Publications by authors named "Martin Weigert"

52 Publications

Deep Learning Enables Individual Xenograft Cell Classification in Histological Images by Analysis of Contextual Features.

J Mammary Gland Biol Neoplasia 2021 May 17. Epub 2021 May 17.

Biomedical Imaging Group, School of Engineering, Ecole Polytechnique Fédéralé de Lausanne (EPFL), Lausanne, Switzerland.

Patient-Derived Xenografts (PDXs) are the preclinical models which best recapitulate inter- and intra-patient complexity of human breast malignancies, and are also emerging as useful tools to study the normal breast epithelium. However, data analysis generated with such models is often confounded by the presence of host cells and can give rise to data misinterpretation. For instance, it is important to discriminate between xenografted and host cells in histological sections prior to performing immunostainings. We developed Single Cell Classifier (SCC), a data-driven deep learning-based computational tool that provides an innovative approach for automated cell species discrimination based on a multi-step process entailing nuclei segmentation and single cell classification. We show that human and murine cell contextual features, more than cell-intrinsic ones, can be exploited to discriminate between cell species in both normal and malignant tissues, yielding up to 96% classification accuracy. SCC will facilitate the interpretation of H&E- and DAPI-stained histological sections of xenografted human-in-mouse tissues and it is open to new in-house built models for further applications. SCC is released as an open-source plugin in ImageJ/Fiji available at the following link: https://github.com/Biomedical-Imaging-Group/SingleCellClassifier .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10911-021-09485-4DOI Listing
May 2021

Deep learning-enhanced light-field imaging with continuous validation.

Nat Methods 2021 May 7;18(5):557-563. Epub 2021 May 7.

Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

Visualizing dynamic processes over large, three-dimensional fields of view at high speed is essential for many applications in the life sciences. Light-field microscopy (LFM) has emerged as a tool for fast volumetric image acquisition, but its effective throughput and widespread use in biology has been hampered by a computationally demanding and artifact-prone image reconstruction process. Here, we present a framework for artificial intelligence-enhanced microscopy, integrating a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction. In our approach, concomitantly acquired, high-resolution two-dimensional light-sheet images continuously serve as training data and validation for the convolutional neural network reconstructing the raw LFM data during extended volumetric time-lapse imaging experiments. Our network delivers high-quality three-dimensional reconstructions at video-rate throughput, which can be further refined based on the high-resolution light-sheet images. We demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity with volumetric imaging rates up to 100 Hz.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-021-01136-0DOI Listing
May 2021

3D FIB-SEM reconstruction of microtubule-organelle interaction in whole primary mouse β cells.

J Cell Biol 2021 Feb;220(2)

Molecular Diabetology, University Hospital and Faculty of Medicine, Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202010039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748794PMC
February 2021

A convolutional neural network segments yeast microscopy images with high accuracy.

Nat Commun 2020 11 12;11(1):5723. Epub 2020 Nov 12.

Laboratory of the Physics of Biological Systems, Institute of Physics, École polytechnique fédérale de Lausanne (EPFL), Lausanne, Switzerland.

The identification of cell borders ('segmentation') in microscopy images constitutes a bottleneck for large-scale experiments. For the model organism Saccharomyces cerevisiae, current segmentation methods face challenges when cells bud, crowd, or exhibit irregular features. We present a convolutional neural network (CNN) named YeaZ, the underlying training set of high-quality segmented yeast images (>10 000 cells) including mutants, stressed cells, and time courses, as well as a graphical user interface and a web application ( www.quantsysbio.com/data-and-software ) to efficiently employ, test, and expand the system. A key feature is a cell-cell boundary test which avoids the need for fluorescent markers. Our CNN is highly accurate, including for buds, and outperforms existing methods on benchmark images, indicating it transfers well to other conditions. To demonstrate how efficient large-scale image processing uncovers new biology, we analyze the geometries of ≈2200 wild-type and cyclin mutant cells and find that morphogenesis control occurs unexpectedly early and gradually.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-19557-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665014PMC
November 2020

Practical sensorless aberration estimation for 3D microscopy with deep learning.

Opt Express 2020 Sep;28(20):29044-29053

Estimation of optical aberrations from volumetric intensity images is a key step in sensorless adaptive optics for 3D microscopy. Recent approaches based on deep learning promise accurate results at fast processing speeds. However, collecting ground truth microscopy data for training the network is typically very difficult or even impossible thereby limiting this approach in practice. Here, we demonstrate that neural networks trained only on simulated data yield accurate predictions for real experimental images. We validate our approach on simulated and experimental datasets acquired with two different microscopy modalities and also compare the results to non-learned methods. Additionally, we study the predictability of individual aberrations with respect to their data requirements and find that the symmetry of the wavefront plays a crucial role. Finally, we make our implementation freely available as open source software in Python.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1364/OE.401933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679184PMC
September 2020

Rod nuclear architecture determines contrast transmission of the retina and behavioral sensitivity in mice.

Elife 2019 12 11;8. Epub 2019 Dec 11.

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and the underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture at the expense of image detail. Here, we show that retinal optical quality improves 2-fold during terminal development, and that this enhancement is caused by nuclear inversion. We further demonstrate that improved retinal contrast transmission, rather than photon-budget or resolution, enhances scotopic contrast sensitivity by 18-27%, and improves motion detection capabilities up to 10-fold in dim environments. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast transmission as a decisive determinant of mammalian visual perception.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.49542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974353PMC
December 2019

CLIJ: GPU-accelerated image processing for everyone.

Nat Methods 2020 01;17(1):5-6

Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-019-0650-1DOI Listing
January 2020

Automated detection of the HER2 gene amplification status in Fluorescence in situ hybridization images for the diagnostics of cancer tissues.

Sci Rep 2019 06 3;9(1):8231. Epub 2019 Jun 3.

Institute of Pathology, University Hospital Carl Gustav Carus (UKD), TU Dresden, Dresden, Germany.

The human epidermal growth factor receptor 2 (HER2) gene amplification status is a crucial marker for evaluating clinical therapies of breast or gastric cancer. We propose a deep learning-based pipeline for the detection, localization and classification of interphase nuclei depending on their HER2 gene amplification state in Fluorescence in situ hybridization (FISH) images. Our pipeline combines two RetinaNet-based object localization networks which are trained (1) to detect and classify interphase nuclei into distinct classes normal, low-grade and high-grade and (2) to detect and classify FISH signals into distinct classes HER2 or centromere of chromosome 17 (CEN17). By independently classifying each nucleus twice, the two-step pipeline provides both robustness and interpretability for the automated detection of the HER2 amplification status. The accuracy of our deep learning-based pipeline is on par with that of three pathologists and a set of 57 validation images containing several hundreds of nuclei are accurately classified. The automatic pipeline is a first step towards assisting pathologists in evaluating the HER2 status of tumors using FISH images, for analyzing FISH images in retrospective studies, and for optimizing the documentation of each tumor sample by automatically annotating and reporting of the HER2 gene amplification specificities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-44643-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546913PMC
June 2019

Content-aware image restoration: pushing the limits of fluorescence microscopy.

Nat Methods 2018 12 26;15(12):1090-1097. Epub 2018 Nov 26.

Center for Systems Biology Dresden, Dresden, Germany.

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-018-0216-7DOI Listing
December 2018

Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms.

Nat Commun 2018 11 5;9(1):4620. Epub 2018 Nov 5.

Institute of Genetics, Technische Universität Dresden, 01062, Dresden, Germany.

Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06497-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218478PMC
November 2018

The influence of body composition on exercise-associated skin temperature changes after resistance training.

J Therm Biol 2018 Jul 2;75:112-119. Epub 2018 Jun 2.

Professorship of Sports Medicine / Sports Biology, Chemnitz University of Technology, Institute of Human Movement Science and Health, 09107 Chemnitz, Germany.

Resistance exercise leads to an increase in skin temperature (T) in the area of the exercised muscle. Infrared thermography seems to be applicable to identify these primary used functional muscles with measuring T changes. The aim of the current study was to investigate the influence of body composition on T patterns after resistance exercise. 38 male subjects (19-32 years, BMI 20.4-55.2 kg/m) participated. Body fat percentage and biceps skinfold thickness were calculated. The subjects were divided into two groups: lean group (LG) with body fat percentage < 25%, obese group (OG) with body fat percentage ≥ 25%. All participants completed three sets with ten repetitions of unilateral biceps curl at 50% of the one repetition maximum. To represent exercise-induced changes of T to rest (T), the algebraic difference of each time point to T was calculated. The resulting delta values (∆) are as follows: immediately after the first, second, and third set (∆T,∆T,∆T), and at 1,2,3,4,5,6,7,8,9,10,15,20,25,30 min after the third set (∆T-∆T). The maximum positive difference to T was defined as ∆T, and the time to reach ∆T was defined as Time to ∆T. LG and OG differed significantly at T (32.8 ± 0.9 vs. 31.1 ± 1.4 °C), ∆T (1.9 ± 0.4 vs. 0.9 ± 0.8 °C), Time to ∆T (4.5 ± 2.0 vs. 17.6 ± 10.2 min) and at ∆T to ∆T (p < 0.005). Correlations between body composition (BMI, body fat percentage, biceps skinfold thickness) and T, ∆T, ∆T, ∆T (-0.47 
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jtherbio.2018.05.009DOI Listing
July 2018

Biobeam-Multiplexed wave-optical simulations of light-sheet microscopy.

PLoS Comput Biol 2018 04 13;14(4):e1006079. Epub 2018 Apr 13.

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Sample-induced image-degradation remains an intricate wave-optical problem in light-sheet microscopy. Here we present biobeam, an open-source software package that enables simulation of operational light-sheet microscopes by combining data from 105-106 multiplexed and GPU-accelerated point-spread-function calculations. The wave-optical nature of these simulations leads to the faithful reproduction of spatially varying aberrations, diffraction artifacts, geometric image distortions, adaptive optics, and emergent wave-optical phenomena, and renders image-formation in light-sheet microscopy computationally tractable.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1006079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5898703PMC
April 2018

Origins and specificity of auto-antibodies in Sm+ SLE patients.

J Autoimmun 2018 06 1;90:94-104. Epub 2018 Mar 1.

Gwen Knapp Center for Lupus and Immunology Research, Department of Pathology, University of Chicago, Chicago, IL, 60637, USA.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease accompanied by production of autoantibodies directed to a variety of self-proteins and nucleic acids. The genetic basis of SLE is also complex with at least 40 susceptibility loci identified. This complexity suggests that there are a variety of SLE manifestations; nevertheless, SLE is treated as a single disease clinically. One unique SLE target is the Smith antigen (Sm), a nuclear ribonucleoprotein complex. Sm response occurs in 25% of patients with SLE. To simplify analysis of the disease and its associated autoantibody repertoire, we focused on this subset [referred to here as "Sm positive", Sm+]. We analyzed the memory B cell repertoire and identified a V region, Vκ4-1, which was significantly overrepresented in the Sm+ SLE subset. Antibodies that express Vκ4-1 are enriched in antinuclear (ANA) positive specificities and often associated with speckled ANA pattern that is a characteristic of Sm binding. In healthy individuals Vκ4-1 B cells are enriched in the unswitched memory population. Unswitched memory B cells resemble mouse marginal zone B cells and this population is decreased in all SLE patients. Moreover, we found a similar decrease in healthy African American donors. African Americans have a significantly higher prevalence of SLE compared to Caucasians. Thus, reduced unswitched memory B cell compartment may represent a new susceptibility marker for SLE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaut.2018.02.008DOI Listing
June 2018

Self-Antigen-Driven Thymic B Cell Class Switching Promotes T Cell Central Tolerance.

Cell Rep 2016 10;17(2):387-398

Committee on Immunology, University of Chicago, Chicago, IL 60637, USA; Department of Medicine, Section of Rheumatology, and Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637, USA. Electronic address:

B cells are unique antigen-presenting cells because their antigen presentation machinery is closely tied to the B cell receptor. Autoreactive thymic B cells can efficiently present cognate self-antigens to mediate CD4 T cell-negative selection. However, the nature of thymocyte-thymic B cell interaction and how this interaction affects the selection of thymic B cell repertoire and, in turn, the T cell repertoire are not well understood. Here we demonstrate that a large percentage of thymic B cells have undergone class switching intrathymically. Thymic B cell class switching requires cognate interaction with specific T cells. Class-switched thymic B cells have a distinct repertoire compared with unswitched thymic B cells or splenic B cells. Particularly, autoreactive B cell specificities preferentially expand in the thymus by undergoing class switching, and these enriched, class-switched autoreactive thymic B cells play an important role in CD4 T cell tolerance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091085PMC
October 2016

Two- and three-dimensional co-culture models of soft tissue healing: pericyte-endothelial cell interaction.

Cell Tissue Res 2016 08 30;365(2):279-93. Epub 2016 Mar 30.

Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Kirrberger Strasse, Building 57, 66421, Homburg, Germany.

The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00441-016-2391-0DOI Listing
August 2016

A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.

Cell 2015 Aug;162(5):1066-77

Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. Electronic address:

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2015.07.047DOI Listing
August 2015

ClearVolume: open-source live 3D visualization for light-sheet microscopy.

Nat Methods 2015 Jun;12(6):480-1

1] Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. [2] Center for Systems Biology Dresden, Dresden, Germany. [3] Faculty of Computer Science, Technische Universität Dresden, Dresden, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3372DOI Listing
June 2015

Sumoylated HSP90 is a dominantly inherited plasma cell dyscrasias risk factor.

J Clin Invest 2015 Jan 8;125(1):316-23. Epub 2014 Dec 8.

Posttranslationally modified proteins serve as autoimmunogenic targets in a wide spectrum of autoimmune diseases. Here, we identified a posttranslationally modified paraprotein target (paratargs) in monoclonal gammopathies of undetermined significance (MGUS), multiple myelomas (MM), and Waldenstrom's macroglobulinemias (WM) using protein macroarrays that were sumoylated and screened for reactivity with paraproteins from MGUS, MM, and WM patients. We found that paraproteins from a proportion of European, African-American, and Japanese patients specifically reacted with the sumoylated heat-shock protein 90 β isoform-α (HSP90-SUMO1, where SUMO indicates small ubiquitin-like modifier), while no reactivity with HSP90-SUMO1 was detected in over 800 controls. HSP90-SUMO1 was present in blood cells from all patients with HSP90-SUMO1-binding paraproteins. We determined that the HSP90-SUMO1 carrier state is autosomal-dominantly inherited and caused by the inability of SUMO peptidase sentrin/SUMO-specific protease 2 (SENP2) to desumoylate HSP90-SUMO1. HSP90-SUMO1 was detected in a small percentage of healthy individuals from all backgrounds; however, only MGUS, MM, and WM patients who were HSP90-SUMO1 carriers produced HSP90-SUMO1-specific paraproteins, suggesting that sumoylated HSP90 promotes pathogenesis of these diseases through chronic antigenic stimulation. This study demonstrates that harboring HSP90-SUMO1 identifies healthy individuals at risk for plasma cell dyscrasias and that dominant inheritance of posttranslationally modified autoantigenic paratargs is one of the strongest molecular defined risk factors for MGUS, MM, and WM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI76802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382232PMC
January 2015

Light chain editors of anti-DNA receptors in human B cells.

J Exp Med 2014 Feb 27;211(2):357-64. Epub 2014 Jan 27.

Gwen Knapp Center for Lupus and Immunology Research, Department of Pathology, University of Chicago, Chicago, IL 60637.

Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20122340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920568PMC
February 2014

Antibodies that bind complex glycosaminoglycans accumulate in the Golgi.

Proc Natl Acad Sci U S A 2013 Jul 1;110(29):11958-63. Epub 2013 Jul 1.

Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

Light (L) chains that edit anti-DNA heavy (H) chains rescue B-cell development by suppressing DNA binding. However, exceptional editor L chains allow B cells to reach splenic compartments even though their B-cell receptors remain autoreactive. Such incompletely edited B cells express multireactive antibodies that accumulate in the Golgi and are released as insoluble, amyloid-like immune complexes. Here, we examine examples of incomplete editing from the analysis of variable to joining (VJ) gene junction of the variable (Vλx) editor L chain. When paired with the anti-DNA heavy chain, VH56R, the Vλx variants yield antibodies with differing specificities, including glycosaminoglycan reactivity. Our results implicate these specificities in the evasion of receptor editing through intracellular sequestration of IgM and the release of insoluble IgM complexes. Our findings can be extrapolated to human L chains and have implications for understanding a latent component of the Ig repertoire that could exert pathogenic and protective functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1308620110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718083PMC
July 2013

Apoptotic marginal zone deletion of anti-Sm/ribonucleoprotein B cells.

Proc Natl Acad Sci U S A 2012 May 30;109(20):7811-6. Epub 2012 Apr 30.

Laboratory of Immunology, Graduate School of Biomedical Sciences, and Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.

CD40L is excessively produced in both human and murine lupus and plays a role in lupus pathogenesis. To address how excess CD40L induces autoantibody production, we crossed CD40L-transgenic mice with the anti-DNA H-chain transgenic mouse lines 3H9 and 56R, well-characterized models for studying B-cell tolerance to nuclear antigens. Excess CD40L did not induce autoantibody production in 3H9 mice in which anergy maintains self-tolerance, nor did it perturb central tolerance, including deletion and receptor editing, of anti-DNA B cells in 56R mice. In contrast, CD40L/56R mice restored a large number of marginal zone (MZ) B cells reactive to Sm/ribonucleoprotein (RNP) and produced autoantibody, whereas these B cells were deleted by apoptosis in MZ of 56R mice. Thus, excess CD40L efficiently blocked tolerance of Sm/RNP-reactive MZ B cells, leading to production of anti-Sm/RNP antibody implicated in the pathogenesis of lupus. These results suggest that self-reactive B cells such as anti-Sm/RNP B cells, which somehow escape tolerance in the bone marrow and migrate to MZ, are tolerized by apoptotic deletion in MZ and that a break in this tolerance may play a role in the pathogenesis of lupus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1204509109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356631PMC
May 2012

B cell receptor light chain repertoires show signs of selection with differences between groups of healthy individuals and SLE patients.

Mol Immunol 2012 Jul 18;51(3-4):273-82. Epub 2012 Apr 18.

Gwen Knapp Center for Lupus and Immunology Research, The University of Chicago, IL 60637, USA.

We have developed a microarray to study the expression of L-chain V genes (V(L) genes) in healthy and SLE patient peripheral κ- and λ-sorted B cells. In all repertoires tested, one V(L) gene accounts for over 10% of all gene V(L) expression, consistent with positive selection acting on L-chains. While a few V(L) genes were highly expressed in all individuals, most V(L) genes were expressed at different levels. Some V(L) genes (5 out of a total of 78) were not detected. We attribute their absence from the repertoire to negative selection. Positive selection and negative selection were also found in SLE repertoires, but expression of V(L) genes was different; the differences point to less regulation of V(L) gene repertoires in SLE. Our data shows that V(L) gene expression is variable and supports a model where the L-chain repertoire is generated by both positive and negative selection on L-chains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2012.03.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264353PMC
July 2012

Model for competition from self during passive immunization, with application to broadly neutralizing antibodies for HIV.

Vaccine 2012 Jan 23;30(3):607-13. Epub 2011 Nov 23.

James Franck Institute, The University of Chicago, Chicago, IL 60637, United States.

We propose a mathematical model to interpret observations concerning the behavior of broadly neutralizing antibodies for chronic HIV in vivo. The model enables us to identify a threshold antibody level that must be achieved to decrease the viral load effectively. Although this threshold has not been reached in existing passive immunization studies, it is within range of humoral immune responses, suggesting that therapeutic vaccines are feasible. In an appendix, we develop a model of passive immunization against influenza, and acute infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2011.11.048DOI Listing
January 2012

Intra-Golgi formation of IgM-glycosaminoglycan complexes promotes Ig deposition.

J Immunol 2011 Sep 12;187(6):3198-207. Epub 2011 Aug 12.

Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. However, it is not known whether intracellular Ag-Ab interactions also contribute to the formation of extracellular immune complexes. In this study, we report that certain murine B cell hybridomas accumulate intracellular IgM and release large, spherical IgM complexes. The complexes (termed "spherons") reach 2 μm in diameter, detach from the cell surface, and settle out of solution. The spherons contain IgM multimers that incorporate the J chain and resist degradation by endoglycosidase H, arguing for IgM passage through the Golgi. Treatment of cells with inhibitors of proteoglycan synthesis, or incubation of spherons with chondroitinase ABC, degrades spherons, indicating that spheron formation and growth depend on interactions between IgM and glycosaminoglycans. This inference is supported by direct binding of IgM to heparin and hyaluronic acid. We conclude that, as a consequence of IgM binding to glycosaminoglycans, multivalent IgM-glycan complexes form in transit of IgM to the cell surface. Intra-Golgi formation of immune complexes could represent a new pathogenic mechanism for immune complex deposition disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1101336DOI Listing
September 2011

Selection of individual VH genes occurs at the pro-B to pre-B cell transition.

J Immunol 2011 Aug 11;187(4):1835-44. Epub 2011 Jul 11.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V(H)), we performed CDR3 spectratyping of ∼ 75-300 rearrangements per individual V(H) in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V(H)s have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V(H)s, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V(H) usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V(H)s. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V(H). High IF clan II/III V(H)s share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V(H)s are subjected to differential selection, that V(H) IF fraction is mainly established through pre-BCR-mediated selection, that it may operate differently in clan I versus II/III V(H)s, and that it has a lasting influence on the Ab repertoire.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1100207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150439PMC
August 2011

Alternative mechanisms of receptor editing in autoreactive B cells.

Proc Natl Acad Sci U S A 2011 Apr 6;108(17):7125-30. Epub 2011 Apr 6.

Department of Pathology, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637.

Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA V(H) regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the V(H). Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and V(H) replacement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1019389108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084116PMC
April 2011

In-silico cell surface modeling reveals mechanism for initial steps of B-cell receptor signal transduction.

Mol Immunol 2009 Sep 23;46(15):3141-50. Epub 2009 Jul 23.

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.

Ligand binding to B-cell receptors (BcRs) on the B-cell surface induces crosslinking and phosphorylation of BCRs by the Src family kinases, followed by initiation of signaling. Although the nature of the earliest events following receptor engagement is currently under intensive investigation, the precise connection between crosslinking and the signaling cascade triggering has thus far remained unclear. Using a novel multiscale, agent-based simulation of B-cell surface dynamics, we present a coherent quantitative analysis of the initial stages of B-cell activation following ligand presentation. The simulation reproduces experimental results of H3 uptake, immunoglobulin secretion, immunoelectron photomicrography and FRET. While merging multiple experimental techniques, the simulation captures all essential events in the first twenty seconds following ligand presentation, and is used to make predictions on subsequent molecular events. We show that B-cell activation is mediated through a positive feedback loop between Lyn and ITAM phosphorylation, but that specificity is achieved through a combination of BcR spatial segregation and BcR selective partitioning within lipid rafts following clustering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molimm.2009.03.027DOI Listing
September 2009