Publications by authors named "Sebastian Scheer"

14 Publications

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c-Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice.

Eur J Immunol 2021 May 7. Epub 2021 May 7.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.
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http://dx.doi.org/10.1002/eji.202048900DOI Listing
May 2021

The Histone Methyltransferase DOT1L Is Essential for Humoral Immune Responses.

Cell Rep 2020 12;33(11):108504

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia; Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Histone modifiers are essential for the ability of immune cells to reprogram their gene expression during differentiation. The recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like) induces oncogenic gene expression in a subset of B cell leukemias. Despite its importance, its role in the humoral immune system is unclear. Here, we demonstrate that DOT1L is a critical regulator of B cell biology. B cell development is defective in Dot1lMb1 mice, culminating in a reduction of peripheral mature B cells. Upon immunization or influenza infection of Dot1lCd23 mice, class-switched antibody-secreting cells are significantly attenuated and germinal centers fail to form. Consequently, DOT1L is essential for B cell memory formation. Transcriptome, pathway, and histological analyses identified a role for DOT1L in reprogramming gene expression for appropriate localization of B cells during the initial stage of the response. Together, these results demonstrate an essential role for DOT1L in generating an effective humoral immune response.
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http://dx.doi.org/10.1016/j.celrep.2020.108504DOI Listing
December 2020

The Methyltransferase DOT1L Controls Activation and Lineage Integrity in CD4 T Cells during Infection and Inflammation.

Cell Rep 2020 12;33(11):108505

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3800, Australia. Electronic address:

CD4 T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-γ under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-γ response, mice with a T-cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and are resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th2 cell lineage commitment and stability and suggest that inhibition of DOT1L may provide a therapeutic strategy to limit type 2 immune responses.
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http://dx.doi.org/10.1016/j.celrep.2020.108505DOI Listing
December 2020

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance.

Cell Rep 2020 10;33(3):108285

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia; Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, 3010, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, 3800, Australia; oNKo-Innate Pty Ltd., 27 Norwood Cres, Moonee Ponds, Victoria, 3039, Australia. Electronic address:

Hhex encodes a homeobox transcriptional regulator important for embryonic development and hematopoiesis. Hhex is highly expressed in NK cells, and its germline deletion results in significant defects in lymphoid development, including NK cells. To determine if Hhex is intrinsically required throughout NK cell development or for NK cell function, we generate mice that specifically lack Hhex in NK cells. NK cell frequency is dramatically reduced, while NK cell differentiation, IL-15 responsiveness, and function at the cellular level remain largely normal in the absence of Hhex. Increased IL-15 availability fails to fully reverse NK lymphopenia following conditional Hhex deletion, suggesting that Hhex regulates developmental pathways extrinsic to those dependent on IL-15. Gene expression and functional genetic approaches reveal that Hhex regulates NK cell survival by directly binding Bcl2l11 (Bim) and repressing expression of this key apoptotic mediator. These data implicate Hhex as a transcriptional regulator of NK cell homeostasis and immunity.
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http://dx.doi.org/10.1016/j.celrep.2020.108285DOI Listing
October 2020

A chemical biology toolbox to study protein methyltransferases and epigenetic signaling.

Nat Commun 2019 01 3;10(1):19. Epub 2019 Jan 3.

Structural Genomics Consortium, University of Toronto, Toronto, ON, M5G 1L7, Canada.

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4 T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.
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http://dx.doi.org/10.1038/s41467-018-07905-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318333PMC
January 2019

Loss of Vascular CD34 Results in Increased Sensitivity to Lung Injury.

Am J Respir Cell Mol Biol 2017 12;57(6):651-661

1 The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild-type (WT) and Cd34 mice by bleomycin administration. We found that Cd34 mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34 mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34 cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced tissue damage.
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http://dx.doi.org/10.1165/rcmb.2016-0386OCDOI Listing
December 2017

The Lysine Methyltransferase G9a in Immune Cell Differentiation and Function.

Front Immunol 2017 11;8:429. Epub 2017 Apr 11.

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

G9a (KMT1C, EHMT2) is a lysine methyltransferase (KMT) whose primary function is to di-methylate lysine 9 of histone H3 (H3K9me2). G9a-dependent H3K9me2 is associated with gene silencing and acts primarily through the recruitment of H3K9me2-binding proteins that prevent transcriptional activation. Gene repression via G9a-dependent H3K9me2 is critically required in embryonic stem (ES) cells for the development of cellular lineages by repressing expression of pluripotency factors. In the immune system, lymphoid cells such as T cells and innate lymphoid cells (ILCs) can differentiate from a naïve state into one of several effector lineages that require both activating and repressive mechanisms to maintain the correct gene expression program. Furthermore, the long-term immunity to re-infection is mediated by memory T cells, which also require specific gene expression and repression to maintain a quiescent state. In this review, we examine the molecular machinery of G9a-dependent functions, address the role of G9a in lymphoid cell differentiation and function, and identify potential functions of T cells and ILCs that may be controlled by G9a. Together, this review will highlight the dynamic nature of G9a-dependent H3K9me2 in the immune system and shed light on the nature of repressive epigenetic modifications in cellular lineage choice.
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http://dx.doi.org/10.3389/fimmu.2017.00429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387087PMC
April 2017

Early-life antibiotic treatment enhances the pathogenicity of CD4 T cells during intestinal inflammation.

J Leukoc Biol 2017 04 29;101(4):893-900. Epub 2016 Dec 29.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada;

The incidence of inflammatory bowel diseases (IBDs) has steadily increased in recent decades-a phenomenon that cannot be explained by genetic mutations alone. Other factors, including the composition of the intestinal microbiome, are potentially important contributors to the increased occurrence of this group of diseases. Previous reports have shown a correlation between early-life antibiotic (Abx) treatment and an increased incidence of IBD. In this report, we investigated the effects of early-life Abx treatments on the pathogenicity of CD4 T cells using an experimental T cell transfer model of IBD. Our results show that CD4 T cells isolated from adult mice that had been treated with Abx during gestation and in early life induced a faster onset of IBD in -deficient mice compared with CD4 T cells of untreated mice. Ex vivo functional analyses of IBD-inducing CD4 T cells did not show significant differences in their immunologic potential ex vivo, despite their in vivo phenotype. However, genome-wide gene-expression analysis revealed that these cells displayed dysregulated expression of genes associated with cell-cycle regulation, metabolism, and cellular stress. Analysis of Abx-treated CD4 T cell donors showed systemically elevated levels of the stress hormone corticosterone throughout life compared with untreated donors. The cohousing of Abx-treated mice with untreated mice decreased serum corticosterone, and a consequent transfer of the cells from cohoused mice into -deficient mice restored the onset and severity of disease to that of untreated animals. Thus, our results suggest that early-life Abx treatment results in a stress response with high levels of corticosterone that influences CD4 T cell function.
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http://dx.doi.org/10.1189/jlb.3MA0716-334RRDOI Listing
April 2017

Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation.

Infect Immun 2016 02 7;84(2):491-501. Epub 2015 Dec 7.

The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia

Immunological cross talk between mucosal tissues such as the intestine and the lung is poorly defined during homeostasis and disease. Here, we show that a low-dose infection with the intestinally restricted helminth parasite Trichuris muris results in the production of Th1 cell-dependent gamma interferon (IFN-γ) and myeloid cell-derived interleukin-10 (IL-10) in the lung without causing overt airway pathology. This cross-mucosal immune response in the lung inhibits the development of papain-induced allergic airway inflammation, an innate cell-mediated type 2 airway inflammatory disease. Thus, we identify convergent and nonredundant roles of adaptive and innate immunity in mediating cross-mucosal suppression of type 2 airway inflammation during low-dose helminth-induced intestinal inflammation. These results provide further insight in identifying novel intersecting immune pathways elicited by gut-to-lung mucosal cross talk.
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http://dx.doi.org/10.1128/IAI.01240-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730564PMC
February 2016

S. mansoni bolsters anti-viral immunity in the murine respiratory tract.

PLoS One 2014 14;9(11):e112469. Epub 2014 Nov 14.

Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.

The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-α, IFN-γ, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-α agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-α-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112469PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232382PMC
December 2015

A novel tool to identify the relative contribution of lymphoid cell types that contribute to IL-10 production during the infection with Schistosoma mansoni: the TIGER index.

J Immunol Methods 2014 Apr 19;406:66-73. Epub 2014 Mar 19.

Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108 Freiburg, Germany.

Introduction: Infection with the trematode helminth Schistosoma mansoni affects more than 200 million people worldwide. Infected patients are thought to show a decreased incidence of asthma and autoimmune diseases, which is, among others, considered a result of an increased production of the immunoregulatory cytokine IL-10. However, the location and the type of cell that is responsible for the highest production of IL-10 in vivo are still unknown.

Aim: Identification of the hierarchy of IL-10 producing cell types in the mesenteric lymph node and spleen during the course of the murine infection with S. mansoni without the need of an external standard.

Methods: We describe the use of the IL-10 reporter mouse TIGER for the study of murine schistosomiasis and introduce a novel tool, which we have called the TIGER index (TI). This index combines data from flow cytometric measurements and cell count analysis and allows identifying the cell type with the highest contribution of IL-10 during the course of infection in the secondary lymphoid organs, sites of extensive immunoregulatory activity in schistosomiasis.

Results: In this paper we have calculated the TI for the mesenteric lymph nodes and the spleen in the course of a chronic infection with S. mansoni. Using the TI, we identified CD4(pos) CD25pos and CD4(pos) CD25(neg) cell populations as the highest producers of IL-10 in the mesenteric lymph node and the spleen in chronic schistosomiasis, respectively, whereas B cells, NK cells and NKT cells showed a lower contribution to IL-10 production throughout the infection.

Conclusion: The TI is a highly useful tool to measure the relative contribution of different cell types, which are responsible for the in vivo production of IL-10 in the secondary lymphoid organs during the infection with S. mansoni. Thus, the strength of the TI ensures the possibility to analyze IL-10 production in a long term experiment without the need of an external standard between each time point of analysis.
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http://dx.doi.org/10.1016/j.jim.2014.03.008DOI Listing
April 2014

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules.

BMC Biotechnol 2009 Oct 31;9:92. Epub 2009 Oct 31.

Dept of Immunotechnology, Lund University, Lund, Sweden.

Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.

Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections.

Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.
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http://dx.doi.org/10.1186/1472-6750-9-92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783032PMC
October 2009

New methods for selective isolation of bacterial DNA from human clinical specimens.

Anaerobe 2010 Feb 20;16(1):47-53. Epub 2009 May 20.

Division of Oral Microbiology and Immunology, Department of Operative and Preventive Dentistry & Periodontology, Medical Faculty, RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52057 Aachen, Germany.

Separation of bacterial DNA from human DNA in clinical samples may have an important impact on downstream applications, involving microbial diagnostic systems. We evaluated two commercially available reagents (MolYsis), Molzym GmbH & Co. KG, Bremen and Pureprove, SIRS-Lab GmbH, Jena, both Germany) for their potential to isolate and purify bacterial DNA from human DNA. We chose oral samples, which usually contain very high amounts of both human and bacterial cells. Three different DNA preparations each were made from eight caries and eight periodontal specimens using the two reagents above and a conventional DNA extraction strategy as reference. Based on target-specific real-time-quantitative PCR assays we compared the reduction of human DNA versus loss of bacterial DNA. Human DNA was monitored by targeting the beta-2-microglobulin gene, while bacteria were monitored by targeting 16S rDNA (total bacteria and Porphyromonas gingivalis) or the glycosyltransferase gene (Streptococcus mutans). We found that in most cases at least 90% of human DNA could successfully be removed, with complete removal in eight of 16 cases using MolYsis, and two (of 16) cases using Pureprove. Conversely, detection of bacterial DNA was possible in all cases with a recovery rate generally ranging from 35% to 50%. In conclusion, both strategies have the potential to reduce background interference from the host DNA which may be of remarkable value for nucleic-acid based microbial diagnostic systems.
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http://dx.doi.org/10.1016/j.anaerobe.2009.04.009DOI Listing
February 2010

Selective isolation of bacterial DNA from human clinical specimens.

J Microbiol Methods 2008 Jan 28;72(1):98-102. Epub 2007 Nov 28.

Division of Oral Microbiology and Immunology, RWTH Aachen University Hospital, Germany.

We evaluated two DNA preparation strategies (MolYsis, Molzym GmbH & Co. KG, Bremen, Germany) and Pureprove, SIRS-Lab GmbH, Jena, Germany) to selectively extract bacterial DNA from human clinical samples. By testing 16 oral samples we found that human DNA could be largely eliminated while detectable levels of bacterial DNA were obtained with all samples. Both approaches hold great potential for microbial diagnostic systems.
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http://dx.doi.org/10.1016/j.mimet.2007.10.007DOI Listing
January 2008