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1.  Characterization of a surrogate murine antibody to model anti-human CD3 therapies 
mAbs  2013;5(4):555-564.
Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.
doi:10.4161/mabs.24736
PMCID: PMC3906309  PMID: 23751612
Monoclonal antibody; anti-CD3; oral antibody; EAE; pharmacodynamics; pharmacokinetics; T cell activation
2.  Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis 
PLoS ONE  2014;9(1):e86844.
Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4+ T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4+ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4+ T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4+ T cells and forkhead box P3 (FoxP3)+ regulatory T (Treg) cells. IFN-γ produced mainly by CD4+ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.
doi:10.1371/journal.pone.0086844
PMCID: PMC3904943  PMID: 24489792
3.  TREM-1 Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance 
PLoS Pathogens  2014;10(1):e1003900.
Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1−/− mice are viable, fertile and show no altered hematopoietic compartment. In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Trem1−/− mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1−/− mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1−/− mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1−/− mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1+/+ controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
Author Summary
Triggering receptor expressed on myeloid cells-1 (TREM-1) is an immune receptor expressed by myeloid cells that has the capacity to augment pro-inflammatory responses in the context of a microbial infection. While a TREM-1-amplified response likely serves the efficient clearance of pathogens, it also bears the potential to cause substantial tissue damage or even death. Hence, TREM-1 appears a possible therapeutic target for tempering deleterious host-pathogen interactions. However, in models of bacterial sepsis controversial findings have been obtained regarding the requirement of TREM-1 for bacterial control - depending on the overall degree of the TREM-1 blockade that was achieved. In order to conclusively investigate harmful versus essential functions of TREM-1 in vivo, we have generated mice deficient in Trem1. Trem1−/− mice were subjected to experimentally-induced intestinal inflammation (as a model of a non-infectious, yet microbial-driven disease) and also analysed following infections with Leishmania major, influenza virus and Legionella pneumophila. Across all models analysed, Trem1−/− mice showed substantially reduced immune-associated disease. We thus describe a previously unanticipated pathogenic role for TREM-1 also during a parasitic and viral infection. Importantly, our data suggest that in certain diseases microbial control can be achieved in the context of blunted inflammation in the absence of TREM-1.
doi:10.1371/journal.ppat.1003900
PMCID: PMC3894224  PMID: 24453980
4.  NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of Acute Respiratory Distress Syndrome 
Reactive oxygen species (ROS) contribute to alveolar cell death in Acute Respiratory Distress Syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.
PMCID: PMC3925898  PMID: 24551274
NOX1; ARDS; cell death; hyperoxia; STAT3; ROS
5.  Antigen Recognition By Autoreactive Cd4+ Thymocytes Drives Homeostasis Of The Thymic Medulla 
PLoS ONE  2012;7(12):e52591.
The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4+ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4+ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4+ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.
doi:10.1371/journal.pone.0052591
PMCID: PMC3531460  PMID: 23300712
6.  The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development 
PLoS Genetics  2012;8(3):e1002606.
The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies.
Author Summary
The Corpus Callosum is the major brain commissure that links the two cerebral hemispheres in mammals. Absence or reduction of the corpus callosum is the most frequent brain malformation observed at birth in humans and leads to cognitive and behavioural deficits. Agenesis of the Corpus Callosum is frequently observed in ciliopathies, a group of human diseases due to defects in cilia assembly or function. However, the cellular origin of this brain malformation in these syndromes remains elusive. RFX3 transcription factor is a key regulator of ciliogenesis in mouse. Here, we show that the Rfx3 mutant mouse shows impaired Corpus Callosum formation. By transplantation experiments, we demonstrate that this is due to defective distribution between the two hemispheres of a transient neuronal population (guidepost neurons) required for routing callosal axons. We show that this abnormal distribution is due to altered FGF8 signalling at early stages of brain development. Our observations show that small but focused signalling defects resulting in specific alterations in the distribution of guidepost neurons are responsible for corpus callosum agenesis.
doi:10.1371/journal.pgen.1002606
PMCID: PMC3315471  PMID: 22479201
7.  Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research 
Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.
doi:10.3389/fimmu.2012.00331
PMCID: PMC3491238  PMID: 23162549
dendritic cell line; in vitro research
8.  The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression 
Diabetes  2010;59(7):1674-1685.
OBJECTIVE
Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing β-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency.
RESEARCH DESIGN AND METHODS
Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of β-cells from endocrine progenitors, and the expression of mature β-cell markers during development in Rfx3−/− and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 β-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes.
RESULTS
Reduced development of insulin-positive cells in Rfx3−/− mice was not due to deficiencies in endocrine progenitors or β-lineage specification, but reflected the accumulation of insulin-positive β-cell precursors and defective β-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated β-cells developed in pancreas-specific Rfx3-deficient embryos. Defective β-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference–mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene.
CONCLUSIONS
Our results show that Rfx3 is required for the differentiation and function of mature β-cells and regulates the β-cell promoter of the glucokinase gene.
doi:10.2337/db09-0986
PMCID: PMC2889767  PMID: 20413507
9.  MHC class II–restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell–mediated autoimmunity 
The Journal of Experimental Medicine  2010;207(9):1891-1905.
Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4+ T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag–specific contacts with CD4+ T cells. These interactions promote the selective expansion of myelin-Ag–specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4+ T cells in vivo.
doi:10.1084/jem.20092627
PMCID: PMC2931160  PMID: 20696698
10.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
Background
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Results
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
Conclusions
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
doi:10.1186/1745-7580-6-10
PMCID: PMC3000836  PMID: 21092113
11.  Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts 
Introduction
Extracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.
Methods
The steady-state mRNA levels of COL1A1, COL1A2, TIMP-1, MMP-1, and MMP-2 were assessed by quantitative PCR in human dermal fibroblasts cultured in the presence of TGF-β, bortezomib, or both. Transient fibroblast transfection was performed with wild-type and mutated COL1A1 and MMP-1 promoters. Chromatin immunoprecipitation, electrophoretic mobility shift assay (EMSA), and DNA pull-down assays were used to assess the binding of c-Jun, SP1, AP2, and Smad2 transcription factors. Immunoblotting and immunofluorescent microscopy were performed for identifying phosphorylated transcription factors and their cellular localization.
Results
Bortezomib decreased the steady-state mRNA levels of COL1A1 and COL1A2, and abrogated SP1 binding to the promoter of COL1A2 in both untreated and TGF-β-activated fibroblasts. Reduced COL1A2 expression was not due to altered TGF-β-induced Smad2 phosphorylation, nuclear translocation, or binding to the COL1A2 promoter. In contrast to collagen, bortezomib specifically increased the steady-state mRNA levels of MMP-1 and enhanced the binding of c-Jun to the promoter of MMP-1. Furthermore, disruption of the proximal AP-1-binding site in the promoter of MMP-1 severely impaired MMP-1 transcription in response to bortezomib.
Conclusions
By altering the binding of at least two transcription factors, c-Jun and SP1, proteasome inhibition results in increased production of MMP-1 and decreased synthesis of type I collagen in human dermal fibroblasts. Thus, the antifibrotic phenotype observed in fibroblasts submitted to proteasome inhibition results from profound modifications in the binding of key transcription factors. This provides a novel rationale for assessing the potential of drugs targeting the proteasome for their anti-fibrotic properties.
doi:10.1186/ar2991
PMCID: PMC2888229  PMID: 20429888
12.  MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses 
PLoS ONE  2010;5(1):e8808.
Background
Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.
Methodology/Principal Findings
We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.
Conclusions/Significance
These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.
doi:10.1371/journal.pone.0008808
PMCID: PMC2808354  PMID: 20098705
13.  New dimensions to CIITA 
Nature immunology  2008;9(7):713-714.
doi:10.1038/ni0708-713
PMCID: PMC2659346  PMID: 18563077
14.  Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site 
Nucleic Acids Research  2009;37(8):2514-2528.
Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence.
doi:10.1093/nar/gkp116
PMCID: PMC2677874  PMID: 19264803
15.  The NLR gene family: An official nomenclature 
Immunity  2008;28(3):285-287.
Summary
Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohn's disease and Blau syndrome.
A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.
doi:10.1016/j.immuni.2008.02.005
PMCID: PMC2630772  PMID: 18341998
16.  Identification of CIITA Regulated Genetic Module Dedicated for Antigen Presentation 
PLoS Genetics  2008;4(4):e1000058.
The class II trans-activator CIITA is a transcriptional co-activator required for the expression of Major Histocompatibility Complex (MHC) genes. Although the latter function is well established, the global target-gene specificity of CIITA had not been defined. We therefore generated a comprehensive list of its target genes by performing genome-wide scans employing four different approaches designed to identify promoters that are occupied by CIITA in two key antigen presenting cells, B cells and dendritic cells. Surprisingly, in addition to MHC genes, only nine new targets were identified and validated by extensive functional and expression analysis. Seven of these genes are known or likely to function in processes contributing to MHC-mediated antigen presentation. The remaining two are of unknown function. CIITA is thus uniquely dedicated for genes implicated in antigen presentation. The finding that CIITA regulates such a highly focused gene expression module sets it apart from all other transcription factors, for which large-scale binding-site mapping has indicated that they exert pleiotropic functions and regulate large numbers of genes.
Author Summary
Most mammalian transcription factors and transcriptional co-activators are believed to regulate the activities of numerous genes fulfilling multiple functions. This pleiotropic role has recently been confirmed directly for several individual factors by large-scale mapping studies aimed at generating comprehensive catalogues of their binding sites in the genome. Until now, all transcription factors, for which such studies have been performed, were found to regulate hundreds or even thousands of genes. We demonstrate, here, that the transcriptional co-activator CIITA (class II transactivator) is an exception to this rule. CIITA is a key regulator of the immune system because it controls the transcription of genes coding for Major Histocompatibility Complex (MHC) class II molecules, which are cell-surface molecules that present peptide antigens to T lymphocytes. To address the possibility that CIITA might exert more widespread functions, we have performed extensive genome-wide searches to establish a comprehensive list of CIITA-regulated genes. Surprisingly, we found that CIITA regulates only a small number of genes, most of which code for proteins implicated directly or indirectly in MHC-mediated antigen presentation. CIITA is thus remarkably dedicated for the regulation of a unique set of functionally related genes constituting a genetic module devoted to a single biological process.
doi:10.1371/journal.pgen.1000058
PMCID: PMC2278383  PMID: 18437201
17.  Transcription-coupled deposition of histone modifications during MHC class II gene activation 
Nucleic Acids Research  2007;35(10):3431-3441.
Posttranslational histone modifications associated with actively expressed genes are generally believed to be introduced primarily by histone-modifying enzymes that are recruited by transcription factors or their associated co-activators. We have performed a comprehensive spatial and temporal analyses of the histone modifications that are deposited upon activation of the MHC class II gene HLA-DRA by the co-activator CIITA. We find that transcription-associated histone modifications are introduced during two sequential phases. The first phase precedes transcription initiation and is characterized exclusively by a rapid increase in histone H4 acetylation over a large upstream domain. All other modifications examined, including the acetylation and methylation of several residues in histone H3, are restricted to short regions situated at or within the 5′ end of the gene and are established during a second phase that is concomitant with ongoing transcription. This second phase is completely abrogated when elongation by RNA polymerase II is blocked. These results provide strong evidence that transcription elongation can play a decisive role in the deposition of histone modification patterns associated with inducible gene activation.
doi:10.1093/nar/gkm214
PMCID: PMC1904273  PMID: 17478518
18.  Expression of RAB4B, a protein governing endocytic recycling, is co-regulated with MHC class II genes 
Nucleic Acids Research  2006;35(2):595-605.
The small GTPase RAB4 regulates endocytic recycling, a process that contributes to Major Histocompatibility Complex (MHC)-mediated antigen presentation by specialized antigen presenting cells (APC) of the immune system. The gene encoding the RAB4B isoform of RAB4 was singled out by two complementary genome-wide screens. One of these consisted of a computer scan to identify genes containing characteristic MHC class II-related regulatory sequences. The second was the use of chromatin immunoprecipitation coupled to microarrays (ChIP-on-chip) to identify novel targets of a transcriptional co-activator called the MHC class II transactivator (CIITA). We show that the RAB4B gene is regulated by a typical MHC class II-like enhancer that is controlled directly by both CIITA and the multiprotein transcription factor complex known as the MHC class II enhanceosome. RAB4B expression is thus activated by the same regulatory machinery that is known to be essential for the expression of MHC class II genes. This molecular link between the transcriptional activation of RAB4B and MHC class II genes implies that APC boost their antigen presentation capacity by increasing RAB4-mediated endocytic recycling.
doi:10.1093/nar/gkl980
PMCID: PMC1802633  PMID: 17175541
19.  The Assembly and Maintenance of Heterochromatin Initiated by Transgene Repeats Are Independent of the RNA Interference Pathway in Mammalian Cells†  
Molecular and Cellular Biology  2006;26(11):4028-4040.
A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.
doi:10.1128/MCB.02189-05
PMCID: PMC1489094  PMID: 16705157
20.  New Functions of the Major Histocompatibility Complex Class II-Specific Transcription Factor RFXANK Revealed by a High-Resolution Mutagenesis Study 
Molecular and Cellular Biology  2005;25(19):8607-8618.
The transcription factors RFX and CIITA are major players in regulation of the expression of all classical and nonclassical major histocompatibility complex class II (MHC-II) genes. RFX nucleates the formation of a multiprotein complex, called the MHC-II enhanceosome, on MHC-II promoters. Assembly of this enhanceosome is an obligatory step for recruitment of the coactivator CIITA and thus for activation of MHC-II gene transcription. We have analyzed the function of the ankyrin repeat-containing protein RFXANK, which forms the heterotrimeric RFX complex together with RFX5 and RFXAP. We discovered that ANKRA2, the closest paralogue of RFXANK, can substitute for RFXANK in the activation of MHC-II genes and that this ability is mediated by its ankyrin repeat domain (ARD). This finding provided the basis for a high-resolution structure-function analysis of the ARD of RFXANK, which allowed us to map the RFX5 interaction domain and residues critical for assembly of the RFX complex. We also found that mutations in the fourth ankyrin repeat of RFXANK abolish assembly of the enhanceosome on MHC-II promoters in vivo but not in vitro, suggesting a new role of RFXANK in facilitating promoter occupation in the context of chromatin.
doi:10.1128/MCB.25.19.8607-8618.2005
PMCID: PMC1265745  PMID: 16166641
21.  Selective Abrogation of Major Histocompatibility Complex Class II Expression on Extrahematopoietic Cells in Mice Lacking Promoter IV of the Class II Transactivator Gene 
MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-γ–induced MHCII expression on a wide variety of non-bone marrow–derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4+ T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-γ–activated cells of the macrophage lineage. pIV−/− mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.
PMCID: PMC2193506  PMID: 11514597
knockout; promoter region; antigen-presenting cells; gene expression regulation; thymic selection
22.  Maturation of Dendritic Cells Is Accompanied by Rapid Transcriptional Silencing of Class II Transactivator (Ciita) Expression 
Cell surface expression of major histocompatibility complex class II (MHCII) molecules is increased during the maturation of dendritic cells (DCs). This enhances their ability to present antigen and activate naive CD4+ T cells. In contrast to increased cell surface MHCII expression, de novo biosynthesis of MHCII mRNA is turned off during DC maturation. We show here that this is due to a remarkably rapid reduction in the synthesis of class II transactivator (CIITA) mRNA and protein. This reduction in CIITA expression occurs in human monocyte-derived DCs and mouse bone marrow–derived DCs, and is triggered by a variety of different maturation stimuli, including lipopolysaccharide, tumor necrosis factor α, CD40 ligand, interferon α, and infection with Salmonella typhimurium or Sendai virus. It is also observed in vivo in splenic DCs in acute myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalitis. The arrest in CIITA expression is the result of a transcriptional inactivation of the MHC2TA gene. This is mediated by a global repression mechanism implicating histone deacetylation over a large domain spanning the entire MHC2TA regulatory region.
PMCID: PMC2193505  PMID: 11514596
MHC class II; class II transactivator; experimental autoimmune encephalitis; bare lymphocyte syndrome; histone deacetylation
23.  Expression of the Three Human Major Histocompatibility Complex Class II Isotypes Exhibits a Differential Dependence on the Transcription Factor RFXAP 
Molecular and Cellular Biology  2001;21(17):5699-5709.
Major histocompatibility complex class II (MHCII) molecules play a pivotal role in the immune system because they direct the development and activation of CD4+ T cells. There are three human MHCII isotypes, HLA-DR, HLA-DQ, and HLA-DP. Key transcription factors controlling MHCII genes have been identified by virtue of the fact that they are mutated in a hereditary immunodeficiency resulting from a lack of MHCII expression. RFXAP—one of the factors affected in this disease—is a subunit of RFX, a DNA-binding complex that recognizes the X box present in all MHCII promoters. To facilitate identification of conserved regions in RFXAP, we isolated the mouse gene. We then delimited conserved domains required to restore endogenous MHCII expression in cell lines lacking a functional RFXAP gene. Surprisingly, we found that 80% of RFXAP is dispensable for the reactivation of DR expression. Only a short C-terminal segment of the protein is essential for this isotype. In contrast, optimal expression of DQ and DP requires a larger C-terminal segment. These results define an RFXAP domain with an MHCII isotype-specific function. Expression of the three MHCII isotypes exhibits a differential requirement for this domain. We show that this is due to a differential dependence on this domain for promoter occupation and recruitment of the coactivator CIITA in vivo.
doi:10.1128/MCB.21.17.5699-5709.2001
PMCID: PMC87290  PMID: 11486010
24.  CIITA Leucine-Rich Repeats Control Nuclear Localization, In Vivo Recruitment to the Major Histocompatibility Complex (MHC) Class II Enhanceosome, and MHC Class II Gene Transactivation 
Molecular and Cellular Biology  2000;20(20):7716-7725.
The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex.
PMCID: PMC86349  PMID: 11003667
25.  A Functionally Essential Domain of RFX5 Mediates Activation of Major Histocompatibility Complex Class II Promoters by Promoting Cooperative Binding between RFX and NF-Y 
Molecular and Cellular Biology  2000;20(10):3364-3376.
Major histocompatibility complex class II (MHC-II) molecules occupy a pivotal position in the adaptive immune system, and correct regulation of their expression is therefore of critical importance for the control of the immune response. Several regulatory factors essential for the transcription of MHC-II genes have been identified by elucidation of the molecular defects responsible for MHC-II deficiency, a hereditary immunodeficiency disease characterized by regulatory defects abrogating MHC-II expression. Three of these factors, RFX5, RFXAP, and RFXANK, combine to form the RFX complex, a regulatory protein that binds to the X box DNA sequence present in all MHC-II promoters. In this study we have undertaken a dissection of the structure and function of RFX5, the largest subunit of the RFX complex. The results define two distinct domains serving two different essential functions. A highly conserved N-terminal region of RFX5 is required for its association with RFXANK and RFXAP, for assembly of the RFX complex in vivo and in vitro, and for binding of this complex to its X box target site in the MHC-II promoter. This N-terminal region is, however, not sufficient for activation of MHC-II expression. This requires an additional domain within the C-terminal region of RFX5. This C-terminal domain mediates cooperative binding between the RFX complex and NF-Y, a transcription factor binding to the Y box sequence of MHC-II promoters. This provides direct evidence that RFX5-mediated cooperative binding between RFX and NF-Y plays an essential role in the transcriptional activation of MHC-II genes.
PMCID: PMC85629  PMID: 10779326

Results 1-25 (26)