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1.  Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis 
The Journal of experimental medicine  2006;203(13):2895-2906.
Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.
doi:10.1084/jem.20061536
PMCID: PMC1850940  PMID: 17178917
2.  Impaired Development of CD4+ CD25+ Regulatory T Cells in the Absence of STAT1: Increased Susceptibility to Autoimmune Disease 
Type I and II interferons (IFNs) exert opposing effects on the progression of multiple sclerosis, even though both IFNs use the signal transducer and activator of transcription 1 (STAT1) as a signaling mediator. Here we report that STAT1-deficient mice expressing a transgenic T cell receptor against myelin basic protein spontaneously develop experimental autoimmune encephalomyelitis with dramatically increased frequency. The heightened susceptibility to this autoimmune disease appears to be triggered by a reduced number as well as a functional impairment of the CD4+ CD25+ regulatory T cells in STAT1-deficient animals. Adoptive transfer of wild-type regulatory T cells into STAT1-deficient hosts is sufficient to prevent the development of autoimmune disease. These results demonstrate an essential role of STAT1 in the maintenance of immunological self-tolerance.
doi:10.1084/jem.20020509
PMCID: PMC1193645  PMID: 14699080
STAT1; EAE; autoimmune disease; regulatory T cells
3.  Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia 
The Journal of experimental medicine  2004;200(11):1395-1405.
Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73−/− mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O2), Cd73−/− mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73−/− mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.
doi:10.1084/jem.20040915
PMCID: PMC1237012  PMID: 15583013
adenosine; inflammation; edema; endothelium; knockout; ANOVA, analysis of variance; APCP, α,β -methylene ADP; E-Ado, etheno-adenosine; E-AMP, etheno-AMP; ES, embryonic stem; NECA, 5′-(N-ethylcarboxamido)-adenosine; 5′-NT, 5′-nucleotidase
4.  Enhancement of clonogenicity of human multiple myeloma by dendritic cells 
The Journal of experimental medicine  2006;203(8):1859-1865.
Infiltration by dendritic cells (DCs) is a common feature of most human tumors. Prior studies evaluating the interaction of DCs with tumors have focused largely on their immunologic properties (for review see Banchereau, J., and R.M. Steinman. 1998. Nature. 392:245–252). In this study, we show that the clonogenicity of several human tumor cell lines and primary tumor cells from myeloma patients is enhanced by their interactions with DCs. Myeloma cells cultured in the presence of DCs have an altered phenotype with an increased proportion of cells lacking terminal plasma cell differentiation marker CD138. DC–tumor interaction also leads to the up-regulation of B cell lymphoma 6 expression in myeloma cells. Effects of DCs on myeloma cells are inhibited by blockade of the receptor activator of NF-kB (RANK)–RANK ligand and B cell–activating factor–APRIL (a proliferation-inducing ligand)-mediated interactions. Together, these data suggest that tumor–DC interactions may directly impact the biology of human tumors, particularly multiple myeloma, and may be a target for therapeutic intervention.
doi:10.1084/jem.20052136
PMCID: PMC2034506  PMID: 16880256
5.  In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain 
The Journal of experimental medicine  2006;203(9):2095-2107.
The microanatomy of immune clearance of infected brain cells remains poorly understood. Immunological synapses are essential anatomical structures that channel information exchanges between T cell–antigen-presenting cells (APC) during the priming and effector phases of T cells' function, and during natural killer–target cell interactions. The hallmark of immunological synapses established by T cells is the formation of the supramolecular activation clusters (SMACs), in which adhesion molecules such as leukocyte function-associated antigen 1 segregate to the peripheral domain of the immunological synapse (p-SMAC), which surrounds the T cell receptor–rich or central SMAC (c-SMAC). The inability so far to detect SMAC formation in vivo has cast doubts on its functional relevance. Herein, we demonstrate that the in vivo formation of SMAC at immunological synapses between effector CD8+ T cells and target cells precedes and mediates clearance of virally infected brain astrocytes.
doi:10.1084/jem.20060420
PMCID: PMC1997281  PMID: 16923851
6.  The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion 
The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites, the infective stage of malaria. Although CSP has been extensively studied as a malaria vaccine candidate, little is known about its structure. Here, we show that CSP is proteolytically cleaved by a papain family cysteine protease of parasite origin. Our data suggest that the highly conserved region I, found just before the repeat region, contains the cleavage site. Cleavage occurs on the sporozoite surface when parasites contact target cells. Inhibitors of CSP processing inhibit cell invasion in vitro, and treatment of mice with E-64, a highly specific cysteine protease inhibitor, completely inhibits sporozoite infectivity in vivo.
doi:10.1084/jem.20040989
PMCID: PMC1995445  PMID: 15630135
7.  Stromal cell–derived factor 1 promotes angiogenesis via a heme oxygenase 1–dependent mechanism 
Stromal cell–derived factor 1 (SDF-1) plays a major role in the migration, recruitment, and retention of endothelial progenitor cells to sites of ischemic injury and contributes to neovascularization. We provide direct evidence demonstrating an important role for heme oxygenase 1 (HO-1) in mediating the proangiogenic effects of SDF-1. Nanomolar concentrations of SDF-1 induced HO-1 in endothelial cells through a protein kinase C ζ–dependent and vascular endothelial growth factor–independent mechanism. SDF-1–induced endothelial tube formation and migration was impaired in HO-1–deficient cells. Aortic rings from HO-1−/− mice were unable to form capillary sprouts in response to SDF-1, a defect reversed by CO, a byproduct of the HO-1 reaction. Phosphorylation of vasodilator-stimulated phosphoprotein was impaired in HO-1−/− cells, an event that was restored by CO. The functional significance of HO-1 in the proangiogenic effects of SDF-1 was confirmed in Matrigel plug, wound healing, and retinal ischemia models in vivo. The absence of HO-1 was associated with impaired wound healing. Intravitreal adoptive transfer of HO-1–deficient endothelial precursors showed defective homing and reendothelialization of the retinal vasculature compared with HO-1 wild-type cells following ischemia. These findings demonstrate a mechanistic role for HO-1 in SDF-1–mediated angiogenesis and provide new avenues for therapeutic approaches in vascular repair.
doi:10.1084/jem.20061609
PMCID: PMC1855437  PMID: 17339405
8.  A Nonimmunogenic Sarcoma Transduced with the cDNA for Interferon γ Elicits CD8+ T Cells against the Wild-type Tumor: Correlation with Antigen Presentation Capability 
The Journal of experimental medicine  1992;175(6):1423-1431.
Summary
To be recognized by CD8+ T lymphocytes, target cells must process and present peptide antigens in the context of major histocompatibility complex (MHC) class I molecules. The nonimmunogenic, low class I–expressing, methylcholanthrene (MCA)-induced murine sarcoma cell line, MCA 101, is a poor presenter of endogenously generated viral antigens to specific CD8+ T lymphocytes and cannot be used to generate tumor infiltrating lymphocytes (TIL). Since interferon γ (IFN- γ) has been shown to upregulate three sets of molecules important for antigen processing and presentation, we retrovirally transduced wild-type MCA 101 (101.WT) tumor with the mIFN-γ cDNA to create the 101.NAT cell line. Unlike 101.WT, some clones of retrovirally transduced 101.NAT tumor expressed high levels of class I, and could be used to generate CD8+ TIL. More importantly, these TIL were therapeutic in vivo against established pulmonary metastases from the wild-type tumor. Although not uniformly cytotoxic amongst several separate cultures, these TIL did specifically release cytokines (IFN-γ and tumor necrosis factor-α) in response to 101.WT targets. 101.WT’s antigerr presentation deficit was also reversed by gene modification with mIFN-γ cDNA. 101.NAT had a greatly improved capacity to present viral antigens to CD8+ cytotoxic T lymphocytes. These findings show that a nonimmunogenic tumor, incapable of generating a CD8+ T cell immune response, could be gene-modified to generate a therapeutically useful immune response against the wild-type tumor. This strategy may be useful in developing treatments for tumor histologies not thought to be susceptible to T cell-based immunotherapy.
PMCID: PMC1974839  PMID: 1588273
9.  Inflammation and the reciprocal production of granulocytes and lymphocytes in bone marrow 
The Journal of experimental medicine  2005;201(11):1771-1780.
The coordinated production of leukocytes in bone marrow is crucial for innate and adaptive immunity. Inflammation alters normal leukocyte production by promoting granulopoiesis over lymphopoiesis, a response that supports the reactive neutrophilia that follows infection. Here we demonstrate that this specialization for granulopoiesis is determined by inflammation-induced reductions of growth and retention factors, most significantly stem cell factor and CXCL12, which act preferentially to inhibit lymphoid development. These hierarchical effects suggest that the normal equilibrium of leukocyte production in bone marrow is determined by lymphopoiesis’ higher demand for specific growth factors and/or retention signals. Inflammation regulates this balance by reducing growth factors that have less impact on developing neutrophils than lymphocytes. We demonstrate that granulopoiesis and lymphopoiesis are coupled specifically in the bone marrow by development in a common niche and propose that the leukopoietic equilibrium is specified by limiting amounts of developmental resources.
doi:10.1084/jem.20041419
PMCID: PMC1952536  PMID: 15939792
10.  Identification of Human Cancers Deficient in Antigen Processing 
Summary
Intracellular antigens must be processed before presentation to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. Using a recombinant vaccinia virus (Vac) to transiently express the Kd molecule, we studied the antigen processing efficiency of 26 different human tumor lines. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to Kd-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by these cell lines from the endoplasmic reticulum to the cell surface. This finding suggested that peptides were not available for binding to nascent MHC molecules in the endoplasmic reticulum. Northern blot analysis of these cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP-7 and LMP-2, as well as the putative peptide transporters TAP-1 and TAP-2. Treatment of cells with interferon γ enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. Potential therapeutic applications of these findings include enhancing antigen processing at the level of the transcription of MHC-encoded proteasome and transporter genes.
PMCID: PMC1950463  PMID: 8426105
11.  Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis 
The Journal of Experimental Medicine  2006;203(13):2895-2906.
Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.
doi:10.1084/jem.20061536
PMCID: PMC1850940  PMID: 17178917
12.  T cell receptor engagement by peptide–MHC ligands induces a conformational change in the CD3 complex of thymocytes 
The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling αβ heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.
doi:10.1084/jem.20042036
PMCID: PMC1868566  PMID: 15728235
13.  Truncation of C-mip (Tc-mip), a new proximal signaling protein, induces c-maf Th2 transcription factor and cytoskeleton reorganization. 
Summary
Several arguments suggest that Minimal Change Nephrotic Syndrome (MCNS) results from yet unknown systemic disorder of T cell function. By screening a cDNA library from T cell relapse, we identified a new Pleckstrin homology (PH) domain-containing protein encoded by a gene located on chromosome 16q24. Its expression was restricted to fetal liver, kidney and PBMC in which two transcripts were identified. The first species (c-mip) was expressed in normal PBMC but weakly detected in PBMC from MCNS patients. The second form, lacking a part of the Nterminal PH domain (Tc-mip, standing for Truncated c-maf inducing protein), was specifically recruited in CD4+ T cells of patients with MCNS. Overexpression of Tc-mip in T cells induced c-maf, transactivateted the IL-4 gene and downregulated IFNγ expression, characteristic of a Th2 commitment. Moreover, the overexpression of Tc-mip induced T cell clustering and a cellular redistribution of the cytoskeleton-associated L-plastin, by a PI3 kinase independent pathway. T-cmip represents therefore the first identified protein, which links proximal signaling to c-maf induction.
doi:10.1084/jem.20030566
PMCID: PMC1865475  PMID: 12939343
Adult; Base Sequence; Child; Cytoskeletal Proteins; genetics; physiology; Cytoskeleton; physiology; ultrastructure; DNA Primers; DNA-Binding Proteins; genetics; metabolism; Humans; Jurkat Cells; Nephrotic Syndrome; genetics; immunology; Polymerase Chain Reaction; Proto-Oncogene Proteins; genetics; metabolism; Proto-Oncogene Proteins c-maf; Recombinant Proteins; genetics; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Signal Transduction; physiology; T-Lymphocyte Subsets; immunology; T-Lymphocytes; immunology; Th2 Cells; immunology; physiology; Transcription Factors; metabolism; Transfection; src Homology Domains
14.  Stromal cell–derived factor 1 promotes angiogenesis via a heme oxygenase 1–dependent mechanism 
Stromal cell–derived factor 1 (SDF-1) plays a major role in the migration, recruitment, and retention of endothelial progenitor cells to sites of ischemic injury and contributes to neovascularization. We provide direct evidence demonstrating an important role for heme oxygenase 1 (HO-1) in mediating the proangiogenic effects of SDF-1. Nanomolar concentrations of SDF-1 induced HO-1 in endothelial cells through a protein kinase C ζ–dependent and vascular endothelial growth factor–independent mechanism. SDF-1–induced endothelial tube formation and migration was impaired in HO-1–deficient cells. Aortic rings from HO-1−/− mice were unable to form capillary sprouts in response to SDF-1, a defect reversed by CO, a byproduct of the HO-1 reaction. Phosphorylation of vasodilator-stimulated phosphoprotein was impaired in HO-1−/− cells, an event that was restored by CO. The functional significance of HO-1 in the proangiogenic effects of SDF-1 was confirmed in Matrigel plug, wound healing, and retinal ischemia models in vivo. The absence of HO-1 was associated with impaired wound healing. Intravitreal adoptive transfer of HO-1–deficient endothelial precursors showed defective homing and reendothelialization of the retinal vasculature compared with HO-1 wild-type cells following ischemia. These findings demonstrate a mechanistic role for HO-1 in SDF-1–mediated angiogenesis and provide new avenues for therapeutic approaches in vascular repair.
doi:10.1084/jem.20061609
PMCID: PMC1855437  PMID: 17339405
15.  In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain 
The Journal of Experimental Medicine  2006;203(9):2095-2107.
The microanatomy of immune clearance of infected brain cells remains poorly understood. Immunological synapses are essential anatomical structures that channel information exchanges between T cell–antigen-presenting cells (APC) during the priming and effector phases of T cells' function, and during natural killer–target cell interactions. The hallmark of immunological synapses established by T cells is the formation of the supramolecular activation clusters (SMACs), in which adhesion molecules such as leukocyte function-associated antigen 1 segregate to the peripheral domain of the immunological synapse (p-SMAC), which surrounds the T cell receptor–rich or central SMAC (c-SMAC). The inability so far to detect SMAC formation in vivo has cast doubts on its functional relevance. Herein, we demonstrate that the in vivo formation of SMAC at immunological synapses between effector CD8+ T cells and target cells precedes and mediates clearance of virally infected brain astrocytes.
doi:10.1084/jem.20060420
PMCID: PMC1997281  PMID: 16923851
16.  Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract 
The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25-deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25-/- mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-γ and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.
doi:10.1084/jem.20051496
PMCID: PMC1800834  PMID: 16606667
17.  Src homology 2-containing 5-inositol phosphatase (SHIP) suppresses an early stage of lymphoid cell development through elevated Interleukin-6 production by myeloid cells in bone marrow 
The Src homology 2-containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP-/- animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP-/- mice. Lineage-negative marrow (Lin-) cells isolated from wild-type mice failed to differentiate into B cells when co-cultured with those of SHIP-/- mice. Furthermore, culture supernatants of SHIP-/- Lin- cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP-/- Lin- cells contained more IL-6 transcripts than wild-type Lin- cells, and neutralizing anti-IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP-/- Lin- cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin- bone marrow cells reproduced the phenotype of SHIP-/- bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.
doi:10.1084/jem.20031193
PMCID: PMC1797415  PMID: 14718513
lymphopoiesis; inflammation; SHIP; interleukin 6
18.  Enhancement of clonogenicity of human multiple myeloma by dendritic cells 
The Journal of Experimental Medicine  2006;203(8):1859-1865.
Infiltration by dendritic cells (DCs) is a common feature of most human tumors. Prior studies evaluating the interaction of DCs with tumors have focused largely on their immunologic properties (for review see Banchereau, J., and R.M. Steinman. 1998. Nature. 392:245–252). In this study, we show that the clonogenicity of several human tumor cell lines and primary tumor cells from myeloma patients is enhanced by their interactions with DCs. Myeloma cells cultured in the presence of DCs have an altered phenotype with an increased proportion of cells lacking terminal plasma cell differentiation marker CD138. DC–tumor interaction also leads to the up-regulation of B cell lymphoma 6 expression in myeloma cells. Effects of DCs on myeloma cells are inhibited by blockade of the receptor activator of NF-kB (RANK)–RANK ligand and B cell–activating factor–APRIL (a proliferation-inducing ligand)-mediated interactions. Together, these data suggest that tumor–DC interactions may directly impact the biology of human tumors, particularly multiple myeloma, and may be a target for therapeutic intervention.
doi:10.1084/jem.20052136
PMCID: PMC2034506  PMID: 16880256
19.  Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection 
The Journal of Experimental Medicine  2006;203(4):1093-1104.
To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.
doi:10.1084/jem.20051659
PMCID: PMC1584282  PMID: 16606669
20.  Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract 
The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25–deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25−/− mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-γ and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.
doi:10.1084/jem.20051496
PMCID: PMC1800834  PMID: 16606667
21.  Memory CD8+ T Cells Provide Innate Immune Protection against Listeria monocytogenes in the Absence of Cognate Antigen 
The Journal of experimental medicine  2003;198(10):1583-1593.
Interferon (IFN)-γ plays an important role in the innate immune response against intracellular bacterial pathogens. It is commonly thought that natural killer cells are the primary source of this cytokine that is involved in activating antibacterial effects in infected cells and polarizing CD4+ T cells toward the Th1 subset. However, here we show that both effector and memory CD8+ T cells have the potential to secrete IFN-γ in response to interleukin (IL)-12 and IL-18 in the absence of cognate antigen. We demonstrate that memory CD8+ T cells specific for the ovalbumin protein secrete IFN-γ rapidly after infection with wild-type Listeria monocytogenes (LM). Furthermore, small numbers of ovalbumin-specific, memory CD8+ T cells can reduce spleen and liver bacterial counts in IFN-γ–deficient mice 3 d after LM infection. Up-regulation of the receptors for IL-12 and IL-18 provides a mechanism for the ability of memory CD8+ T cells to respond in this antigen nonspecific manner. Thus, CD8+ T cells play an important role in the innate immune response against intracellular pathogens by rapidly secreting IFN-γ in response to IL-12 and IL-18.
doi:10.1084/jem.20031051
PMCID: PMC1592647  PMID: 14623912
innate immunity; T lymphocytes; cytokines; Listeria monocytogenes; IFN-γ; CFSE, carboxyfluorescein diacetate, succinimidyl ester; LM, Listeria monocytogenes; VSV, vesicular stomatitis virus; VV, vaccinia virus
22.  Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection 
The Journal of experimental medicine  2006;203(4):1093-1104.
To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.
doi:10.1084/jem.20051659
PMCID: PMC1584282  PMID: 16606669
ASC, apoptosis-associated specklike protein; CFP, crude flagellar preparation; LDH, lactate dehydrogenase; LLO, listeriolysin O; LRR, leucine-rich repeat; MOI, multiplicity of infection; NOD, nucleotide-binding oligomerization domain; PAMP, pathogen-associated molecular pattern; PE, postexponential; TLR, Toll-like receptor
23.  Mutation of the phospholipase C-γ1–binding site of LAT affects both positive and negative thymocyte selection 
The Journal of experimental medicine  2005;201(7):1125-1134.
Linker for activation of T cells (LAT) is a scaffolding adaptor protein that is critical for T cell development and function. A mutation of LAT (Y136F) that disrupts phospholipase C-γ1 activation and subsequent calcium influx causes a partial block in T cell development and leads to a severe lymphoproliferative disease in homozygous knock-in mice. One possible contribution to the fatal disease of LAT Y136F knock-in mice could be from autoreactive T cells generated in these mice because of altered thymocyte selection. To examine the impact of the LAT Y136F mutation on thymocyte positive and negative selection, we bred this mutation onto the HY T cell receptor (TCR) transgenic, recombination activating gene-2 knockout background. Female mice with this genotype showed a severe defect in positive selection, whereas male mice exhibited a phenotype resembling positive selection (i.e., development and survival of CD8hi HY TCR-specific T cells) instead of negative selection. These results support the hypothesis that in non-TCR transgenic, LAT Y136F knock-in mice, altered thymocyte selection leads to the survival and proliferation of autoreactive T cells that would otherwise be negatively selected in the thymus.
doi:10.1084/jem.20041869
PMCID: PMC1538971  PMID: 15795236
DN, double negative; DP, double positive; LAT, linker for activation of T cells; PLC, phospholipase C; SP, single positive
24.  Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent 
The Journal of Experimental Medicine  2005;202(11):1539-1547.
Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide–specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti–IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene–deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell–driven response to allergen in vivo by an IL-10–dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.
doi:10.1084/jem.20051166
PMCID: PMC1350743  PMID: 16314435
25.  Differential anti-tumor immunity mediated by NKT cell subsets in vivo1 
The Journal of experimental medicine  2005;202(9):1279-1288.
We have previously shown that NKT cell-deficient TCR Jα18−/− mice are more susceptible to methylcholanthrene (MCA)-induced sarcomas, and that normal tumor surveillance can be restored by adoptive transfer of wildtype (WT) liver-derived NKT cells. Liver-derived NKT cells were used in these studies because of their relative abundance in this organ, and it was assumed that they were representative of NKT cells from other sites. In this study, we compared NKT cells from liver, thymus and spleen for their ability to mediate rejection of the sarcoma cell line (MCA-1) in vivo, and found this was a specialized function of liver-derived NKT cells. Furthermore, when CD4+ and CD4− liver-derived NKT cells were administered separately, MCA-1 rejection was mediated primarily by the CD4− fraction. Very similar results were achieved using the B16F10 melanoma metastasis model, which requires NKT cell stimulation with α-galactosylceramide (α-GalCer). The impaired ability of thymus-derived NKT cells was due in part to their production of Interleukin (IL)-4, as tumor immunity was clearly enhanced following transfer of IL-4-deficient thymus-derived NKT cells. This is the first study to demonstrate the existence of functionally distinct NKT cell subsets in vivo and may shed light on the long appreciated paradox that NKT cells function as immunosuppressive cells in some disease models, while promoting cell-mediated immunity in others.
doi:10.1084/jem.20050953
PMCID: PMC1459911  PMID: 16275765
CD1d; NKT; α-galactosylceramide; thymus; tumor

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