Publications by authors named "Marcus R Clark"

60 Publications

Artificial Intelligence and Cellular Segmentation in Tissue Microscopy Images.

Am J Pathol 2021 Jun 12. Epub 2021 Jun 12.

Department of Radiology and Committee on Medical Physics, University of Chicago, Chicago, IL, USA, 60637. Electronic address:

With applications in object detection, image feature extraction, image classification, and image segmentation, artificial intelligence is enabling high-throughput analysis of image data in a variety of biomedical imaging disciplines, ranging from radiology and pathology to cancer biology and immunology. Specifically, a growth in research surrounding deep learning has led to widespread application of computer vision techniques to analyze and mine data from biomedical images. The availability of open-source software packages and the development of novel, trainable deep neural network architectures has led to an increase in accuracy of cell detection and segmentation algorithms. By automating cellular segmentations, it is now possible to mine quantifiable cellular and spatio-cellular features from microscopy images, providing insight into the organization of cells in various pathologies. This mini-review provides an overview of the current state-of-the-art deep learning and artificial intelligence methods for segmentation and data mining of cells in microscopy images of tissue.
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http://dx.doi.org/10.1016/j.ajpath.2021.05.022DOI Listing
June 2021

Compartments and Connections Within the Germinal Center.

Front Immunol 2021 31;12:659151. Epub 2021 Mar 31.

Gwen Knapp Center for Lupus and Immunology Research, Section of Rheumatology, Department of Medicine, University of Chicago, Chicago, IL, United States.

Protective high affinity antibody responses emerge through an orchestrated developmental process that occurs in germinal centers (GCs). While GCs have been appreciated since 1930, a wealth of recent progress provides new insights into the molecular and cellular dynamics governing humoral immunity. In this review, we highlight advances that demonstrate that fundamental GC B cell function, selection, proliferation and SHM occur within distinct cell states. The resulting new model provides new opportunities to understand the evolution of immunity in infectious, autoimmune and neoplastic diseases.
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http://dx.doi.org/10.3389/fimmu.2021.659151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045557PMC
March 2021

Quantifying the effects of biopsy fixation and staining panel design on automatic instance segmentation of immune cells in human lupus nephritis.

J Biomed Opt 2021 Jan;26(2)

University of Chicago, Committee on Medical Physics, Department of Radiology, Chicago, Illinois, United States.

Significance: Lupus nephritis (LuN) is a chronic inflammatory kidney disease. The cellular mechanisms by which LuN progresses to kidney failure are poorly characterized. Automated instance segmentation of immune cells in immunofluorescence images of LuN can probe these cellular interactions.

Aim: Our specific goal is to quantify how sample fixation and staining panel design impact automated instance segmentation and characterization of immune cells.

Approach: Convolutional neural networks (CNNs) were trained to segment immune cells in fluorescence confocal images of LuN biopsies. Three datasets were used to probe the effects of fixation methods on cell features and the effects of one-marker versus two-marker per cell staining panels on CNN performance.

Results: Networks trained for multi-class instance segmentation on fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) samples stained with a two-marker panel had sensitivities of 0.87 and 0.91 and specificities of 0.82 and 0.88, respectively. Training on samples with a one-marker panel reduced sensitivity (0.72). Cell size and intercellular distances were significantly smaller in FFPE samples compared to fresh frozen (Kolmogorov-Smirnov, p  ≪  0.0001).

Conclusions: Fixation method significantly reduces cell size and intercellular distances in LuN biopsies. The use of two markers to identify cell subsets showed improved CNN sensitivity relative to using a single marker.
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http://dx.doi.org/10.1117/1.JBO.26.2.022910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791891PMC
January 2021

Cellular aspects of the pathogenesis of lupus nephritis.

Curr Opin Rheumatol 2021 Mar;33(2):197-204

Section of Rheumatology, Department of Medicine and Gwenn Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, USA.

Purpose Of Review: Lupus nephritis is a common severe manifestation of systemic lupus erythematosus. Despite recent advances in therapeutics and understanding of its pathogenesis, there are still substantial unmet needs. This review discusses recent discoveries in these areas, especially the role of tubulointerstitial inflammation (TII) in lupus nephritis.

Recent Findings: Non-white ethnicity is still a major risk and poor prognostic factor in lupus nephritis. TII and fibrosis have been found to be associated with worse renal outcome but the current lupus nephritis treatment guidelines and trials are based on the degree of glomerular inflammation. In combination with mycophenolate mofetil, a B-cell-targeted therapy (belimumab) and a calcineurin inhibitor (voclosporin) have shown efficacy in recent lupus nephritis trials. However, response rates have been modest. While lupus glomerulonephritis results from immune complex deposition derived from systemic autoantibodies, TII arises from complex processes associated with in situ adaptive cell networks. These include local antibody production, and cognate or antigen-induced interactions between T follicular helper cells, and likely other T-cell populations, with antigen presenting cells including B cells, myeloid dendritic cells and plasmacytoid dendritic cells.

Summary: Better understanding of the pathogenesis of TII will identify novel therapeutic targets predicted to improve outcomes in our patients with lupus nephritis.
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http://dx.doi.org/10.1097/BOR.0000000000000777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905798PMC
March 2021

Machine Learning to Quantify Humoral Selection in Human Lupus Tubulointerstitial Inflammation.

Front Immunol 2020 27;11:593177. Epub 2020 Nov 27.

Gwen Knapp Center for Lupus and Immunology Research, Section of Rheumatology and Department of Medicine, University of Chicago, Chicago, IL, United States.

In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation (SHM) and selection increase affinity for vimentin. Indeed, germline reversion of several antibodies demonstrated that higher affinity AVAs can be selected from both low affinity B cell germline clones and even those that are strongly reactive with other autoantigens. While we demonstrated affinity maturation, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a consequence of increasing polyreactivity or even non-specific binding. Therefore, it was unclear if there was also selection for increased specificity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity . Furthermore, reversion of single predicted amino acids in antibody variable regions indicated that we could use CytoSkaler to capture both negative and positive selection events. More broadly, our data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens.
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http://dx.doi.org/10.3389/fimmu.2020.593177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731665PMC
November 2020

Control of Early B Cell Development by the RNA N-Methyladenosine Methylation.

Cell Rep 2020 06;31(13):107819

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

The RNA N-methyladenosine (mA) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA mA methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic mA reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA mA methylation and its reader proteins in early B cell development.
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http://dx.doi.org/10.1016/j.celrep.2020.107819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371152PMC
June 2020

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

J Autoimmun 2020 09 30;113:102469. Epub 2020 Apr 30.

Neurovascular Surgery Program, Section of Neurosurgery, The University of Chicago Medicine and Biological Sciences, 5842 S. Maryland Ave, Chicago, IL, 60637, United States. Electronic address:

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.
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http://dx.doi.org/10.1016/j.jaut.2020.102469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483292PMC
September 2020

Novel specialized cell state and spatial compartments within the germinal center.

Nat Immunol 2020 06 27;21(6):660-670. Epub 2020 Apr 27.

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL, USA.

Within germinal centers (GCs), complex and highly orchestrated molecular programs must balance proliferation, somatic hypermutation and selection to both provide effective humoral immunity and to protect against genomic instability and neoplastic transformation. In contrast to this complexity, GC B cells are canonically divided into two principal populations, dark zone (DZ) and light zone (LZ) cells. We now demonstrate that, following selection in the LZ, B cells migrated to specialized sites within the canonical DZ that contained tingible body macrophages and were sites of ongoing cell division. Proliferating DZ (DZp) cells then transited into the larger DZ to become differentiating DZ (DZd) cells before re-entering the LZ. Multidimensional analysis revealed distinct molecular programs in each population commensurate with observed compartmentalization of noncompatible functions. These data provide a new three-cell population model that both orders critical GC functions and reveals essential molecular programs of humoral adaptive immunity.
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http://dx.doi.org/10.1038/s41590-020-0660-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255947PMC
June 2020

Kidney tissue hypoxia dictates T cell-mediated injury in murine lupus nephritis.

Sci Transl Med 2020 04;12(538)

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.

The kidney is a frequent target of autoimmune injury, including in systemic lupus erythematosus; however, how immune cells adapt to kidney's unique environment and contribute to tissue damage is unknown. We found that renal tissue, which normally has low oxygen tension, becomes more hypoxic in lupus nephritis. In the injured mouse tissue, renal-infiltrating CD4 and CD8 T cells express hypoxia-inducible factor-1 (HIF-1), which alters their cellular metabolism and prevents their apoptosis in hypoxia. HIF-1-dependent gene-regulated pathways were also up-regulated in renal-infiltrating T cells in human lupus nephritis. Perturbation of these environmental adaptations by selective HIF-1 blockade inhibited infiltrating T cells and reversed tissue hypoxia and injury in murine models of lupus. The results suggest that targeting HIF-1 might be effective for treating renal injury in autoimmune diseases.
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http://dx.doi.org/10.1126/scitranslmed.aay1620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055156PMC
April 2020

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.

Nat Commun 2019 10 18;10(1):4768. Epub 2019 Oct 18.

Department of Pathology, New York University School of Medicine, New York University, New York, NY, USA.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.
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http://dx.doi.org/10.1038/s41467-019-12824-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802180PMC
October 2019

CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis.

Nat Immunol 2019 10 2;20(10):1393-1403. Epub 2019 Sep 2.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL, USA.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.
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http://dx.doi.org/10.1038/s41590-019-0468-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754289PMC
October 2019

Transcription factories in Igκ allelic choice and diversity.

Adv Immunol 2019 19;141:33-49. Epub 2018 Dec 19.

Section of Rheumatology and Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL, United States. Electronic address:

The vertebrate immune system is tasked with the challenge of responding to any pathogen the organism might encounter, and retaining memory of that pathogen in case of future infection. Recognition and memory of pathogens are encoded within the adaptive immune system and production of T and B lymphocytes with diverse antigen receptor repertoires. In B lymphocytes, diversity is generated by sequential recombination between Variable (V), Diversity (D) and Joining (J) gene segments in the immunoglobulin heavy chain gene (Igh) and subsequent V-J recombination in immunoglobulin light chain genes (Igκ followed by Igλ). However, the process by which particular V, D and J segments are selected during recombination, and stochasticity is maintained to ensure antibody repertoire diversity, is still unclear. In this review, we focus on Igκ and recent findings regarding the relationships between gene structure, the generation of diversity and allelic choice. Surprisingly, the nuclear environment in which each Igκ allele resides, including transcription factories assembled on the nuclear matrix, plays critical roles in both gene regulation and in shaping the diversity of Vκ genes accessible to recombination. These findings provide a new paradigm for understanding Igκ recombination and Vκ diversity in the context of B lymphopoiesis.
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http://dx.doi.org/10.1016/bs.ai.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699622PMC
March 2020

Quantifying in situ adaptive immune cell cognate interactions in humans.

Nat Immunol 2019 04 18;20(4):503-513. Epub 2019 Feb 18.

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL, USA.

Two-photon excitation microscopy (TPEM) has revolutionized the understanding of adaptive immunity. However, TPEM usually requires animal models and is not amenable to the study of human disease. The recognition of antigen by T cells requires cell contact and is associated with changes in T cell shape. We postulated that by capturing these features in fixed tissue samples, we could quantify in situ adaptive immunity. Therefore, we used a deep convolutional neural network to identify fundamental distance and cell-shape features associated with cognate help (cell-distance mapping (CDM)). In mice, CDM was comparable to TPEM in discriminating cognate T cell-dendritic cell (DC) interactions from non-cognate T cell-DC interactions. In human lupus nephritis, CDM confirmed that myeloid DCs present antigen to CD4 T cells and identified plasmacytoid DCs as an important antigen-presenting cell. These data reveal a new approach with which to study human in situ adaptive immunity broadly applicable to autoimmunity, infection, and cancer.
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http://dx.doi.org/10.1038/s41590-019-0315-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474677PMC
April 2019

BRWD1 orchestrates epigenetic landscape of late B lymphopoiesis.

Nat Commun 2018 09 24;9(1):3888. Epub 2018 Sep 24.

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, USA.

Transcription factor (TF) networks determine cell fate in hematopoiesis. However, how TFs cooperate with other regulatory mechanisms to instruct transcription remains poorly understood. Here we show that in small pre-B cells, the lineage restricted epigenetic reader BRWD1 closes early development enhancers and opens the enhancers of late B lymphopoiesis to TF binding. BRWD1 regulates over 7000 genes to repress proliferative and induce differentiation programs. However, BRWD1 does not regulate the expression of TFs required for B lymphopoiesis. Hypogammaglobulinemia patients with BRWD1 mutations have B-cell transcriptional profiles and enhancer landscapes similar to those observed in Brwd1 mice. These data indicate that, in both mice and humans, BRWD1 is a master orchestrator of enhancer accessibility that cooperates with TF networks to drive late B-cell development.
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http://dx.doi.org/10.1038/s41467-018-06165-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155124PMC
September 2018

Regulated Capture of Vκ Gene Topologically Associating Domains by Transcription Factories.

Cell Rep 2018 08;24(9):2443-2456

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

Expression of vast repertoires of antigen receptors by lymphocytes, with each cell expressing a single receptor, requires stochastic activation of individual variable (V) genes for transcription and recombination. How this occurs remains unknown. Using single-cell RNA sequencing (scRNA-seq) and allelic variation, we show that individual pre-B cells monoallelically transcribe divergent arrays of Vκ genes, thereby opening stochastic repertoires for subsequent Vκ-Jκ recombination. Transcription occurs upon translocation of Vκ genes to RNA polymerase II arrayed on the nuclear matrix in transcription factories. Transcription is anchored by CTCF-bound sites or E2A-loaded Vκ promotors and continues over large genomic distances delimited only by topological associating domains (TADs). Prior to their monoallelic activation, Vκ loci are transcriptionally repressed by cyclin D3, which prevents capture of Vκ gene containing TADs by transcription factories. Cyclin D3 also represses protocadherin, olfactory, and other monoallelically expressed genes, suggesting a widely deployed mechanism for coupling monoallelic gene activation with cell cycle exit.
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http://dx.doi.org/10.1016/j.celrep.2018.07.091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310487PMC
August 2018

PI3Kδ: Too much of a good thing.

Nat Immunol 2018 09;19(9):910-911

Department of Medicine, Section of Rheumatology, Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL, USA.

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http://dx.doi.org/10.1038/s41590-018-0183-2DOI Listing
September 2018

Humoral Immunity to Vimentin in HLA-DRB1*03 Patients With Pulmonary Sarcoidosis.

Front Immunol 2018 9;9:1516. Epub 2018 Jul 9.

Respiratory Medicine Unit, Department of Medicine, Solna and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital Solna, Stockholm, Sweden.

Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of , the Vα2.3/Vβ22 T-cell receptor (TCR), and Löfgren's syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with . Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03 patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4 T-cells expressing the Vα2.3/Vβ22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (Vim) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03 patient BALF contained higher concentrations of anti-Vim antibodies than BALF from both HCs and HLA-DRB1*03 patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked recognition of vimentin by both T- and B-cells in HLA-DRB1*03 sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus.
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http://dx.doi.org/10.3389/fimmu.2018.01516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046378PMC
July 2018

Igβ ubiquitination activates PI3K signals required for endosomal sorting.

J Exp Med 2017 Dec 15;214(12):3775-3790. Epub 2017 Nov 15.

Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, Departments of Medicine and Pathology, University of Chicago, Chicago, IL

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.
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http://dx.doi.org/10.1084/jem.20161868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716028PMC
December 2017

EZH2 Regulates the Developmental Timing of Effectors of the Pre-Antigen Receptor Checkpoints.

J Immunol 2017 06 10;198(12):4682-4691. Epub 2017 May 10.

Committee on Immunology, The University of Chicago, Chicago, IL 60637;

The histone methyltransferase EZH2 is required for B and T cell development; however, the molecular mechanisms underlying this requirement remain elusive. In a murine model of lymphoid-specific EZH2 deficiency we found that EZH2 was required for proper development of adaptive, but not innate, lymphoid cells. In adaptive lymphoid cells EZH2 prevented the premature expression of and the consequent stabilization of p53, an effector of the pre-Ag receptor checkpoints. Deletion of in EZH2-deficient lymphocytes prevented p53 stabilization, extended lymphocyte survival, and restored differentiation resulting in the generation of mature B and T lymphocytes. Our results uncover a crucial role for EZH2 in adaptive lymphocytes to control the developmental timing of effectors of the pre-Ag receptor checkpoints.
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http://dx.doi.org/10.4049/jimmunol.1700319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527689PMC
June 2017

Bcl-2 as a Therapeutic Target in Human Tubulointerstitial Inflammation.

Arthritis Rheumatol 2016 11;68(11):2740-2751

University of Chicago, Chicago, Illinois.

Objective: In lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ adaptive immune cell networks that amplify local tissue damage. Since conventional therapy appears ineffective for severe TII, and these patients often progress to renal failure, understanding in situ mechanisms might reveal new therapeutic targets. This study was undertaken to assess whether dysregulated apoptotic regulators maintain local adaptive immunity and drive inflammation in TII.

Methods: This study utilized novel computational approaches that, when applied to multicolor confocal images, quantified apoptotic regulator protein expression in selected lymphocyte subsets. This approach was validated using laser-capture microdissection (LCM) coupled to quantitative polymerase chain reaction (qPCR). Furthermore, the consequences of dysregulated apoptotic mediator expression were explored in a murine model of lupus nephritis.

Results: Analyses of renal biopsy tissue from patients with lupus nephritis and those with mixed cellular renal allograft rejection revealed that the B cell lymphoma 2 protein (Bcl-2) was frequently expressed in infiltrating lymphocytes, whereas expression of myeloid cell leukemia 1 was low. In contrast, the reciprocal pattern of expression was observed in tonsil germinal centers. These results were consistent with RNA expression data obtained using LCM and qPCR. Bcl-2 was also highly expressed in tubulointerstitial infiltrates in (NZB × NZW)F1 (NZB/NZW) mice. Furthermore, treatment of NZB/NZW mice with ABT-199, a selective oral inhibitor of Bcl-2, prolonged survival and prevented proteinuria and development of TII in a lupus prevention model. Interestingly, glomerular immune complexes were partially ameliorated by ABT-199 treatment, and serum anti-double-stranded DNA antibody titers were unaffected.

Conclusion: These data demonstrate that Bcl-2 is an attractive therapeutic target in patients with lupus nephritis who manifest TII.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5083145PMC
http://dx.doi.org/10.1002/art.39744DOI Listing
November 2016

Overview of pathophysiology and treatment of human lupus nephritis.

Curr Opin Rheumatol 2016 09;28(5):460-7

Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, USA.

Purpose Of Review: Despite recent developments and treatment successes, the outcome, and prognosis of patients with lupus nephritis (LuN) have not greatly changed since the 1980s. This review covers the application of new concepts to the understanding of renal inflammation and the study of new pharmacologic agents to improve patient outcomes.

Recent Findings: Studies have shown that the presence of anti-vimentin antibodies and T follicular helper cells in patient biopsies is associated with more severe interstitial inflammation, which has been tied to faster disease progression and onset of end-stage renal disease. Additionally, data regarding the role of serum IgE antidouble-stranded DNA antibodies in LuN by means of mediating IFN1 production by plasmacytoid dendritic cells are highlighted. Finally, a thorough review of completed and currently open clinical trials of therapeutic agents is provided.

Summary: Current management of LuN is guided almost exclusively by glomerular involvement. Based on the data provided in this review, we argue that renal tubulointerstitial inflammation is no less important and represents an overlooked feature in the current clinical approach to patients. Tubulointerstitial inflammation is driven by both adaptive and innate immune mechanisms that are still poorly understood. Studying these pathogenic processes promises to reveal new therapeutic opportunities for those LuN patients with the worst prognosis.

Video Abstract: Alternate video abstract introduction (see Video, Supplemental Digital Content 1, with introduction by two of the authors - VL and KT). Abstract Video: http://links.lww.com/COR/A35.
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http://dx.doi.org/10.1097/BOR.0000000000000319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965286PMC
September 2016

Self-reactive IgE exacerbates interferon responses associated with autoimmunity.

Nat Immunol 2016 Feb 21;17(2):196-203. Epub 2015 Dec 21.

Research Department, MedImmune, Gaithersburg, Maryland, USA.

Canonically, immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE), IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs), a type of cell of the immune system linked to viral defense, which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE, followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.
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http://dx.doi.org/10.1038/ni.3326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718782PMC
February 2016

The Pathogenesis and Therapeutic Implications of Tubulointerstitial Inflammation in Human Lupus Nephritis.

Semin Nephrol 2015 Sep;35(5):455-64

Department of Pathology, University of Chicago, Chicago, IL.

Nephritis is a common complication of systemic lupus erythematosus for which current therapies often prove inadequate. Current lupus nephritis classification systems emphasize glomerular acuity and scarring. However, tubulointerstitial inflammation (TII) and scarring are much better predictors of progression to renal failure. It now is becoming clear that the immunologic features, and probable underlying mechanisms, are very different in lupus glomerulonephritis and TII at the time of biopsy. Although glomerulonephritis is a manifestation of systemic autoimmunity, TII is associated with local in situ adaptive immune cell networks predicted to amplify local inflammation and tissue damage. In addition, poorly defined networks of innate immune cells and effectors likely contribute to the severity of local inflammation. Defining these in situ immune mechanisms should lead to a better understanding of prognostically meaningful lupus nephritis subsets and show novel therapeutic opportunities.
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http://dx.doi.org/10.1016/j.semnephrol.2015.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653081PMC
September 2015

Histone reader BRWD1 targets and restricts recombination to the Igk locus.

Nat Immunol 2015 Oct 24;16(10):1094-103. Epub 2015 Aug 24.

Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois, USA.

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.
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http://dx.doi.org/10.1038/ni.3249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575638PMC
October 2015

RAG Represents a Widespread Threat to the Lymphocyte Genome.

Cell 2015 Aug 30;162(4):751-65. Epub 2015 Jul 30.

Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011, USA; Howard Hughes Medical Institute, 295 Congress Avenue, New Haven, CT 06511, USA. Electronic address:

The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of "cryptic" recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes.
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http://dx.doi.org/10.1016/j.cell.2015.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537821PMC
August 2015

Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis.

Arthritis Rheumatol 2014 Dec;66(12):3359-70

University of Chicago, Chicago, Illinois.

Objective: In lupus nephritis (LN), severe tubulointerstitial inflammation (TII) predicts progression to renal failure. Severe TII is associated with tertiary lymphoid neogenesis and in situ antigen-driven clonal B cell selection. The autoantigen(s) driving in situ B cell selection in TII are not known. This study was undertaken to identify the dominant driving autoantigen(s).

Methods: Single CD38+ or Ki-67+ B cells were laser captured from 7 biopsy specimens that were diagnostic for LN. Eighteen clonally expanded immunoglobulin heavy- and light-chain variable region pairs were cloned and expressed as monoclonal antibodies. Seven more antibodies were cloned from flow-sorted CD38+ cells from an eighth biopsy specimen. Antigen characterization was performed using a combination of confocal microscopy, enzyme-linked immunosorbent assay, screening protoarrays, immunoprecipitation, and mass spectrometry. Serum IgG titers to the dominant antigen in 48 LN and 35 non-nephritic lupus samples were determined using purified antigen-coated arrays. Autoantigen expression on normal and LN kidney was localized by immunohistochemistry and immunofluorescence.

Results: Eleven of 25 antibodies reacted with cytoplasmic structures, 4 reacted with nuclei, and none reacted with double-stranded DNA. Vimentin was the only autoantigen identified by both mass spectrometry and protoarray. Ten of the 11 anticytoplasmic TII antibodies directly bound vimentin. Vimentin was highly expressed by tubulointerstitial inflammatory cells, and the TII antibodies tested preferentially bound inflamed tubulointerstitium. Finally, high titers of serum antivimentin antibodies were associated with severe TII (P = 0.0001).

Conclusion: Vimentin, an antigenic feature of inflammation, is a dominant autoantigen targeted in situ in LN TII. This adaptive autoimmune response likely feeds forward to worsen TII and renal damage.
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http://dx.doi.org/10.1002/art.38888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264660PMC
December 2014

Immune complex formation and in situ B-cell clonal expansion in human cerebral cavernous malformations.

J Neuroimmunol 2014 Jul 10;272(1-2):67-75. Epub 2014 May 10.

Neurovascular Surgery Program, Section of Neurosurgery, Department of Surgery, University of Chicago Medicine and Biological Sciences, USA. Electronic address:

Cerebral cavernous malformations (CCMs) represent clusters of dilated vascular channels, predisposing to hemorrhagic stroke and seizures. They are associated with defective blood brain barrier, hemorrhages of different ages and a robust inflammatory cell infiltrate. We report for the first time evidence of co-localized IgG and complement membrane attack complexes in CCM lesions. CD4(+) and CD8(+) T-cells are aggregated with CD20(+) B-cells. And IgG repertoire analyses demonstrate in situ B-cell clonal expansion and antigen-driven affinity maturation in CCMs. These results suggest an organ-intrinsic adaptive immune response in CCMs that should be further characterized as a potential therapeutic target.
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http://dx.doi.org/10.1016/j.jneuroim.2014.04.016DOI Listing
July 2014

Balancing Proliferation with Igκ Recombination during B-lymphopoiesis.

Front Immunol 2014 2;5:139. Epub 2014 Apr 2.

Department of Medicine, Section of Rheumatology, Gwen Knapp Center for Lupus and Immunology Research, The University of Chicago , Chicago, IL , USA.

The essential events of B-cell development are the stochastic and sequential rearrangement of immunoglobulin heavy (Igμ) and then light chain (Igκ followed by Igλ) loci. The counterpoint to recombination is proliferation, which both maintains populations of pro-B cells undergoing Igμ recombination and expands the pool of pre-B cells expressing the Igμ protein available for subsequent Igκ recombination. Proliferation and recombination must be segregated into distinct and mutually exclusive developmental stages. Failure to do so risks aberrant gene translocation and leukemic transformation. Recent studies have demonstrated that proliferation and recombination are each affected by different and antagonistic receptors. The IL-7 receptor drives proliferation while the pre-B-cell antigen receptor, which contains Igμ and surrogate light chain, enhances Igκ accessibility and recombination. Remarkably, the principal downstream proliferative effectors of the IL-7R, STAT5 and cyclin D3, directly repress Igκ accessibility through very divergent yet complementary mechanisms. Conversely, the pre-B-cell receptor represses cyclin D3 leading to cell cycle exit and enhanced Igκ accessibility. These studies reveal how cell fate decisions can be directed and reinforced at each developmental transition by single receptors. Furthermore, they identify novel mechanisms of Igκ repression that have implications for gene regulation in general.
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http://dx.doi.org/10.3389/fimmu.2014.00139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980108PMC
June 2014

Cell distance mapping identifies functional T follicular helper cells in inflamed human renal tissue.

Sci Transl Med 2014 Apr;6(230):230ra46

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

T follicular helper (TFH) cells are critical for B cell activation in germinal centers and are often observed in human inflamed tissue. However, it is difficult to know if they contribute in situ to inflammation. Expressed markers define TFH subsets associated with distinct functions in vitro. However, such markers may not reflect in situ function. The delivery of T cell help to B cells requires direct cognate recognition. We hypothesized that by visualizing and quantifying such interactions, we could directly assess TFH cell competency in situ. Therefore, we developed computational tools to quantify spatial relationships between different cell subtypes in tissue [cell distance mapping (CDM)]. Analysis of inflamed human tissues indicated that measurement of internuclear distances between TFH and B cells could be used to discriminate between apparent cognate and noncognate interactions. Furthermore, only cognate-competent TFH cell populations expressed high levels of Bcl-6 and interleukin-21. These data suggest that CDM can be used to identify adaptive immune cell networks driving in situ inflammation. Such knowledge should help identify diseases, and disease subsets, that may benefit from therapeutic targeting of specific T cell-antigen-presenting cell interactions.
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http://dx.doi.org/10.1126/scitranslmed.3008146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129446PMC
April 2014