Publications by authors named "John J Priatel"

34 Publications

The Role of Autoimmunity-Related Gene in the B Cell Receptor-Mediated HLA Class II Pathway.

J Immunol 2020 Aug 8;205(4):945-956. Epub 2020 Jul 8.

Department of Immunology, Erasmus MC, 3015 CN Rotterdam, the Netherlands;

C-type lectin is located next to , the master transcription factor of HLA class II (HLA-II), at a susceptibility locus for several autoimmune diseases, including multiple sclerosis (MS). We previously found that promotes the biogenesis of HLA-II peptide-loading compartments (MIICs) in myeloid cells. Given the emerging role of B cells as APCs in these diseases, in this study, we addressed whether and how is involved in the BCR-dependent HLA-II pathway. was coexpressed with surface class II-associated invariant chain peptides (CLIP) in human EBV-positive and not EBV-negative B cell lines. Stable knockdown of in EBV-positive Raji B cells resulted in an upregulation of surface HLA-DR and CD74 (invariant chain), whereas CLIP was slightly but significantly reduced. In addition, IgM-mediated uptake was decreased, and MIICs were less clustered in -silenced Raji cells, implying that controls both HLA-DR/CD74 and BCR/Ag processing in MIICs. In primary B cells, was only induced under CLIP-stimulating conditions in vitro and was predominantly expressed in CLIP naive populations. Finally, CLIP-loaded HLA-DR molecules were abnormally enriched, and coregulation with was abolished in blood B cells of patients who rapidly develop MS. These findings demonstrate that CLEC16A participates in the BCR-dependent HLA-II pathway in human B cells and that this regulation is impaired during MS disease onset. The abundance of CLIP already on naive B cells of MS patients may point to a chronically induced stage and a new mechanism underlying B cell-mediated autoimmune diseases such as MS.
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http://dx.doi.org/10.4049/jimmunol.1901409DOI Listing
August 2020

Cutting Edge: Intestinal Mucus Limits the Clonal Deletion of Developing T Cells Specific for an Oral Antigen.

J Immunol 2020 Jul 15;205(2):329-334. Epub 2020 Jun 15.

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;

A layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium. The MUC2 mucin is the primary constituent of intestinal mucus and plays critical protective roles against luminal microbes and other noxious agents. In this study, we investigated whether MUC2 helps maintain CD8 T cell tolerance toward intestinal luminal Ags by gavaging wild-type and mice with a model Ag and monitoring immune responses posttreatment. We report that orally delivered OVA rapidly disseminates through the blood of (but not control) mice and causes immune activation of Ag-specific CD8 T cells at both local and distal sites. Further, the administration of oral OVA to mice led to its presentation by thymic dendritic cells and the deletion of Ag-specific thymocytes. Collectively, our findings suggest that intestinal mucus helps limit the shaping of the TCR repertoire of developing thymocytes by intestinal luminal Ags.
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http://dx.doi.org/10.4049/jimmunol.1900687DOI Listing
July 2020

Deep Phenotyping by Mass Cytometry and Single-Cell RNA-Sequencing Reveals LYN-Regulated Signaling Profiles Underlying Monocyte Subset Heterogeneity and Lifespan.

Circ Res 2020 05 10;126(10):e61-e79. Epub 2020 Mar 10.

From the Department of Microbiology and Immunology (M.E.R., M.B., J.A.F.D.S., R.A.C., W.C., A.W., I.M., K.W.H.), University of British Columbia, Vancouver, Canada.

Rationale: Monocytes are key effectors of the mononuclear phagocyte system, playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into 2 major subsets termed conventional monocytes and patrolling monocytes (pMo) but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology; however, LYN's role in regulating specific monocyte subset homeostasis has not been investigated.

Objective: We sought to comprehensively profile monocytes to elucidate the underlying heterogeneity within monocytes and dissect how deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis.

Methods And Results: Mass cytometric analysis of monocyte subsets and signaling pathway activation patterns in conventional monocytes and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN's role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of -deficient pMos. Furthermore, single-cell RNA sequencing revealed marked perturbations in the gene expression profiles of monocytes with upregulation of genes involved in pMo development, survival, and function. deficiency also led to a significant increase in aorta-associated pMos and protected mice from high-fat diet-induced atherosclerosis.

Conclusions: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315708DOI Listing
May 2020

Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation.

Elife 2019 06 3;8. Epub 2019 Jun 3.

Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.
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http://dx.doi.org/10.7554/eLife.44574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591008PMC
June 2019

An allosteric MALT1 inhibitor is a molecular corrector rescuing function in an immunodeficient patient.

Nat Chem Biol 2019 03 28;15(3):304-313. Epub 2019 Jan 28.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.
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http://dx.doi.org/10.1038/s41589-018-0222-1DOI Listing
March 2019

Prevention of autoimmune diabetes and islet allograft rejection by beta cell expression of XIAP: Insight into possible mechanisms of local immunomodulation.

Mol Cell Endocrinol 2018 12 5;477:48-56. Epub 2018 Jun 5.

Departments of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada; Surgery, BC Children's Hospital, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Overexpression of the X-linked inhibitor of apoptosis (XIAP) prevents islet allograft rejection. We constructed an adeno-associated virus expressing XIAP driven by the rat insulin promoter (dsAAV8-RIP-XIAP) for long-term beta-cell gene expression in vivo. Pancreatic delivery of dsAAV8-RIP-XIAP prevented autoimmune diabetes in 70% of non-obese diabetic (NOD) mice, associated with decreased insulitis. Islets from Balb/c mice transduced with dsAAV8-RIP-XIAP were protected following transplantation into streptozotocin (STZ)-diabetic Bl/6 recipients, associated with decreased graft infiltration. Interestingly, dsAAV8-RIP-XIAP transduction induced expression of lactate dehydrogenase (LDHA) and monocarboxylate transporter 1 (MCT1), two genes normally suppressed in beta cells and involved in production and release of lactate, a metabolite known to suppress local immune responses. Transduction of Balb/c islets with AAV8-RIP-LDHA-MCT1 tended to prolong allograft survival following transplant into STZ-diabetic Bl/6 recipients. These findings suggest that XIAP has therapeutic potential in autoimmune diabetes and raise the possibility that local lactate production may play a role in XIAP-mediated immunomodulation.
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http://dx.doi.org/10.1016/j.mce.2018.05.015DOI Listing
December 2018

The importance of functional validation after next-generation sequencing: evaluation of a novel CARD11 variant.

Pediatr Allergy Immunol 2018 09 25;29(6):663-668. Epub 2018 Jun 25.

Department of Pediatrics, British Columbia Children's Hospital, University of British Columbia, Vancouver, BC, Canada.

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http://dx.doi.org/10.1111/pai.12930DOI Listing
September 2018

2B4-SAP signaling is required for the priming of naive CD8 T cells by antigen-expressing B cells and B lymphoma cells.

Oncoimmunology 2017;6(2):e1267094. Epub 2016 Dec 27.

British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

Mutations in gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8 T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. CD8 T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, CD8 T cells responded equivalently to wild-type CD8 T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8 T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8 T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8 T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.
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http://dx.doi.org/10.1080/2162402X.2016.1267094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5353922PMC
December 2016

JAK1 gain-of-function causes an autosomal dominant immune dysregulatory and hypereosinophilic syndrome.

J Allergy Clin Immunol 2017 06 19;139(6):2016-2020.e5. Epub 2017 Jan 19.

Department of Pediatrics, British Columbia Children's Hospital, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2016.12.957DOI Listing
June 2017

The paracaspase MALT1 cleaves HOIL1 reducing linear ubiquitination by LUBAC to dampen lymphocyte NF-κB signalling.

Nat Commun 2015 Nov 3;6:8777. Epub 2015 Nov 3.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.
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http://dx.doi.org/10.1038/ncomms9777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659944PMC
November 2015

CCL22 Prevents Rejection of Mouse Islet Allografts and Induces Donor-Specific Tolerance.

Cell Transplant 2015 ;24(10):2143-54

Department of Pathology and Laboratory Medicine, University of British Columbia and Child and Family Research Institute, Vancouver, BC, Canada.

Manipulation of regulatory T cell (Treg) migration by islet expression of the chemokine CCL22 prevents diabetes in NOD mice and delays recurrent autoimmunity in syngeneic islet transplants. We sought to determine whether attracting Tregs with CCL22 also prevents islet allograft rejection. Isolated Bl/6 mouse islets were transduced overnight with adenovirus expressing CCL22 (Ad-CCL22) downstream of the CMV promoter. Islets were transplanted under the renal capsule of Balb/c recipients made diabetic by streptozotocin. To assess immunologic tolerance, graft-bearing kidneys from recipients of CCL22-expressing islet grafts were removed, and mice received a second transplant of naive islets from the same donor strain or third-party islets into the contralateral kidney. Adenoviral expression of CCL22 conferred prolonged protection of islet allografts in MHC-mismatched, diabetic recipients, maintaining normoglycemia in 75% of recipients for at least 80 days. Increased frequency of Treg cells was observed in islet grafts transduced with Ad-CCL22 compared with untreated grafts. Normoglycemic recipients of CCL22-expressing islet grafts showed complete absence of antidonor antibodies and no lymphocyte proliferation after exposure to donor splenocytes. After removal of the primary graft at day 80, mice that received a second transplant with untreated islets from the same donor strain did not reject the grafts, suggesting the development of tolerance. Expression of CCL22 recruits Treg cells to transplanted islets, prevents activation of alloreactive T-cells and islet allograft failure and induces alloantigen-specific tolerance. Manipulation of Treg cells by CCL22 in transplanted islets may be a novel therapeutic strategy for diabetes.
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http://dx.doi.org/10.3727/096368914X685249DOI Listing
August 2016

CD1d Expression and Invariant NKT Cell Responses in Herpesvirus Infections.

Front Immunol 2015 25;6:312. Epub 2015 Jun 25.

Department of Pathology, Sidra Medical and Research Center , Doha , Qatar.

Invariant natural killer T (iNKT) cells are a highly conserved subset of unconventional T lymphocytes that express a canonical, semi-invariant T cell receptor and surface markers shared with the natural killer cell lineage. iNKT cells recognize exogenous and endogenous glycolipid antigens restricted by non-polymorphic CD1d molecules, and are highly responsive to the prototypical agonist, α-galactosylceramide. Upon activation, iNKT cells rapidly coordinate signaling between innate and adaptive immune cells through the secretion of proinflammatory cytokines, leading to the maturation of antigen-presenting cells, and expansion of antigen-specific CD4+ and CD8+ T cells. Because of their potent immunoregulatory properties, iNKT cells have been extensively studied and are known to play a pivotal role in mediating immune responses against microbial pathogens including viruses. Here, we review evidence that herpesviruses manipulate CD1d expression to escape iNKT cell surveillance and establish lifelong latency in humans. Collectively, published findings suggest that iNKT cells play critical roles in anti-herpesvirus immune responses and could be harnessed therapeutically to limit viral infection and viral-associated disease.
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http://dx.doi.org/10.3389/fimmu.2015.00312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4479820PMC
July 2015

Multiple sclerosis-associated CLEC16A controls HLA class II expression via late endosome biogenesis.

Brain 2015 Jun 29;138(Pt 6):1531-47. Epub 2015 Mar 29.

2 Department of Neurology and MS Center ErasMS, Erasmus MC, University Medical Center, 's-Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands

C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in monocyte-derived dendritic cells, in which it strongly co-localized with human leukocyte antigen class II. Treatment of these professional antigen-presenting cells with vitamin D, a key protective environmental factor in multiple sclerosis, downmodulated CLEC16A in parallel with human leukocyte antigen class II. Knockdown of CLEC16A in distinct types of model and primary antigen-presenting cells resulted in severely impaired cytoplasmic distribution and formation of human leucocyte antigen class II-positive late endosomes, as determined by immunofluorescence and electron microscopy. Mechanistically, CLEC16A participated in the molecular machinery of human leukocyte antigen class II-positive late endosome formation and trafficking to perinuclear regions, involving the dynein motor complex. By performing co-immunoprecipitations, we found that CLEC16A directly binds to two critical members of this complex, RILP and the HOPS complex. CLEC16A silencing in antigen-presenting cells disturbed RILP-mediated recruitment of human leukocyte antigen class II-positive late endosomes to perinuclear regions. Together, we identify CLEC16A as a pivotal gene in multiple sclerosis that serves as a direct regulator of the human leukocyte antigen class II pathway in antigen-presenting cells. These findings are a first step in coupling multiple sclerosis-associated genes to the regulation of the strongest genetic factor in multiple sclerosis, human leukocyte antigen class II.
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http://dx.doi.org/10.1093/brain/awv080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614123PMC
June 2015

SLAM-SAP signaling promotes differentiation of IL-17-producing T cells and progression of experimental autoimmune encephalomyelitis.

J Immunol 2014 Dec 31;193(12):5841-53. Epub 2014 Oct 31.

Child and Family Research Institute, BC Children's Hospital, Vancouver, British Columbia V5Z 4H4, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada; Department of Pathology, Sidra Medical and Research Center, Doha, Qatar

IL-17 plays critical roles in host defenses, combating bacterial and fungal infections, as well as the pathogenesis of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). The signaling adaptor SAP is essential for normal immune homeostasis and mutations within SH2D1A, the locus encoding this protein, result in serious and sometimes fatal syndromes, including X-linked lymphoproliferative disease and severe cases of common variable immunodeficiency. However, the precise cellular basis of how SAP deficiency contributes to immune dysfunction remains incompletely understood. In this study, we found that CD4 and CD8 T cells lacking SAP had a diminished capacity to differentiate into IL-17-producing Th17 and T cytotoxic (Tc17) cells relative to wild-type lymphocytes. The use of costimulating SLAM Abs was found to augment the differentiation of IL-17-secreting effectors in wild-type but not Sh2d1a(-/-) splenic T cells under IL-17-polarizing conditions. In addition, SAP's regulation of IL-17-secreting T cells was shown to be a T cell-intrinsic role, as purified naive Sh2d1a(-/-) CD4 and CD8 T cells were inherently defective at converting into Th17 and Tc17 cells in vitro and in vivo. Furthermore, Sh2d1a(-/-) mice were protected from EAE and exhibited greatly decreased numbers of CNS-infiltrating Th17 and Tc17 effector T cells and reduced disease severity. Collectively, these results suggest that SLAM-SAP signaling drives the differentiation and function of Th17 and Tc17 cells in vitro and in vivo and contributes to the pathogenesis of autoimmunity in EAE.
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http://dx.doi.org/10.4049/jimmunol.1301435DOI Listing
December 2014

Lyn deficiency leads to increased microbiota-dependent intestinal inflammation and susceptibility to enteric pathogens.

J Immunol 2014 Nov 22;193(10):5249-63. Epub 2014 Oct 22.

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;

The Lyn tyrosine kinase governs the development and function of various immune cells, and its dysregulation has been linked to malignancy and autoimmunity. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating the intestinal microbiota and inflammatory responses as well as protection from enteric pathogens. Lyn(-/-) mice were highly susceptible to dextran sulfate sodium (DSS) colitis, characterized by significant wasting, rectal bleeding, colonic pathology, and enhanced barrier permeability. Increased DSS susceptibility in Lyn(-/-) mice required the presence of T but not B cells and correlated with dysbiosis and increased IFN-γ(+) and/or IL-17(+) colonic T cells. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, associated with altered intestinal production of IL-22 and IgA, and was transmissible to wild-type mice, resulting in increased susceptibility to DSS. Lyn deficiency also resulted in an inability to control infection by the enteric pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Lyn(-/-) mice exhibited profound cecal inflammation, bacterial dissemination, and morbidity following S. Typhimurium challenge and greater colonic inflammation throughout the course of C. rodentium infection. These results identify Lyn as a key regulator of the mucosal immune system, governing pathophysiology in multiple models of intestinal disease.
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http://dx.doi.org/10.4049/jimmunol.1302832DOI Listing
November 2014

Natural killer T cell strategies to combat Epstein-Barr virus infection.

Oncoimmunology 2014;3:e28329. Epub 2014 Apr 9.

Child and Family Research Institute; Immunity in Health and Disease; University of British Columbia; Vancouver, BC Canada ; Department of Pathology and Laboratory Medicine; University of British Columbia; Vancouver, BC Canada ; Department of Pathology; Sidra Medical and Research Center; Doha, Qatar.

Epstein-Barr virus (EBV) infection results in rapid loss of CD1d expression from the surface of infected B cells, thus enabling the virus to evade immune recognition by natural killer T (NKT) cells. Using pharmacologic means to boost CD1d expression, potent NKT cell effector functions can be elicited toward EBV-infected B cells, suggesting the promise of novel strategies to target EBV-associated diseases such as some B-cell malignancies.
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http://dx.doi.org/10.4161/onci.28329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063158PMC
February 2021

Combined immunodeficiency associated with homozygous MALT1 mutations.

J Allergy Clin Immunol 2014 May 12;133(5):1458-62, 1462.e1-7. Epub 2013 Dec 12.

Department of Pediatrics, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2013.10.045DOI Listing
May 2014

Innate immune control of EBV-infected B cells by invariant natural killer T cells.

Blood 2013 Oct 23;122(15):2600-8. Epub 2013 Aug 23.

Child and Family Research Institute, Immunity in Health and Disease, and Department of Pathology and Laboratory Medicine, University of British Columbia and British Columbia Children's Hospital, Vancouver, BC, Canada; and.

Individuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.
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http://dx.doi.org/10.1182/blood-2013-01-480665DOI Listing
October 2013

Lyn-dependent signaling regulates the innate immune response by controlling dendritic cell activation of NK cells.

J Immunol 2012 May 9;188(10):5094-105. Epub 2012 Apr 9.

Department of Microbiology and Immunology, I3 Research Group, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

The innate immune response is a first line of defense against invading pathogens; however, the magnitude of this response must be tightly regulated, as hyper- or suboptimal responses can be detrimental to the host. Systemic inflammation resulting from bacterial infection can lead to sepsis, which remains a serious problem with high mortality rates. Lyn tyrosine kinase plays a key role in adaptive immunity, although its role in innate immunity remains unclear. In this study, we show that Lyn gain-of-function (Lyn(up/up)) mice display enhanced sensitivity to endotoxin and succumb to upregulated proinflammatory cytokine production at a dose well tolerated by control animals. Endotoxin sensitivity in Lyn(up/up) mice depends on dendritic cells (DCs) and NK cells and occurs though a mechanism involving increased maturation and activation of the DC compartment, leading to elevated production of IFN-γ by NK cells. We further show that modulation of endotoxin-induced signal transduction in DCs by Lyn involves the phosphatases Src homology 2 domain-containing phosphatase-1 and SHIP-1. Collectively, we demonstrate that Lyn regulates DC physiology such that alterations in Lyn-dependent signaling have profound effects on the nature and magnitude of inflammatory responses. Our studies highlight how perturbations in signaling pathways controlling DC/NK cell-regulated responses to microbial products can profoundly affect the magnitude of innate immune responses.
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http://dx.doi.org/10.4049/jimmunol.1103395DOI Listing
May 2012

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions.

J Immunol 2012 Feb 16;188(4):1638-46. Epub 2012 Jan 16.

Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.
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http://dx.doi.org/10.4049/jimmunol.1103026DOI Listing
February 2012

NKT cells are required for complete Freund's adjuvant-mediated protection from autoimmune diabetes.

J Immunol 2011 Sep 15;187(6):2898-904. Epub 2011 Aug 15.

Child & Family Research Institute, Immunity in Health and Disease, BC Children's Hospital, Vancouver, British Columbia V5Z 4H4, Canada.

Autoimmune diabetes in NOD mice can be prevented by application of Ags derived from Mycobacterium tuberculosis in the form of bacillus Calmette-Guérin or CFA. Disease protection by CFA is associated with a reduction in the numbers of pathogenic β-cell specific, self-reactive CTLs, a phenomenon dependent on the presence and function of NK cells. However, the mechanisms by which NK cells are activated and recruited by heat-killed M. tuberculosis within CFA are unclear. In this study, we report that CFA-mediated NK cell activation and mobilization is dependent on CD1d expression. The administration of M. tuberculosis from CFA results in rapid NKT cell activation and IFN-γ secretion both in vitro and in vivo. CFA-induced NKT cell activation is intact in MyD88(-/-) mice suggesting that the mechanism is independent of TLR signaling. Furthermore, CD1d expression was found to be essential for both M. tuberculosis-triggered NKT cell activation and CFA-mediated protection of NOD mice from diabetes. Collectively, these findings reveal hitherto previously unidentified roles for NKT cells in the adjuvant-promoting effects of CFA on innate and adaptive immunity.
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http://dx.doi.org/10.4049/jimmunol.1002551DOI Listing
September 2011

The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis.

Immunity 2011 Sep 11;35(3):349-60. Epub 2011 Aug 11.

The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

The transport of calcium ions (Ca(2+)) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca(2+) concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca(2+) "stores" and "store-operated" Ca(2+) entry via plasma membrane channels. However, the identity of molecular components conducting Ca(2+) currents within developing and mature T cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca(2+) channel Ca(V)1.4 plays a cell-intrinsic role in the function, development, and survival of naive T cells. Plasma membrane Ca(V)1.4 was found to be essential for modulation of intracellular Ca(2+) stores and T cell receptor (TCR)-induced rises in cytosolic-free Ca(2+), impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) pathways. Collectively, these studies revealed that Ca(V)1.4 functions in controlling naive T cell homeostasis and antigen-driven T cell immune responses.
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http://dx.doi.org/10.1016/j.immuni.2011.07.011DOI Listing
September 2011

Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling.

Diabetes 2011 Mar 26;60(3):857-66. Epub 2011 Jan 26.

Department of Pathology and Laboratory Medicine, University of British Columbia, Child and Family Research Institute, Immunity in Health and Disease, British Columbia's Children's Hospital, Vancouver, British Columbia, Canada.

Objective: Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children.

Research Design And Methods: Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray.

Results: NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I-related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase-AKT pathway.

Conclusions: These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.
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http://dx.doi.org/10.2337/db09-1706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046846PMC
March 2011

Cutting edge: Increased IL-17-secreting T cells in children with new-onset type 1 diabetes.

J Immunol 2010 Oct 1;185(7):3814-8. Epub 2010 Sep 1.

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

CD4(+)FOXP3(+) regulatory T cells are essential for immune tolerance, and murine studies suggest that their dysfunction can lead to type 1 diabetes (T1D). Human studies assessing regulatory T cell dysfunction in T1D have relied on analysis of FOXP3-expressing cells. Recently, distinct subsets of CD4(+)FOXP3(+) T cells with differing function were identified. Notably, CD45RA(-)CD25(int)FOXP3(low) T cells lack suppressive function and secrete the proinflammatory cytokine IL-17. Therefore, we evaluated whether the relative fractions of CD4(+)FOXP3(+) subsets are altered in new-onset T1D subjects. We report that children with new-onset T1D have an increased proportion of CD45RA(-)CD25(int)FOXP3(low) cells that are not suppressive and secrete significantly more IL-17 than other FOXP3(+) subsets. Moreover, these T1D subjects had a higher proportion of both CD4(+) and CD8(+) T cells that secrete IL-17. The bias toward IL-17-secreting T cells in T1D suggests a role for this proinflammatory cytokine in the pathogenesis of disease.
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http://dx.doi.org/10.4049/jimmunol.1001860DOI Listing
October 2010

RasGRP1 regulates antigen-induced developmental programming by naive CD8 T cells.

J Immunol 2010 Jan 9;184(2):666-76. Epub 2009 Dec 9.

Child and Family Research Institute, Vancouver, British Columbia, Canada.

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1(-/-) 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.
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http://dx.doi.org/10.4049/jimmunol.0803521DOI Listing
January 2010

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice.

J Immunol 2008 May;180(9):5973-82

Department of Microbiology and Immunology, Life Science Centre, University of British Columbia, 2350 Health Science Mall, Vancouver, BC, Canada.

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.
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http://dx.doi.org/10.4049/jimmunol.180.9.5973DOI Listing
May 2008

Critical role for IFN-gamma in natural killer cell-mediated protection from diabetes.

Eur J Immunol 2008 Jan;38(1):82-9

Department of Pathology and Laboratory Medicine, British Columbia Children's Hospital, University of British Columbia and Child and Family Research Institute, Vancouver, Canada.

Autoimmune diabetes in nonobese diabetic (NOD) mice can be prevented by a single injection of complete Freund's adjuvant (CFA), but the mechanisms mediating protection remains unclear. We previously showed that NOD mice immunized with CFA have a markedly reduced incidence of diabetes that is associated with a significant decrease in the number of beta-cell-specific, autoreactive cytotoxic T lymphocytes and, furthermore, that the effect of CFA is mediated by natural killer (NK) cells. In this study, we report one mechanism by which NK cells regulate the onset of diabetes. Administration of CFA produced a rapid increase in NK cell frequency and function, including cytotoxicity and IFN-gamma secretion. By co-transferring NK cells from IFN-gamma-deficient (or wild-type) NOD mice and spleen cells from diabetic NOD mice to NOD/SCID recipients, we show that IFN-gamma secretion by NK cells significantly influences the effect of CFA protection. In contrast, NK cytotoxicity does not appear to participate in CFA-mediated protection from diabetes. Our findings demonstrate that NK cells mediate the protective effects of CFA through secretion of IFN-gamma.
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http://dx.doi.org/10.1002/eji.200737189DOI Listing
January 2008

Chronic immunodeficiency in mice lacking RasGRP1 results in CD4 T cell immune activation and exhaustion.

J Immunol 2007 Aug;179(4):2143-52

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.

The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1(-/-) mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1(-/-) CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1(-/-) CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1(-/-) CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1(-/-) mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1(-/-) mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.
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http://dx.doi.org/10.4049/jimmunol.179.4.2143DOI Listing
August 2007

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice.

J Exp Med 2007 Mar 26;204(3):619-31. Epub 2007 Feb 26.

Department of Microbiology and Immunology, University of British Columbia Vancouver, British Columbia, Canada V6T 1Z3.

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.
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http://dx.doi.org/10.1084/jem.20061871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2137912PMC
March 2007

RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development.

J Immunol 2006 Aug;177(3):1470-80

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.
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http://dx.doi.org/10.4049/jimmunol.177.3.1470DOI Listing
August 2006