Publications by authors named "Lionel Feigenbaum"

97 Publications

NK cells prevent T cell lymphoma development in T cell receptor-transgenic mice.

Cell Immunol 2020 06 27;352:104081. Epub 2020 Feb 27.

Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Mice that express a single transgenic T cell receptor have a low incidence of T cell lymphoma development. We investigated whether this tumor development is restricted by surveillance mechanisms that are exerted by IL-15-dependent cells. Lymphoma incidence was increased to between 30 and 60% when TCR transgenes were expressed in IL-15-deficient mice. Mice in which NK cells had been depleted genetically or with neutralizing antibodies allowed lymphoma growth while the absence of CD8 T cells was without consequence. Half of the emerged T cell lymphomas carried Notch1 mutations. The distinct phenotype of the lymphomas involved expression of PD1, CD30, CD24, the stress receptor ligand Mult1 and MHC class I down-regulation. NK cells were able to directly lyse lymphoma cells, and neutralizations of Mult1 and class I expression prevented NK cell degranulation. Together these data support an involvement of NK cells in tumor surveillance of nascent T cell lymphomas.
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http://dx.doi.org/10.1016/j.cellimm.2020.104081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217732PMC
June 2020

Visualizing the Selectivity and Dynamics of Interferon Signaling In Vivo.

Cell Rep 2019 Dec;29(11):3539-3550.e4

Immunology and Host Defense Group, Discipline of Infectious Diseases and Immunology, Faculty of Medicine and Health, The University of Sydney, NSW 2006, Australia; Centenary Institute, The University of Sydney, NSW 2050, Australia. Electronic address:

Interferons (IFN) are pleiotropic cytokines essential for defense against infection, but the identity and tissue distribution of IFN-responsive cells in vivo are poorly defined. In this study, we generate a mouse strain capable of reporting IFN-signaling activated by all three types of IFNs and investigate the spatio-temporal dynamics and identity of IFN-responding cells following IFN injection and influenza virus infection. Despite ubiquitous expression of IFN receptors, cellular responses to IFNs are highly heterogenous in vivo and are determined by anatomical site, cell type, cellular preference to individual IFNs, and activation status. Unexpectedly, type I and II pneumocytes, the primary target of influenza infection, exhibit striking differences in the strength and temporal dynamics of IFN signaling associated with differential susceptibility to the viral infection. Our findings suggest that time- and cell-type-dependent integration of distinct IFN signals govern the specificity and magnitude of IFN responses in vivo.
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http://dx.doi.org/10.1016/j.celrep.2019.11.021DOI Listing
December 2019

Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis.

J Clin Invest 2017 Nov 9;127(11):3954-3969. Epub 2017 Oct 9.

Research and Development, Biogen, Cambridge, Massachusetts, USA.

Uromodulin-associated kidney disease (UAKD) is caused by mutations in the uromodulin (UMOD) gene that result in a misfolded form of UMOD protein, which is normally secreted by nephrons. In UAKD patients, mutant UMOD is poorly secreted and accumulates in the ER of distal kidney epithelium, but its role in disease progression is largely unknown. Here, we modeled UMOD accumulation in mice by expressing the murine equivalent of the human UMOD p.Cys148Trp point mutation (UmodC147W/+ mice). Like affected humans, these UmodC147W/+ mice developed spontaneous and progressive kidney disease with organ failure over 24 weeks. Analysis of diseased kidneys and purified UMOD-producing cells revealed early activation of the PKR-like ER kinase/activating transcription factor 4 (PERK/ATF4) ER stress pathway, innate immune mediators, and increased apoptotic signaling, including caspase-3 activation. Unexpectedly, we also detected autophagy deficiency. Human cells expressing UMOD p.Cys147Trp recapitulated the findings in UmodC147W/+ mice, and autophagy activation with mTOR inhibitors stimulated the intracellular removal of aggregated mutant UMOD. Human cells producing mutant UMOD were susceptible to TNF-α- and TRAIL-mediated apoptosis due to increased expression of the ER stress mediator tribbles-3. Blocking TNF-α in vivo with the soluble recombinant fusion protein TNFR:Fc slowed disease progression in UmodC147W/+ mice by reducing active caspase-3, thereby preventing tubule cell death and loss of epithelial function. These findings reveal a targetable mechanism for disease processes involved in UAKD.
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http://dx.doi.org/10.1172/JCI93817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663352PMC
November 2017

H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation.

Hum Mol Genet 2017 01;26(2):354-366

Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.
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http://dx.doi.org/10.1093/hmg/ddw392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075457PMC
January 2017

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function.

Nat Immunol 2015 May 6;16(5):517-24. Epub 2015 Apr 6.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.
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http://dx.doi.org/10.1038/ni.3146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406853PMC
May 2015

Expression of the SNARE protein SNAP-23 is essential for cell survival.

PLoS One 2015 23;10(2):e0118311. Epub 2015 Feb 23.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118311PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338070PMC
January 2016

TSLP expression: analysis with a ZsGreen TSLP reporter mouse.

J Immunol 2015 Feb 24;194(3):1372-80. Epub 2014 Dec 24.

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;

Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays a central role in induction of allergic inflammatory responses. Its principal targets have been reported to be dendritic cells and/or CD4 T cells; epithelial cells are a principal source. We report in this study the development of a reporter mouse (TSLP-ZsG) in which a ZsGreen (ZsG)-encoding construct has been inserted by recombineering into a bacterial artificial chromosome immediately at the translation initiating ATG of TSLP. The expression of ZsG by mice transgenic for the recombinant BAC appears to be a faithful surrogate for TSLP expression, particularly in keratinocytes and medullary thymic epithelial cells. Limited ZsG and TSLP mRNA was observed in bone marrow-derived mast cells, basophils, and dendritic cells. Using the TSLP-ZsG reporter mouse, we show that TNF-α and IL-4/IL-13 are potent inducers of TSLP expression by keratinocytes and that local activation of Th2 and Th1 cells induces keratinocyte TSLP expression. We suggest that the capacity of TSLP to both induce Th2 differentiation and to be induced by activated Th2 cells raises the possibility that TSLP may be involved in a positive feedback loop to enhance allergic inflammatory conditions.
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http://dx.doi.org/10.4049/jimmunol.1400519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297686PMC
February 2015

Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance.

Nat Immunol 2015 Feb 15;16(2):197-206. Epub 2014 Dec 15.

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

Regulatory T cells (Treg cells) can express the transcription factors T-bet and GATA-3, but the function of this expression and whether such cells represent stable subsets is still unknown. By using various reporter tools, we found that the expression of T-bet and GATA-3 in Treg cells was dynamically influenced by the cytokine environment. Treg cell-specific deletion of the gene encoding either T-bet (Tbx21) or GATA-3 (Gata3) alone did not result in loss of Treg cell function; however, mice with combined deficiency in both genes in Treg cells developed severe autoimmune-like diseases. Loss of Treg cell function correlated with upregulation of expression of the transcription factor RORγt and reduced expression of the transcription factor Foxp3. Thus, in the steady state, activated Treg cells transiently upregulated either T-bet or GATA-3 to maintain T cell homeostasis.
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http://dx.doi.org/10.1038/ni.3053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297509PMC
February 2015

B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity.

Cell 2014 Dec 4;159(7):1524-37. Epub 2014 Dec 4.

Genomics and Immunity, NIAMS, NIH, Bethesda, MD 20892, USA; Center of Cancer Research, NCI, NIH, Bethesda, MD 20892, USA. Electronic address:

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.
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http://dx.doi.org/10.1016/j.cell.2014.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272762PMC
December 2014

"Glowing head" mice: a genetic tool enabling reliable preclinical image-based evaluation of cancers in immunocompetent allografts.

PLoS One 2014 4;9(11):e109956. Epub 2014 Nov 4.

Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland, United States of America.

Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the "Glowing Head" or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109956PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219677PMC
July 2015

The Notch pathway inhibits TGFβ signaling in breast cancer through HEYL-mediated crosstalk.

Cancer Res 2014 Nov 12;74(22):6509-18. Epub 2014 Sep 12.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233182PMC
November 2014

Activated T cells secrete an alternatively spliced form of common γ-chain that inhibits cytokine signaling and exacerbates inflammation.

Immunity 2014 Jun 5;40(6):910-23. Epub 2014 Jun 5.

Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

The common γ-chain (γc) plays a central role in signaling by IL-2 and other γc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of γc mRNA that results in protein expression and secretion of the γc extracellular domain. The soluble form of γc (sγc) is present in serum and directly binds to IL-2Rβ and IL-7Rα proteins on T cells to inhibit cytokine signaling and promote inflammation. sγc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sγc. Thus, sγc expression is a naturally occurring immunomodulator that regulates γc cytokine signaling and controls T cell activation and differentiation.
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http://dx.doi.org/10.1016/j.immuni.2014.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143255PMC
June 2014

The transcription factor ThPOK suppresses Runx3 and imposes CD4(+) lineage fate by inducing the SOCS suppressors of cytokine signaling.

Nat Immunol 2014 Jul 1;15(7):638-45. Epub 2014 Jun 1.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4(+) lineage fate and Runx3 promoting the CD8(+) lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8(+) lineage fate, nor is it understood why ThPOK itself imposes the CD4(+) lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4(+) lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4(+) lineage fate in the thymus.
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http://dx.doi.org/10.1038/ni.2917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693509PMC
July 2014

CtIP-mediated resection is essential for viability and can operate independently of BRCA1.

J Exp Med 2014 Jun 19;211(6):1027-36. Epub 2014 May 19.

Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3' ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.
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http://dx.doi.org/10.1084/jem.20131939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042650PMC
June 2014

A mouse model of HIES reveals pro- and anti-inflammatory functions of STAT3.

Blood 2014 May 14;123(19):2978-87. Epub 2014 Mar 14.

Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD;

Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.
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http://dx.doi.org/10.1182/blood-2013-09-523167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014840PMC
May 2014

Lck availability during thymic selection determines the recognition specificity of the T cell repertoire.

Cell 2013 Sep;154(6):1326-41

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire.
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http://dx.doi.org/10.1016/j.cell.2013.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792650PMC
September 2013

Ikaros is required to survive positive selection and to maintain clonal diversity during T-cell development in the thymus.

Blood 2013 Oct 1;122(14):2358-68. Epub 2013 Aug 1.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; and.

The zinc-finger protein Ikaros is a key player in T-cell development and a potent tumor suppressor in thymocytes. To understand the molecular basis of its function, we disabled Ikaros activity in vivo using a dominant negative Ikaros transgene (DN-IkTg). In DN-IkTg mice, T-cell development was severely suppressed, and positively selected thymocytes clonally expanded, resulting in a small thymus with a heavily skewed T-cell receptor (TCR) repertoire. Notably, DN-IkTg induced vigorous proliferation concomitant to downregulation of antiapoptotic factor expression such as Bcl2. Ikaros activity was required during positive selection, and specifically at the CD4(+)CD8(lo) intermediate stage of thymocyte differentiation, where it prevented persistent TCR signals from inducing aberrant proliferation and expansion. In particular, DN-IkTg induced the accumulation of CD4 single-positive (SP) thymocytes with a developmentally transitional phenotype, and it imposed a developmental arrest accompanied by massive apoptosis. Thus, we identified an in vivo requirement for Ikaros function, which is to suppress the proliferative potential of persistent TCR signals and to promote the survival and differentiation of positively selected thymocytes.
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http://dx.doi.org/10.1182/blood-2012-12-472076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790506PMC
October 2013

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα.

Proc Natl Acad Sci U S A 2013 Aug 31;110(33):13534-9. Epub 2013 Jul 31.

Metabolism Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-1374, USA.

Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic β-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, β-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.
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http://dx.doi.org/10.1073/pnas.1312911110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746870PMC
August 2013

Mouse prostate epithelial luminal cells lineage originate in the basal layer where the primitive stem/early progenitor cells reside: implications for identifying prostate cancer stem cells.

Biomed Res Int 2013 1;2013:913179. Epub 2013 Jun 1.

Department of Biology and Center for Cancer Research, Tuskegee University, Tuskegee, AL 36088, USA.

Prostate stem cells are thought to be responsible for generation of all prostate epithelial cells and for tissue maintenance. The lineage relationship between basal and luminal cells in the prostate is not well clarified. We developed a mouse model to trace cell fate and a mouse model with a slowly cycling cell label to provide insight into this question. The results obtained indicate that putative mouse prostate stem cells are likely to reside in the basal layer.
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http://dx.doi.org/10.1155/2013/913179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683430PMC
December 2013

Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals.

Immunity 2013 Jun 6;38(6):1116-28. Epub 2013 Jun 6.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Puma⁺⁺⁺p-Bim⁺⁺p-JNK⁺⁺DUSP6⁻) and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ∼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3⁺CD25⁻ Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.
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http://dx.doi.org/10.1016/j.immuni.2013.02.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700677PMC
June 2013

Global regulation of promoter melting in naive lymphocytes.

Cell 2013 May;153(5):988-99

Laboratory of Pathology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA.

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.
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http://dx.doi.org/10.1016/j.cell.2013.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684982PMC
May 2013

Normal viability of Kai1/Cd82 deficient mice.

Mol Carcinog 2014 Aug 8;53(8):610-24. Epub 2013 Feb 8.

Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University College of Human Medicine, Grand Rapids, Michigan; Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.

The KAI1/CD82 tetraspanin is a widely expressed cell surface molecule thought to organize diverse cellular signaling processes. KAI1/CD82 suppresses metastasis but not tumorigenicity, establishing it as one of a class of metastasis suppressor genes. In order to further assess its functions, we have characterized the phenotypic properties of Kai1/Cd82 deleted mice, including viability, fertility, lymphocyte composition, blood chemistry and tissue histopathology, and of their wild-type and heterozygote littermates. Interestingly, Kai1/Cd82(-/-) showed no obvious genotype associated defects in any of these processes and displayed no genotype associated histopathologic abnormalities after 12 or 18 months of life. Expression profiles of non-immortal, wild-type and Kai1/Cd82(-/-) mouse embryo fibroblast (MEFs) indicated distinct sex-specific and genotype-specific profiles. These data identify 191 and 1,271 differentially expressed transcripts (by twofold at P < 0.01) based on Kai1/CD82 genotype status in female and male MEFs, respectively. Differentially expressed genes in male MEFs were surprisingly enriched for cell division related processes, suggesting that Kai1/Cd82 may functionally affect these processes. This suggests that Kai/Cd82 has an unappreciated role in the early establishment of proliferation and division when challenged with a new environment that might play a role in adaptability to new metastatic sites.
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http://dx.doi.org/10.1002/mc.22009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706324PMC
August 2014

Thpok-independent repression of Runx3 by Gata3 during CD4+ T-cell differentiation in the thymus.

Eur J Immunol 2013 Apr 12;43(4):918-28. Epub 2013 Feb 12.

Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda 20892-4259, MD, USA.

CD4(+) helper T cells are essential for immune responses and differentiate in the thymus from CD4(+) CD8(+) "double-positive" (DP) thymocytes. The transcription factor Runx3 inhibits CD4(+) T-cell differentiation by repressing Cd4 gene expression; accordingly, Runx3 is not expressed in DP thymocytes or developing CD4(+) T cells. The transcription factor Thpok is upregulated in CD4-differentiating thymocytes and required to repress Runx3. However, how Runx3 is controlled at early stages of CD4(+) T-cell differentiation, before the onset of Thpok expression, remains unknown. Here we show that Gata3, a transcription factor preferentially and transiently upregulated by CD4(+) T-cell precursors, represses Runx3 and binds the Runx3 locus in vivo. Accordingly, we show that high-level Gata3 expression and expression of Runx3 are mutually exclusive. Furthermore, whereas Runx3 represses Cd4, we show that Gata3 promotes Cd4 expression in Thpok-deficient thymocytes. Thus, in addition to its previously documented role in promoting CD4-lineage gene-expression, Gata3 represses CD8-lineage gene expression. These findings identify Gata3 as a critical pivot of CD4-CD8 lineage differentiation.
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http://dx.doi.org/10.1002/eji.201242944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319927PMC
April 2013

NLRP1 inflammasome activation induces pyroptosis of hematopoietic progenitor cells.

Immunity 2012 Dec 6;37(6):1009-23. Epub 2012 Dec 6.

The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia.

Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1β but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.
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http://dx.doi.org/10.1016/j.immuni.2012.08.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275304PMC
December 2012

High-avidity T cells are preferentially tolerized in the tumor microenvironment.

Cancer Res 2013 Jan 30;73(2):595-604. Epub 2012 Nov 30.

Tumor Immunity and Tolerance Section, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Frederick National Laboratory for Cancer Research, NCI, Frederick, MD 21702, USA.

One obstacle in eliciting potent antitumor immune responses is the induction of tolerance to tumor antigens. TCR(lo) mice bearing a TCR transgene specific for the melanoma antigen tyrosinase-related protein-2 (TRP-2, Dct) harbor T cells that maintain tumor antigen responsiveness but lack the ability to control melanoma outgrowth. We used this model to determine whether higher avidity T cells could control tumor growth without becoming tolerized. As a part of the current study, we developed a second TRP-2-specific TCR transgenic mouse line (TCR(hi)) that bears higher avidity T cells and spontaneously developed autoimmune depigmentation. In contrast to TCR(lo) T cells, which were ignorant of tumor-derived antigen, TCR(hi) T cells initially delayed subcutaneous B16 melanoma tumor growth. However, persistence in the tumor microenvironment resulted in reduced IFN-γ production and CD107a (Lamp1) mobilization, hallmarks of T-cell tolerization. IFN-γ expression by TCR(hi) T cells was critical for upregulation of MHC-I on tumor cells and control of tumor growth. Blockade of PD-1 signals prevented T-cell tolerization and restored tumor immunity. Depletion of tumor-associated dendritic cells (TADC) reduced tolerization of TCR(hi) T cells and enhanced their antitumor activity. In addition, TADCs tolerized TCR(hi) T cells but not TCR(lo) T cells in vitro. Our findings show that T-cell avidity is a critical determinant of not only tumor control but also susceptibility to tolerization in the tumor microenvironment. For this reason, care should be exercised when considering T-cell avidity in designing cancer immunotherapeutics.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-1123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222512PMC
January 2013

Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus.

J Exp Med 2012 Nov 29;209(12):2263-76. Epub 2012 Oct 29.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

The thymus generates T cells with diverse specificities and functions. To assess the contribution of cytokine receptors to the differentiation of T cell subsets in the thymus, we constructed conditional knockout mice in which IL-7Rα or common cytokine receptor γ chain (γ(c)) genes were deleted in thymocytes just before positive selection. We found that γ(c) expression was required to signal the differentiation of MHC class I (MHC-I)-specific thymocytes into CD8(+) cytotoxic lineage T cells and into invariant natural killer T cells but did not signal the differentiation of MHC class II (MHC-II)-specific thymocytes into CD4(+) T cells, even into regulatory Foxp3(+)CD4(+) T cells which require γ(c) signals for survival. Importantly, IL-7 and IL-15 were identified as the cytokines responsible for CD8(+) cytotoxic T cell lineage specification in vivo. Additionally, we found that small numbers of aberrant CD8(+) T cells expressing Runx3d could arise without γ(c) signaling, but these cells were developmentally arrested before expressing cytotoxic lineage genes. Thus, γ(c)-transduced cytokine signals are required for cytotoxic lineage specification in the thymus and for inducing the differentiation of MHC-I-selected thymocytes into functionally mature T cells.
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http://dx.doi.org/10.1084/jem.20121505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501363PMC
November 2012

The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses.

Immunity 2012 Oct 4;37(4):660-73. Epub 2012 Oct 4.

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.
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http://dx.doi.org/10.1016/j.immuni.2012.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717271PMC
October 2012

Loss of ATM kinase activity leads to embryonic lethality in mice.

J Cell Biol 2012 Aug;198(3):295-304

Laboratory of Genome Integrity, Frederick Cancer Research and Development Center; National Cancer Institute, National Institutes of Health, Bethesda, MD 20814, USA.

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.
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http://dx.doi.org/10.1083/jcb.201204035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413361PMC
August 2012

CD8 lineage-specific regulation of interleukin-7 receptor expression by the transcriptional repressor Gfi1.

J Biol Chem 2012 Oct 3;287(41):34386-99. Epub 2012 Aug 3.

Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA.

Interleukin-7 receptor α (IL-7Rα) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7Rα expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7Rα up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7Rα up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7Rα transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8(+), and not CD4(+) T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.
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http://dx.doi.org/10.1074/jbc.M112.378687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464544PMC
October 2012

Clonal deletion and the fate of autoreactive thymocytes that survive negative selection.

Nat Immunol 2012 Apr 29;13(6):569-78. Epub 2012 Apr 29.

Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4(+)CD8(lo) intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4(-)CD8(-) double-negative thymocytes positive for the T cell antigen receptor αβ subtype (TCRαβ) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα(+) intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα(+) IELs as the developmental fate of autoreactive thymocytes that survive negative selection.
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http://dx.doi.org/10.1038/ni.2292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362677PMC
April 2012