be obtained. All mouse models developed ataxia, but the onset was earlier in global and neural-selective (Nestin) Mfp2
-/- knockout mice as compared to Purkinje cell (PC)-selective Mfp2 knockouts.At the histological level, this was associated with developmental anomalies in global and Nestin-Mfp2-/- mice, including aberrant wiring of PCs by parallel and climbing fibers and altered electrical properties of PCs. In all mouse models, dystrophy of PC axons with swellings initiating in the deep cerebellar nuclei and evolving to the proximal axon, preceded death of PCs. These degenerative features are in part mediated by deficient peroxisomal β-oxidation within PCs but are accelerated when MFP2 is also absent from other neural cell types. The metabolic causes of the diverse cerebellar pathologies remain unknown.In conclusion, peroxisomal β-oxidation is required both for the development and for the maintenance of the cerebellum. This is mediated by PC autonomous and nonautonomous mechanisms.- Published
- 2020
- Full Text
- View/download PDF
24. Autonomous Purkinje cell axonal dystrophy causes ataxia in peroxisomal multifunctional protein-2 deficiency.
- Author
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De Munter S, Bamps D, Malheiro AR, Kumar Baboota R, Brites P, and Baes M
- Subjects
- Aging, Animals, Astrocytes pathology, Astrocytes physiology, Axons pathology, Cerebellar Ataxia etiology, Cerebellar Ataxia pathology, Disease Models, Animal, Gliosis pathology, Gliosis physiopathology, Mice, Transgenic, Microglia pathology, Microglia physiology, Neurodegenerative Diseases pathology, Peroxisomal Disorders pathology, Peroxisomal Multifunctional Protein-2 genetics, Purkinje Cells pathology, Axons physiology, Cerebellar Ataxia physiopathology, Neurodegenerative Diseases physiopathology, Peroxisomal Disorders physiopathology, Peroxisomal Multifunctional Protein-2 deficiency, Purkinje Cells physiology
- Abstract
Peroxisomes play a crucial role in normal neurodevelopment and in the maintenance of the adult brain. This depends largely on intact peroxisomal β-oxidation given the similarities in pathologies between peroxisome biogenesis disorders and deficiency of multifunctional protein-2 (MFP2), the central enzyme of this pathway. Recently, adult patients diagnosed with cerebellar ataxia were shown to have mild mutations in the MFP2 gene, hydroxy-steroid dehydrogenase (17 beta) type 4 (HSD17B4). Cerebellar atrophy also develops in MFP2 deficient mice but the cellular origin of the degeneration is unexplored. In order to investigate whether peroxisomal β-oxidation is essential within Purkinje cells, the sole output neurons of the cerebellum, we generated and characterized a mouse model with Purkinje cell selective deletion of the MFP2 gene. We show that selective loss of MFP2 from mature cerebellar Purkinje neurons causes a late-onset motor phenotype and progressive Purkinje cell degeneration, thereby mimicking ataxia and cerebellar deterioration in patients with mild HSD17B4 mutations. We demonstrate that swellings on Purkinje cell axons coincide with ataxic behavior and precede neurodegeneration. Loss of Purkinje cells occurs in a characteristic banded pattern, proceeds in an anterior to posterior fashion and is accompanied by progressive astro- and microgliosis. These data prove that the peroxisomal β-oxidation pathway is required within Purkinje neurons to maintain their axonal integrity, independent of glial dysfunction., (© 2018 International Society of Neuropathology.)
- Published
- 2018
- Full Text
- View/download PDF
25. Nanobody Based Dual Specific CARs.
- Author
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De Munter S, Ingels J, Goetgeluk G, Bonte S, Pille M, Weening K, Kerre T, Abken H, and Vandekerckhove B
- Subjects
- Humans, Jurkat Cells, Leukemia, B-Cell genetics, Leukemia, B-Cell immunology, Leukemia, B-Cell pathology, Leukemia, B-Cell therapy, Lymphoma, B-Cell genetics, Lymphoma, B-Cell immunology, Lymphoma, B-Cell pathology, Lymphoma, B-Cell therapy, T-Lymphocytes pathology, Transduction, Genetic, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell immunology, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Single-Domain Antibodies genetics, Single-Domain Antibodies immunology, T-Lymphocytes immunology
- Abstract
Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity., Competing Interests: The authors declare no conflict of interest.
- Published
- 2018
- Full Text
- View/download PDF
26. The checkpoint for agonist selection precedes conventional selection in human thymus.
- Author
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Verstichel G, Vermijlen D, Martens L, Goetgeluk G, Brouwer M, Thiault N, Van Caeneghem Y, De Munter S, Weening K, Bonte S, Leclercq G, Taghon T, Kerre T, Saeys Y, Van Dorpe J, Cheroutre H, and Vandekerckhove B
- Abstract
The thymus plays a central role in self-tolerance, partly by eliminating precursors with a T cell receptor (TCR) that binds strongly to self-antigens. However, the generation of self-agonist-selected lineages also relies on strong TCR signaling. How thymocytes discriminate between these opposite outcomes remains elusive. Here, we identified a human agonist-selected PD-1
+ CD8αα+ subset of mature CD8αβ+ T cells that displays an effector phenotype associated with agonist selection. TCR stimulation of immature post-β-selection thymocyte blasts specifically gives rise to this innate subset and fixes early T cell receptor alpha variable (TRAV) and T cell receptor alpha joining (TRAJ) rearrangements in the TCR repertoire. These findings suggest that the checkpoint for agonist selection precedes conventional selection in the human thymus., (Copyright © 2017, American Association for the Advancement of Science.)- Published
- 2017
- Full Text
- View/download PDF
27. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities.
- Author
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Van Caeneghem Y, De Munter S, Tieppo P, Goetgeluk G, Weening K, Verstichel G, Bonte S, Taghon T, Leclercq G, Kerre T, Debets R, Vermijlen D, Abken H, and Vandekerckhove B
- Abstract
Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5
+ CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+ CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3- AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3- AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.- Published
- 2017
- Full Text
- View/download PDF
28. Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions.
- Author
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De Munter S, Görnemann J, Derua R, Lesage B, Qian J, Heroes E, Waelkens E, Van Eynde A, Beullens M, and Bollen M
- Subjects
- Biotinylation, Genetic Complementation Test, HEK293 Cells, Humans, Reproducibility of Results, Biological Assay methods, Dimerization, Protein Interaction Mapping
- Abstract
The biotin identification (BioID) protocol uses a mutant of the biotin ligase BirA (BirA*) fused to a protein-of-interest to biotinylate proximate proteins in intact cells. Here, we show that two inactive halves of BirA* separately fused to a catalytic and regulatory subunit of protein phosphatase PP1 reconstitute a functional BirA* enzyme upon heterodimerization of the phosphatase subunits. We also demonstrate that this BirA* fragment complementation approach, termed split-BioID, can be used to screen for substrates and other protein interactors of PP1 holoenzymes. Split-BioID is a novel and versatile tool for the identification of (transient) interactors of protein dimers., (© 2016 Federation of European Biochemical Societies.)
- Published
- 2017
- Full Text
- View/download PDF
29. Early-onset Purkinje cell dysfunction underlies cerebellar ataxia in peroxisomal multifunctional protein-2 deficiency.
- Author
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De Munter S, Verheijden S, Vanderstuyft E, Malheiro AR, Brites P, Gall D, Schiffmann SN, and Baes M
- Subjects
- Animals, Axons metabolism, Cerebellar Ataxia metabolism, Mice, Knockout, Peroxisomal Multifunctional Protein-2 deficiency, Cerebellum metabolism, Peroxisomal Multifunctional Protein-2 metabolism, Purkinje Cells metabolism, Synapses physiology
- Abstract
The cerebellar pathologies in peroxisomal diseases underscore that these organelles are required for the normal development and maintenance of the cerebellum, but the mechanisms have not been resolved. Here we investigated the origins of the early-onset coordination impairment in a mouse model with neural selective deficiency of multifunctional protein-2, the central enzyme of peroxisomal β-oxidation. At the age of 4weeks, Nestin-Mfp2(-/-) mice showed impaired motor learning on the accelerating rotarod and underperformed on the balance beam test. The gross morphology of the cerebellum and Purkinje cell arborization were normal. However, electrophysiology revealed a reduced Purkinje cell firing rate, a decreased excitability and an increased membrane capacitance. The distribution of climbing and parallel fiber synapses on Purkinje cells was immature and was accompanied by an increased spine length. Despite normal myelination, Purkinje cell axon degeneration was evident from the occurrence of axonal swellings containing accumulated organelles. In conclusion, the electrical activity, axonal integrity and wiring of Purkinje cells are exquisitely dependent on intact peroxisomal β-oxidation in neural cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
30. The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism.
- Author
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Kumar GS, Gokhan E, De Munter S, Bollen M, Vagnarelli P, Peti W, and Page R
- Subjects
- Carrier Proteins chemistry, Cell Cycle, Cell Cycle Proteins chemistry, Crystallography, X-Ray, Humans, Ki-67 Antigen chemistry, Magnetic Resonance Spectroscopy, Models, Molecular, Nuclear Proteins chemistry, Protein Conformation, Protein Phosphatase 1 chemistry, Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Ki-67 Antigen metabolism, Nuclear Proteins metabolism, Protein Multimerization, Protein Phosphatase 1 metabolism
- Abstract
Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes., Competing Interests: The authors declare that no competing interests exist.
- Published
- 2016
- Full Text
- View/download PDF
31. Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch.
- Author
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Qian J, Beullens M, Huang J, De Munter S, Lesage B, and Bollen M
- Subjects
- Anaphase, CDC2 Protein Kinase, Cell Line, Tumor, Chromosomes metabolism, Cyclin-Dependent Kinases metabolism, Fluorescent Antibody Technique, HEK293 Cells, HeLa Cells, Humans, Microscopy, Confocal, Nuclear Envelope metabolism, Phosphoric Monoester Hydrolases, Prometaphase, Aurora Kinase B metabolism, Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Cyclin-Dependent Kinases genetics, Mitosis genetics, Nuclear Proteins metabolism, Protein Phosphatase 1 metabolism, Protein Phosphatase 2 metabolism, beta Karyopherins metabolism
- Abstract
RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates.
- Published
- 2015
- Full Text
- View/download PDF
32. The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth.
- Author
-
Winkler C, De Munter S, Van Dessel N, Lesage B, Heroes E, Boens S, Beullens M, Van Eynde A, and Bollen M
- Subjects
- Cell Death, Endoribonucleases genetics, HeLa Cells, Humans, Neoplasm Proteins genetics, Neoplasms genetics, Phosphoprotein Phosphatases genetics, Protein Phosphatase 1 genetics, Protein Phosphatase 1 metabolism, RNA-Binding Proteins genetics, Endoribonucleases metabolism, Mitosis, Neoplasm Proteins antagonists & inhibitors, Neoplasm Proteins metabolism, Neoplasms enzymology, Phosphoprotein Phosphatases metabolism, Protein Phosphatase 1 antagonists & inhibitors, RNA-Binding Proteins metabolism
- Abstract
The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe., (© 2015. Published by The Company of Biologists Ltd.)
- Published
- 2015
- Full Text
- View/download PDF
33. Peroxisomal Disorders: A Review on Cerebellar Pathologies.
- Author
-
De Munter S, Verheijden S, Régal L, and Baes M
- Subjects
- Animals, Cerebellum growth & development, Cerebellum physiopathology, Humans, Oxidation-Reduction, Peroxisomes physiology, Peroxisomal Disorders physiopathology
- Abstract
Peroxisomes are organelles with diverse metabolic tasks including essential roles in lipid metabolism. They are of utmost importance for the normal functioning of the nervous system as most peroxisomal disorders are accompanied with neurological symptoms. Remarkably, the cerebellum exquisitely depends on intact peroxisomal function both during development and adulthood. In this review, we cover all aspects of cerebellar pathology that were reported in peroxisome biogenesis disorders and in diseases caused by dysfunction of the peroxisomal α-oxidation, β-oxidation or ether lipid synthesis pathways. We also discuss the phenotypes of mouse models in which cerebellar pathologies were recapitulated and search for connections with the metabolic abnormalities. It becomes increasingly clear that besides the most severe forms of peroxisome dysfunction that are associated with developmental cerebellar defects, milder impairments can give rise to ataxia later in life., (© 2015 International Society of Neuropathology.)
- Published
- 2015
- Full Text
- View/download PDF
34. Precise Anatomic Localization of Accumulated Lipids in Mfp2 Deficient Murine Brains Through Automated Registration of SIMS Images to the Allen Brain Atlas.
- Author
-
Škrášková K, Khmelinskii A, Abdelmoula WM, De Munter S, Baes M, McDonnell L, Dijkstra J, and Heeren RM
- Abstract
Mass spectrometry imaging (MSI) is a powerful tool for the molecular characterization of specific tissue regions. Histochemical staining provides anatomic information complementary to MSI data. The combination of both modalities has been proven to be beneficial. However, direct comparison of histology based and mass spectrometry-based molecular images can become problematic because of potential tissue damages or changes caused by different sample preparation. Curated atlases such as the Allen Brain Atlas (ABA) offer a collection of highly detailed and standardized anatomic information. Direct comparison of MSI brain data to the ABA allows for conclusions to be drawn on precise anatomic localization of the molecular signal. Here we applied secondary ion mass spectrometry imaging at high spatial resolution to study brains of knock-out mouse models with impaired peroxisomal β-oxidation. Murine models were lacking D-multifunctional protein (MFP2), which is involved in degradation of very long chain fatty acids. SIMS imaging revealed deposits of fatty acids within distinct brain regions. Manual comparison of the MSI data with the histologic stains did not allow for an unequivocal anatomic identification of the fatty acids rich regions. We further employed an automated pipeline for co-registration of the SIMS data to the ABA. The registration enabled precise anatomic annotation of the brain structures with the revealed lipid deposits. The precise anatomic localization allowed for a deeper insight into the pathology of Mfp2 deficient mouse models.
- Published
- 2015
- Full Text
- View/download PDF
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