Publications by authors named "Malay Mandal"

40 Publications

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

It Takes Three Receptors to Raise a B Cell.

Trends Immunol 2020 07 22;41(7):629-642. Epub 2020 May 22.

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

As the unique source of diverse immunoglobulin repertoires, B lymphocytes are an indispensable part of humoral immunity. B cell progenitors progress through sequential and mutually exclusive states of proliferation and recombination, coordinated by cytokines and chemokines. Mutations affecting the crucial pre-B cell checkpoint result in immunodeficiency, autoimmunity, and leukemia. This checkpoint was previously modeled by the signaling of two opposing receptors, IL-7R and the pre-BCR. We provide an update to this model in which three receptors, IL-7R, pre-BCR, and CXCR4, work in concert to coordinate both the proper positioning of B cell progenitors in the bone marrow (BM) microenvironment and their progression through the pre-B checkpoint. Furthermore, signaling initiated by all three receptors directly instructs cell fate and developmental progression.
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http://dx.doi.org/10.1016/j.it.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331465PMC
July 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

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

Monitoring Changes of Cardio-Respiratory Parameters During 2000m Rowing Performance.

Int J Exerc Sci 2019 1;12(2):483-490. Epub 2019 Mar 1.

Department of Exercise Physiology, Sports Science, Sports Authority of India, NSSC, Bangalore, INDIA.

The purpose of this study was to characterize the kinetics of cardio-respiratory parameters of elite male rowers during 2000m rowing time trial. 16 lightweight category (LWC) and 11 open category (OC) elite male rowers attending National camp were included in the study. Pulmonary gas exchange and heart rate (HR) during 2000m rowing ergometer test was determined through breath-by-breath analysis, with a portable metabolic gas analyzer and HR monitor. Time to completion, HR, oxygen uptake (V̇O2), minute ventilation (V̇E) and respiratory exchange ratio (RER) were recorded at 500m, 1000m, 1500m and 2000m intervals. No significant (p>0.05) difference was observed in the HR kinetics during 2000m rowing between the groups. However, split HR during the entire course was on the higher side in OC than LWC. Relative V̇O2 at 1000m (p<0.01), 1500m (p<0.05) and 2000m (p<0.01) was significantly less in OC rowers compared to LWC. However, V̇E was significantly higher for the OC group at 1500m (p<0.05) and 2000m (p<0.01) whereas RER was only significantly higher at 2000m (p<0.05). %change in absolute and relative V̇O2, V̇E and RER at each 500m interval showed no significant difference among the groups. OC rowers had taken significantly less time (p<0.05) to complete first 500m, 500m to 1000m and last 500m distance than LWC rowers. This detailed insight of rower's physiological responses can help coaches and support staff to determine the physiological working capacity of rowers at different levels, predicting performance and provided normative ranges for developing a representative physiological profile of elite Indian rowers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413856PMC
March 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

Publisher Correction: A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

Nat Commun 2017 11 30;8(1):1933. Epub 2017 Nov 30.

Committee on Immunology, University of Chicago, Chicago, IL, 60637, USA.

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 11 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.
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http://dx.doi.org/10.1038/s41467-017-01780-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709372PMC
November 2017

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

A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

Nat Commun 2017 10 16;8(1):863. Epub 2017 Oct 16.

Committee on Immunology, University of Chicago, Chicago, IL, 60637, USA.

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-helper effector programs during the development of multiple innate lymphocyte lineages, including natural killer T cell, innate lymphoid cell, mucosal-associated invariant T cell and γδ lineages. PLZF is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood. Here, by systematic CRISPR/Cas9-assisted deletions of chromatin accessible regions within the Zbtb16 locus in mouse, we identify a critical enhancer controlling PLZF expression exclusively in innate lymphoid lineages. Multiple sites within this enhancer express canonical motifs for the TF Runx1, which is essential for the development of these lineages. Notably, some regulatory sites control the kinetic rather than the overall level of PLZF expression. Thus, our comprehensive, unbiased analysis of regulatory elements in vivo reveals critical mechanisms of Zbtb16 regulation shared between innate and innate-like lymphoid lineages. Zbtb16-encoded transcription factor PLZF directs the differentiation of multiple innate and innate-like cell lineages, but how Zbtb16 itself is regulated remains unclear. Here the authors show, using CRISPR gene editing, ATAC-seq and ChIP-seq, that specific Runx1-bound enhancer elements critically modulate lineage-dependent expressions of PLZF.
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http://dx.doi.org/10.1038/s41467-017-00882-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643357PMC
October 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

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

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

Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling.

Nat Rev Immunol 2014 Feb 31;14(2):69-80. Epub 2013 Dec 31.

1] Department of Discovery Immunology, Genentech Inc. [2] Division of Immunobiology and the Center for Systems Immunology, Cincinnati Children's Hospital Medical Center, Ohio 45229-3039, USA.

The development of B cells is dependent on the sequential DNA rearrangement of immunoglobulin loci that encode subunits of the B cell receptor. The pathway navigates a crucial checkpoint that ensures expression of a signalling-competent immunoglobulin heavy chain before commitment to rearrangement and expression of an immunoglobulin light chain. The checkpoint segregates proliferation of pre-B cells from immunoglobulin light chain recombination and their differentiation into B cells. Recent advances have revealed the molecular circuitry that controls two rival signalling systems, namely the interleukin-7 (IL-7) receptor and the pre-B cell receptor, to ensure that proliferation and immunoglobulin recombination are mutually exclusive, thereby maintaining genomic integrity during B cell development.
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http://dx.doi.org/10.1038/nri3570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276135PMC
February 2014

Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression.

J Exp Med 2012 Nov 29;209(12):2199-213. Epub 2012 Oct 29.

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

Ubiquitously expressed D-type cyclins are required for hematopoiesis but are dispensable in other cell lineages. Furthermore, within different hematopoietic progenitor populations the D-type cyclins play nonredundant roles. The basis of this lineage and developmental specificity is unknown. In pro-B cells we demonstrate four distinct nuclear D-type cyclin compartments, including one cyclin D3 fraction associated with CDK4 and another phosphoinositide 3-kinase-regulated fraction not required for proliferation. A third fraction of cyclin D3 was associated with the nuclear matrix and repression of >200 genes including the variable (V) gene segments Igkv1-117, Iglv1, and Igh-VJ558. Consistent with different subnuclear compartments and functions, distinct domains of cyclin D3 mediated proliferation and Igk V gene segment repression. None of the cyclin D3 nuclear compartments overlapped with cyclin D2, which was distributed, unbound to CDK4, throughout the nucleus. Furthermore, compartmentalization of the cyclins appeared to be lineage restricted because in fibroblasts, cyclin D2 and cyclin D3 occupied a single nuclear compartment and neither bound CDK4 efficiently. These data suggest that subnuclear compartmentalization enables cyclin D3 to drive cell cycle progression and repress V gene accessibility, thereby ensuring coordination of proliferation with immunoglobulin recombination.
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http://dx.doi.org/10.1084/jem.20120800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501354PMC
November 2012

A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation.

Nat Immunol 2012 Jan 22;13(3):300-7. Epub 2012 Jan 22.

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, USA.

The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.
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http://dx.doi.org/10.1038/ni.2210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028049PMC
January 2012

Epigenetic repression of the Igk locus by STAT5-mediated recruitment of the histone methyltransferase Ezh2.

Nat Immunol 2011 Oct 30;12(12):1212-20. Epub 2011 Oct 30.

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

During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.
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http://dx.doi.org/10.1038/ni.2136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233979PMC
October 2011

Ikaros and Aiolos inhibit pre-B-cell proliferation by directly suppressing c-Myc expression.

Mol Cell Biol 2010 Sep 21;30(17):4149-58. Epub 2010 Jun 21.

Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198-5805, USA.

Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell receptor and is dependent on cyclin D3 and c-Myc. We have shown previously that interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to suppress pre-B-cell proliferation. However, the molecular mechanisms through which Ikaros and Aiolos exert their growth inhibitory effect remain to be determined. Here, we provide evidence that Aiolos and Ikaros bind to the c-Myc promoter in vivo and directly suppress c-Myc expression in pre-B cells. We further show that downregulation of c-Myc is critical for the growth-inhibitory effect of Ikaros and Aiolos. Ikaros and Aiolos also induce expression of p27 and downregulate cyclin D3 in pre-B cells, and the growth-inhibitory effect of Ikaros and Aiolos is compromised in the absence of p27. A time course analysis further reveals that downregulation of c-Myc by Ikaros and Aiolos precedes p27 induction and cyclin D3 downregulation. Moreover, downregulation of c-Myc by Ikaros and Aiolos is necessary for the induction of p27 and downregulation of cyclin D3. Collectively, our studies identify a pre-B-cell receptor signaling induced inhibitory network, orchestrated by Ikaros and Aiolos, which functions to terminate pre-B-cell expansion.
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http://dx.doi.org/10.1128/MCB.00224-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937562PMC
September 2010

Ras orchestrates exit from the cell cycle and light-chain recombination during early B cell development.

Nat Immunol 2009 Oct 6;10(10):1110-7. Epub 2009 Sep 6.

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

Signals through the pre-B cell antigen receptor (pre-BCR) and interleukin 7 receptor (IL-7R) coordinate pre-B cell population expansion with subsequent recombination of the locus encoding immunoglobulin kappa-chain (Igk). Although many 'downstream' effectors of each receptor are known, how they integrate to mediate development has remained unclear. Here we report that pre-BCR-mediated activation of the Ras-MEK-Erk signaling pathway silenced transcription of Ccnd3 (encoding cyclin D3) and coordinated exit from the cell cycle with induction of the transcription factor E2A and the initiation of Igk recombination. IL-7R-mediated activation of the transcription factor STAT5 opposed this pathway by promoting Ccnd3 expression and concomitantly inhibiting Igk transcription by binding to the Igk intronic enhancer and preventing E2A recruitment. Our data show how pre-BCR signaling poises pre-B cells to undergo differentiation after escape from IL-7R signaling.
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http://dx.doi.org/10.1038/ni.1785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057509PMC
October 2009

Beta-catenin/Tcf determines the outcome of thymic selection in response to alphabetaTCR signaling.

J Immunol 2009 Sep 28;183(6):3873-84. Epub 2009 Aug 28.

Molecular Oncology Research Institute, Tufts New England Medical Center, Boston, MA 02111, USA.

Thymic maturation of T cells depends on the intracellular interpretation of alphabetaTCR signals by processes that are poorly understood. In this study, we report that beta-catenin/Tcf signaling was activated in double-positive thymocytes in response to alphabetaTCR engagement and impacted thymocyte selection. TCR engagement combined with activation of beta-catenin signaled thymocyte deletion, whereas Tcf-1 deficiency rescued from negative selection. Survival/apoptotis mediators including Bim, Bcl-2, and Bcl-x(L) were alternatively influenced by stabilization of beta-catenin or ablation of Tcf-1, and Bim-mediated beta-catenin induced thymocyte deletion. TCR activation in double-positive cells with stabilized beta-catenin triggered signaling associated with negative selection, including sustained overactivation of Lat and Jnk and a transient activation of Erk. These observations are consistent with beta-catenin/Tcf signaling acting as a switch that determines the outcome of thymic selection downstream the alphabetaTCR cascade.
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http://dx.doi.org/10.4049/jimmunol.0901369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695211PMC
September 2009

Regulation of lymphocyte progenitor survival by the proapoptotic activities of Bim and Bid.

Proc Natl Acad Sci U S A 2008 Dec 16;105(52):20840-5. Epub 2008 Dec 16.

Department of Medicine, University of Chicago, Chicago, IL 60637, USA.

On their entry into the thymus, developing lymphocyte progenitors depend on signaling from the pre-T cell receptor (pre-TCR), which orchestrates differentiation, cell proliferation, and survival. The exact mechanism of pre-TCR-mediated suppression of T cell death remains unclear and controversial. Here, we identify Bim and Bid, 2 members of the BH3-only group of the BCL2 family, as important regulators of pre-T cell death. Both factors are highly expressed in proapoptotic thymocytes and their expression is suppressed on signaling through the pre-TCR. Their expression is directly regulated by the transcription factors FoxO3a and p53. Bid expression and p53 activity are related to the ongoing rearrangement of the TCR loci and induced DNA damage responses. Bim expression and FoxO3a nuclear translocation are directly controlled by the pre-TCR by means of its downstream kinase Akt/PKB. Interestingly, deletion of either gene on a pre-TCR(-/-) background rescues survival, but fails to induce further progenitor differentiation uncoupling the 2 processes.
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http://dx.doi.org/10.1073/pnas.0807557106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634950PMC
December 2008

Targeting the NF-kappaB signaling pathway in Notch1-induced T-cell leukemia.

Nat Med 2007 Jan 17;13(1):70-7. Epub 2006 Dec 17.

Department of Medicine, Section of Rheumatology, University of Chicago, 5841 South Maryland Avenue Chicago, Illinois 60637, USA.

T-cell acute lymphoblastic leukemia (T-ALL), unlike other ALL types, is only infrequently associated with chromosomal aberrations, but it was recently shown that most individuals with T-ALL carry activating mutations in the NOTCH1 gene. However, the signaling pathways and target genes responsible for Notch1-induced neoplastic transformation remain undefined. We report here that constitutively active Notch1 activates the NF-kappaB pathway transcriptionally and via the IkappaB kinase (IKK) complex, thereby causing increased expression of several well characterized target genes of NF-kappaB in bone marrow hematopoietic stem cells and progenitors. Our observations demonstrate that the NF-kappaB pathway is highly active in established human T-ALL and that inhibition of the pathway can efficiently restrict tumor growth both in vitro and in vivo. These findings identify NF-kappaB as one of the major mediators of Notch1-induced transformation and suggest that the NF-kappaB pathway is a potential target of future therapies of T-ALL.
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http://dx.doi.org/10.1038/nm1524DOI Listing
January 2007

Fluorometric and isothermal titration calorimetric studies on binding interaction of a telechelic polymer with sodium alkyl sulfates of varying chain length.

Langmuir 2006 Apr;22(8):3514-20

Department of Chemistry, Jadavpur University, Calcutta-700 032, India.

Steady-state fluorescence measurements and isothermal titration calorimetric experiments have been performed to study the interaction between a telechelic polymer, pyrene-end-capped poly(ethylene oxide) (PYPY), and sodium alkyl sulfate surfactants having decyl, dodecyl, and tetradecyl hydrocarbon tails. Fluorometric results suggest polymer-surfactant interaction in the very low range of polymer concentrations. The relative variation in the excimer to monomer pyrene emission intensities with varying surfactant concentration reveals that initial addition of surfactant favors intramolecular preassociation until the surfactant molecules start binding with the ethylene oxide (EO) chain. With the growing number of surfactant aggregates along the EO chain, the association becomes hindered due to the polyelectrolyte effect. The results from microcalorimetric titrations in the low concentration range of PYPY solution (approximately 10(-6) M) with alkyl sulfates suggest two kinds of surfactant-polymer interactions, one with the polymer hydrophobic end groups and the other with the ethylene oxide backbone. The overall polymer-surfactant interaction starts at a much lower surfactant concentration for the hydrophobically modified polymers compared to that in the case of unsubstituted poly(ethylene oxide) homopolymer. From the experiments critical aggregation concentration values and the second critical concentration where free micelles start forming have been determined. An endeavor has been made to unveil the mechanism underlying the corresponding associations of the surfactants with the polymer.
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http://dx.doi.org/10.1021/la053370fDOI Listing
April 2006

Surfactant-induced modulation of fluorosensor activity: a simple way to maximize the sensor efficiency.

J Am Chem Soc 2006 Mar;128(10):3126-7

Department of Chemistry, Jadavpur University, Calcutta 700 032, India.

Tuning of the sensory capability of a potentially bioactive indoloquinolizine system, namely, 3-acetyl-4-oxo-6,7-dihydro-12H-indolo-[2,3-a]-quinolizine (AODIQ), is described in a biomimicking micellar nanocage. It has been shown that surfactant concentration dictates the sensing behavior of the fluorophore toward physiologically essential trace metals, such as Cu2+. This is a simple, efficient, and general technique that allows one to utilize the sensor to its maximum efficiency.
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http://dx.doi.org/10.1021/ja058359gDOI Listing
March 2006

Hedgehog signaling controls thymocyte progenitor homeostasis and differentiation in the thymus.

Nat Immunol 2006 Apr 5;7(4):418-26. Epub 2006 Mar 5.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, Illinois 60637, USA.

Commitment of hematopoietic progenitors to the T cell lineage requires the integration of multiple signaling pathways. Evidence has suggested involvement of hedgehog (Hh) signaling in T cell differentiation through its signal transducer smoothened (Smo). However, the precise function of the Hh pathway remains controversial, mainly because T cell-specific in vivo genetic models have not been used. Using pre-T cell-specific, mature T cell-specific and poly(I).poly(C)-inducible deletions of Smo and antagonists of Smo signaling, we report here that Hh is an essential positive regulator of T cell progenitor differentiation. Furthermore, we localize Hh function to a stage preceding pre-T cell receptor signaling, connect Smo signaling to the activity of the Gli1 and Gli2 transcription factors and demonstrate that Hh affects regulators of thymocyte survival and proliferation.
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http://dx.doi.org/10.1038/ni1313DOI Listing
April 2006

Regulation of T-cell progenitor survival and cell-cycle entry by the pre-T-cell receptor.

Immunol Rev 2006 Feb;209:159-69

University of Chicago, Department of Medicine, Section of Rheumatology, Committees of Immunology, Cancer and Developmental Biology, Chicago, IL 60637, USA.

Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. Although multiple reports have addressed several aspects of pre-TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR-beta locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T-cell leukemia and lymphomas. In this review, we integrate pre-TCR signaling pathways and study their effects on the regulation of T-cell progenitor cell-cycle entry and cell survival. We also connect aberrant pre-TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.
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http://dx.doi.org/10.1111/j.0105-2896.2006.00343.xDOI Listing
February 2006

The BCL2A1 gene as a pre-T cell receptor-induced regulator of thymocyte survival.

J Exp Med 2005 Feb;201(4):603-14

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637, USA.

The pre-T cell receptor (TCR) is expressed early during T cell development and imposes a tight selection for differentiating T cell progenitors. Pre-TCR-expressing cells are selected to survive and differentiate further, whereas pre-TCR(-) cells are "negatively" selected to die. The mechanisms of pre-TCR-mediated survival are poorly understood. Here, we describe the induction of the antiapoptotic gene BCL2A1 (A1) as a potential mechanism regulating inhibition of pre-T cell death. We characterize in detail the signaling pathway involved in A1 induction and show that A1 expression can induce pre-T cell survival by inhibiting activation of caspase-3. Moreover, we show that in vitro "knockdown" of A1 expression can compromise survival even in the presence of a functional pre-TCR. Finally, we suggest that pre-TCR-induced A1 overexpression can contribute to T cell leukemia in both mice and humans.
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http://dx.doi.org/10.1084/jem.20041924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2213063PMC
February 2005

Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane.

Biochim Biophys Acta 2004 Sep;1674(2):158-74

Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.
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http://dx.doi.org/10.1016/j.bbagen.2004.06.013DOI Listing
September 2004