Publications by authors named "Philippe Krebs"

50 Publications

GPR15 facilitates recruitment of regulatory T cells to promote colorectal cancer.

Cancer Res 2021 Mar 16. Epub 2021 Mar 16.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen

Colorectal cancer (CRC) is one of the most frequent malignancies worldwide. Despite considerable progress in early detection and treatment, there is still an unmet need for novel anti-tumor therapies, particularly in advanced CRC. Regulatory T cells (Tregs) are increased in the peripheral blood and tumor tissue of CRC patients. Recently, transient ablation of tumor-associated Tregs was shown to foster CD8+ T cell-mediated anti-tumoral immunity in murine CRC models. However, before considering therapies, targeting Tregs in cancer patients and detailed knowledge of the phenotype and features of tumor-associated Tregs is indispensable. Here we demonstrate in a murine model of inflammation-induced CRC that tumor-associated Tregs are mainly of thymic origin and equipped with a specific set of molecules strongly associated with enhanced migratory properties. Particularly, a dense infiltration of Tregs in mouse and human CRC lesions correlated with increased expression of the orphan chemoattractant receptor GPR15 on these cells. Comprehensive gene expression analysis revealed that tumor-associated GPR15+ Tregs have a Th17-like phenotype, thereby producing IL-17 and TNF-α. Gpr15 deficiency repressed Treg infiltration in CRC, which paved the way for enhanced anti-tumoral CD8+ T cell immunity and reduced tumorigenesis. In conclusion, GPR15 represents a promising novel target for modifying T cell-mediated anti-tumoral immunity in CRC.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2133DOI Listing
March 2021

Interleukin-33 signaling exacerbates experimental infectious colitis by enhancing gut permeability and inhibiting protective Th17 immunity.

Mucosal Immunol 2021 Mar 2. Epub 2021 Mar 2.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

A wide range of microbial pathogens is capable of entering the gastrointestinal tract, causing infectious diarrhea and colitis. A finely tuned balance between different cytokines is necessary to eradicate the microbial threat and to avoid infection complications. The current study identified IL-33 as a critical regulator of the immune response to the enteric pathogen Citrobacter rodentium. We observed that deficiency of the IL-33 signaling pathway attenuates bacterial-induced colitis. Conversely, boosting this pathway strongly aggravates the inflammatory response and makes the mice prone to systemic infection. Mechanistically, IL-33 mediates its detrimental effect by enhancing gut permeability and by limiting the induction of protective T helper 17 cells at the site of infection, thus impairing host defense mechanisms against the enteric pathogen. Importantly, IL-33-treated infected mice supplemented with IL-17A are able to resist the otherwise strong systemic spreading of the pathogen. These findings reveal a novel IL-33/IL-17A crosstalk that controls the pathogenesis of Citrobacter rodentium-driven infectious colitis. Manipulating the dynamics of cytokines may offer new therapeutic strategies to treat specific intestinal infections.
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http://dx.doi.org/10.1038/s41385-021-00386-7DOI Listing
March 2021

Integrin Alpha E (CD103) Limits Virus-Induced IFN-I Production in Conventional Dendritic Cells.

Front Immunol 2020 27;11:607889. Epub 2021 Jan 27.

Institute of Immunology, Medical Faculty, University of Duisburg-Essen, Essen, Germany.

Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E (, CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection.
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http://dx.doi.org/10.3389/fimmu.2020.607889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873973PMC
January 2021

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Cell Stem Cell 2021 Apr 9;28(4):637-652.e8. Epub 2020 Dec 9.

Department of Hematology, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands; Department of Cell Biology, Institute for Biomedical Engineering, Faculty of Medicine, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany; Oncode Institute, Erasmus Medical Center, Rotterdam 3015GD, the Netherlands. Electronic address:

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.
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http://dx.doi.org/10.1016/j.stem.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024900PMC
April 2021

The multifaceted role of TRAIL signaling in cancer and immunity.

FEBS J 2020 Nov 20. Epub 2020 Nov 20.

Institute of Pathology, University of Bern, Switzerland.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can lead to the induction of apoptosis in tumor or infected cells. However, activation of TRAIL signaling may also trigger nonapoptotic pathways in cancer and in nontransformed cells, that is, immune cells. Here, we review the current knowledge on noncanonical TRAIL signaling. The biological outcomes of TRAIL signaling in immune and malignant cells are presented and explained, with a focus on the role of TRAIL for natural killer (NK) cell function. Furthermore, we highlight the technical difficulties in dissecting the precise molecular mechanisms involved in the switch between apoptotic and nonapoptotic TRAIL signaling. Finally, we discuss the consequences thereof for a therapeutic manipulation of TRAIL in cancer and possible approaches to bypass these difficulties.
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http://dx.doi.org/10.1111/febs.15637DOI Listing
November 2020

Cleft lip and cleft palate in knockout mice is associated with alterations in epithelial-mesenchymal crosstalk.

Development 2020 04 30;147(21). Epub 2020 Apr 30.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.
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http://dx.doi.org/10.1242/dev.187369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225129PMC
April 2020

Non-apoptotic TRAIL function modulates NK cell activity during viral infection.

EMBO Rep 2020 01 19;21(1):e48789. Epub 2019 Nov 19.

Institute of Pathology, University of Bern, Bern, Switzerland.

The role of death receptor signaling for pathogen control and infection-associated pathogenesis is multifaceted and controversial. Here, we show that during viral infection, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) modulates NK cell activity independently of its pro-apoptotic function. In mice infected with lymphocytic choriomeningitis virus (LCMV), Trail deficiency led to improved specific CD8 T-cell responses, resulting in faster pathogen clearance and reduced liver pathology. Depletion experiments indicated that this effect was mediated by NK cells. Mechanistically, TRAIL expressed by immune cells positively and dose-dependently modulates IL-15 signaling-induced granzyme B production in NK cells, leading to enhanced NK cell-mediated T cell killing. TRAIL also regulates the signaling downstream of IL-15 receptor in human NK cells. In addition, TRAIL restricts NK1.1-triggered IFNγ production by NK cells. Our study reveals a hitherto unappreciated immunoregulatory role of TRAIL signaling on NK cells for the granzyme B-dependent elimination of antiviral T cells.
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http://dx.doi.org/10.15252/embr.201948789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945065PMC
January 2020

Keep calm: the intestinal barrier at the interface of peace and war.

Cell Death Dis 2019 11 7;10(11):849. Epub 2019 Nov 7.

Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern, Switzerland.

Epithelial barriers have to constantly cope with both harmless and harmful stimuli. The epithelial barrier therefore serves as a dynamic and not static wall to safeguard its proper physiological function while ensuring protection. This is achieved through multiple defence mechanisms involving various cell types - epithelial and non-epithelial - that work in an integrated manner to build protective barriers at mucosal sites. Damage may nevertheless occur, due to pathogens, physical insults or dysregulated immune responses, which trigger a physiologic acute or a pathologic chronic inflammatory cascade. Inflammation is often viewed as a pathological condition, particularly due to the increasing prevalence of chronic inflammatory (intestinal) diseases. However, inflammation is also necessary for wound healing. The aetiology of chronic inflammatory diseases is incompletely understood and identification of the underlying mechanisms would reveal additional therapeutic approaches. Resolution is an active host response to end ongoing inflammation but its relevance is under-appreciated. Currently, most therapies aim at dampening inflammation at damaged mucosal sites, yet these approaches do not efficiently shut down the inflammation process nor repair the epithelial barrier. Therefore, future treatment strategies should also promote the resolution phase. Yet, the task of repairing the barrier can be an arduous endeavour considering its multiple integrated layers of defence - which is advantageous for damage prevention but becomes challenging to repair at multiple levels. In this review, using the intestines as a model epithelial organ and barrier paradigm, we describe the consequences of chronic inflammation and highlight the importance of the mucosae to engage resolving processes to restore epithelial barrier integrity and function. We further discuss the contribution of pre-mRNA alternative splicing to barrier integrity and intestinal homeostasis. Following discussions on current open questions and challenges, we propose a model in which resolution of inflammation represents a key mechanism for the restoration of epithelial integrity and function.
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http://dx.doi.org/10.1038/s41419-019-2086-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838056PMC
November 2019

The IL-33/ST2 pathway shapes the regulatory T cell phenotype to promote intestinal cancer.

Mucosal Immunol 2019 07 5;12(4):990-1003. Epub 2019 Jun 5.

Institute of Pathology, University of Bern, Bern, Switzerland.

The composition of immune infiltrates strongly affects the prognosis of patients with colorectal cancer (CRC). Interleukin (IL)-33 and regulatory T cells (Tregs) in the tumor microenvironment have been separately implicated in CRC; however their contribution to intestinal carcinogenesis is still controversial. Here, we reveal that IL-33 signaling promotes CRC by changing the phenotype of Tregs. In mice with CRC, tumor-infiltrating Tregs preferentially upregulate IL-33 receptor (ST2), and IL-33/ST2 signaling positively correlates with tumor number and size. Transcriptomic and flow cytometry analyses demonstrate that ST2 expression induces a more activated and migratory phenotype in FOXP3 Tregs, which favors their accumulation in the tumor environment. Consequently, genetic ablation of St2 reduces Treg infiltration and concomitantly enhances the frequencies of effector CD8 T cells, thereby restraining CRC. Mechanistically, IL-33 curtails IL-17 production by FOXP3 Tregs and inhibits Th17 differentiation. In humans, numbers of activated ST2-expressing Tregs are increased in blood and tumor lesions of CRC patients, suggesting a similar mode of regulation. Together, these data indicate a central role of IL-33/ST2 signaling in shaping an immunosuppressive environment during intestinal tumorigenesis. Blockade of this pathway may provide a strategy to modulate the composition of CRC immune infiltrates.
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http://dx.doi.org/10.1038/s41385-019-0176-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746527PMC
July 2019

Targeting CD47 in Anaplastic Thyroid Carcinoma Enhances Tumor Phagocytosis by Macrophages and Is a Promising Therapeutic Strategy.

Thyroid 2019 07 10;29(7):979-992. Epub 2019 May 10.

1Institute of Pathology, University of Bern, Bern, Switzerland.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a "don't eat me" signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47-signal regulatory protein alpha signaling axis in ATC is not well understood. This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice. Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages . Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies. Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.
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http://dx.doi.org/10.1089/thy.2018.0555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648226PMC
July 2019

TREM-1 promotes intestinal tumorigenesis.

Sci Rep 2017 11 1;7(1):14870. Epub 2017 Nov 1.

Institute of Pathology, University of Bern, Bern, Switzerland.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune responses. Increasing evidence suggests a role for TREM-1 not only in acute pathogen-induced reactions but also in chronic and non-infectious inflammatory disorders, including various types of cancer. Here, we demonstrate that genetic deficiency in Trem1 protects from colorectal cancer. In particular, Trem1 mice exhibited reduced tumor numbers and load in an experimental model of inflammation-driven tumorigenesis. Gene expression analysis of Trem1 versus Trem1 tumor tissue demonstrated distinct immune signatures. Whereas Trem1 tumors showed an increased abundance of transcripts linked to adaptive immunity, Trem1 tumors were characterized by overexpression of innate pro-inflammatory genes associated with tumorigenesis. Compared to adjacent tumor-free colonic mucosa, expression of Trem1 was increased in murine and human colorectal tumors. Unexpectedly, TREM-1 was not detected on tumor-associated Ly6C MHC class II macrophages. In contrast, TREM-1 was highly expressed by tumor-infiltrating neutrophils which represented the predominant myeloid population in Trem1 but not in Trem1 tumors. Collectively, our findings demonstrate a clear role of TREM-1 for intestinal tumorigenesis and indicate TREM-1-expressing neutrophils as critical players in colorectal tumor development.
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http://dx.doi.org/10.1038/s41598-017-14516-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665947PMC
November 2017

The ESRP1-GPR137 axis contributes to intestinal pathogenesis.

Elife 2017 10 4;6. Epub 2017 Oct 4.

Institute of Pathology, University of Bern, Bern, Switzerland.

Aberrant alternative pre-mRNA splicing (AS) events have been associated with several disorders. However, it is unclear whether deregulated AS directly contributes to disease. Here, we reveal a critical role of the AS regulator epithelial splicing regulator protein 1 (ESRP1) for intestinal homeostasis and pathogenesis. In mice, reduced ESRP1 function leads to impaired intestinal barrier integrity, increased susceptibility to colitis and altered colorectal cancer (CRC) development. Mechanistically, these defects are produced in part by modified expression of ESRP1-specific isoforms differently activating the Wnt pathway. In humans, is downregulated in inflamed biopsies from inflammatory bowel disease patients. ESRP1 loss is an adverse prognostic factor in CRC. Furthermore, generation of dependent isoforms is altered in CRC and expression of a specific isoform predicts CRC patient survival. These findings indicate a central role of ESRP1-regulated AS for intestinal barrier integrity. Alterations in ESRP1 function or expression contribute to intestinal pathology.
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http://dx.doi.org/10.7554/eLife.28366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665647PMC
October 2017

Autophagy Inhibition Improves Sunitinib Efficacy in Pancreatic Neuroendocrine Tumors via a Lysosome-dependent Mechanism.

Mol Cancer Ther 2017 11 20;16(11):2502-2515. Epub 2017 Jul 20.

Institute of Pathology, University of Bern, Bern, Switzerland.

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNET) is an unmet medical need. The antiangiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. In addition, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis , whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chloroquine. Sunitinib also induced lysosomal membrane permeabilization, which further increased in the presence of chloroquine or knockdown of lysosome-associated membrane protein (LAMP2). Both combinations led to cell death. Our data indicate that chloroquine increases sunitinib efficacy in PanNET treatment via autophagy inhibition and lysosomal membrane permeabilization. We suggest that adding chloroquine to sunitinib treatment will increase efficacy of PanNET treatment and that such patients should be included in respective ongoing clinical trials. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0136DOI Listing
November 2017

The Role of IL-33-Dependent Inflammation in the Tumor Microenvironment.

Front Immunol 2016 9;7:682. Epub 2017 Jan 9.

Institute of Pathology, University of Bern , Bern , Switzerland.

There is compelling evidence that inflammation contributes to tumorigenesis. Inflammatory mediators within the tumor microenvironment can either promote an antitumor immune response or support tumor pathogenesis. Therefore, it is critical to determine the relative contribution of tumor-associated inflammatory pathways to cancer development. Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines that is released upon tissue stress or damage to operate as an alarmin. IL-33 has been primarily implicated in the induction of type-2 immune responses. However, recent findings have shown a role of IL-33 in several cancers where it may exert multiple functions. In this review, we will present the current knowledge on the role of IL-33 in the microenvironment of different tumors. We will highlight which cells produce and which cells are activated by IL-33 in cancer. Furthermore, we will explain how IL-33 modulates the tumor-associated inflammatory microenvironment to restrain or promote tumorigenesis. Finally, we will discuss the issues to be addressed first before potentially targeting the IL-33 pathway for cancer therapy.
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http://dx.doi.org/10.3389/fimmu.2016.00682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220330PMC
January 2017

Cytokine-Induced Modulation of Colorectal Cancer.

Front Oncol 2016 19;6:96. Epub 2016 Apr 19.

Institute of Pathology, University of Bern , Bern , Switzerland.

The emergence of novel immunomodulatory cancer therapies over the last decade, above all immune checkpoint blockade, has significantly advanced tumor treatment. For colorectal cancer (CRC), a novel scoring system based on the immune cell infiltration in tumors has greatly improved disease prognostic evaluation and guidance to more specific therapy. These findings underline the relevance of tumor immunology in the future handling and therapeutic approach of malignant disease. Inflammation can either promote or suppress CRC pathogenesis and inflammatory mediators, mainly cytokines, critically determine the pro- or anti-tumorigenic signals within the tumor environment. Here, we review the current knowledge on the cytokines known to be critically involved in CRC development and illustrate their mechanisms of action. We also highlight similarities and differences between CRC patients and murine models of CRC and point out cytokines with an ambivalent role for intestinal cancer. We also identify some of the future challenges in the field that should be addressed for the development of more effective immunomodulatory therapies.
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http://dx.doi.org/10.3389/fonc.2016.00096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835502PMC
May 2016

The IL-33/ST2 pathway contributes to intestinal tumorigenesis in humans and mice.

Oncoimmunology 2016;5(1):e1062966. Epub 2015 Jun 26.

Institute of Pathology, University of Bern , Bern, Switzerland.

Colorectal cancer (CRC) develops through a multistep process and is modulated by inflammation. However, the inflammatory pathways that support intestinal tumors at different stages remain incompletely understood. Interleukin (IL)-33 signaling plays a role in intestinal inflammation, yet its contribution to the pathogenesis of CRC is unknown. Using immunohistochemistry on 713 resected human CRC specimens, we show here that IL-33 and its receptor ST2 are expressed in low-grade and early-stage human CRCs, and to a lesser extent in higher-grade and more advanced-stage tumors. In a mouse model of CRC, ST2-deficiency protects from tumor development. Moreover, bone marrow (BM) chimera studies indicate that engagement of the IL-33/ST2 pathway on both the radio-resistant and radio-sensitive compartment is essential for CRC development. Mechanistically, activation of IL-33/ST2 signaling compromises the integrity of the intestinal barrier and triggers the production of pro-tumorigenic IL-6 by immune cells. Together, this data reveals a tumor-promoting role of IL-33/ST2 signaling in CRC.
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http://dx.doi.org/10.1080/2162402X.2015.1062966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760343PMC
June 2015

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection.

Proc Natl Acad Sci U S A 2015 Oct 5;112(42):E5706-14. Epub 2015 Oct 5.

Department of Genetics, The Scripps Research Institute, La Jolla, CA 92037; Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390

Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrate's C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.
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http://dx.doi.org/10.1073/pnas.1515619112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620900PMC
October 2015

IL-33 signaling contributes to the pathogenesis of myeloproliferative neoplasms.

J Clin Invest 2015 Jul 26;125(7):2579-91. Epub 2015 May 26.

Myeloproliferative neoplasms (MPNs) are characterized by the clonal expansion of one or more myeloid cell lineage. In most cases, proliferation of the malignant clone is ascribed to defined genetic alterations. MPNs are also associated with aberrant expression and activity of multiple cytokines; however, the mechanisms by which these cytokines contribute to disease pathogenesis are poorly understood. Here, we reveal a non-redundant role for steady-state IL-33 in supporting dysregulated myelopoiesis in a murine model of MPN. Genetic ablation of the IL-33 signaling pathway was sufficient and necessary to restore normal hematopoiesis and abrogate MPN-like disease in animals lacking the inositol phosphatase SHIP. Stromal cell-derived IL-33 stimulated the secretion of cytokines and growth factors by myeloid and non-hematopoietic cells of the BM, resulting in myeloproliferation in SHIP-deficient animals. Additionally, in the transgenic JAK2V617F model, the onset of MPN was delayed in animals lacking IL-33 in radio-resistant cells. In human BM, we detected increased numbers of IL-33-expressing cells, specifically in biopsies from MPN patients. Exogenous IL-33 promoted cytokine production and colony formation by primary CD34+ MPN stem/progenitor cells from patients. Moreover, IL-33 improved the survival of JAK2V617F-positive cell lines. Together, these data indicate a central role for IL-33 signaling in the pathogenesis of MPNs.
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http://dx.doi.org/10.1172/JCI77347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563674PMC
July 2015

ENU-induced phenovariance in mice: inferences from 587 mutations.

BMC Res Notes 2012 Oct 24;5:577. Epub 2012 Oct 24.

Department of Genetics, The Scripps Research Institute, La Jolla, CA 92037, USA.

Background: We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.

Findings: In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.

Conclusions: Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.
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http://dx.doi.org/10.1186/1756-0500-5-577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3532239PMC
October 2012

Slc15a4, a gene required for pDC sensing of TLR ligands, is required to control persistent viral infection.

PLoS Pathog 2012 Sep 13;8(9):e1002915. Epub 2012 Sep 13.

Department of Genetics, The Scripps Research Institute, La Jolla, California, USA.

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.
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http://dx.doi.org/10.1371/journal.ppat.1002915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441671PMC
September 2012

Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice.

Gastroenterology 2012 Aug 19;143(2):418-28. Epub 2012 Apr 19.

La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA.

Background & Aims: Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells.

Methods: We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments.

Results: Specific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vβ7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation.

Conclusions: Intestinal microbes can affect iNKT cell phenotypes and functions in mice.
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http://dx.doi.org/10.1053/j.gastro.2012.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404247PMC
August 2012

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet.

Proc Natl Acad Sci U S A 2011 Dec 21;108(49):19678-82. Epub 2011 Nov 21.

Department of Genetics, The Scripps Research Institute, La Jolla, CA 92037, USA.

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2-3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention.
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http://dx.doi.org/10.1073/pnas.1117835108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241770PMC
December 2011

Mutation of the gastric hydrogen-potassium ATPase alpha subunit causes iron-deficiency anemia in mice.

Blood 2011 Dec 5;118(24):6418-25. Epub 2011 Oct 5.

Department of Genetics, The Scripps Research Institute, La Jolla, CA, USA.

Iron is an essential component of heme and hemoglobin, and therefore restriction of iron availability directly limits erythropoiesis. In the present study, we report a defect in iron absorption that results in iron-deficiency anemia, as revealed by an N-ethyl-N-nitrosourea-induced mouse phenotype called sublytic. Homozygous sublytic mice develop hypochromic microcytic anemia with reduced osmotic fragility of RBCs. The sublytic phenotype stems from impaired gastrointestinal iron absorption caused by a point mutation of the gastric hydrogen-potassium ATPase α subunit encoded by Atp4a, which results in achlorhydria. The anemia of sublytic homozygotes can be corrected by feeding with a high-iron diet or by parenteral injection of iron dextran; rescue can also be achieved by providing acidified drinking water to sublytic homozygotes. These findings establish the necessity of the gastric proton pump for iron absorption and effective erythropoiesis.
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http://dx.doi.org/10.1182/blood-2011-04-350082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3236123PMC
December 2011

Disruption of MyD88 signaling suppresses hemophagocytic lymphohistiocytosis in mice.

Blood 2011 Jun 6;117(24):6582-8. Epub 2011 May 6.

Department of Genetics, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory disorder with a poor prognosis for affected individuals. To find a means of suppressing the clinical phenotype, we investigated the cellular and molecular mechanisms leading to HLH in Unc13d(jinx/jinx) mice, in which cytolytic function of NK and CD8(+) T cells is impaired. Unc13d(jinx/jinx) mutants infected with lymphochoriomeningitis virus (LCMV) present typical clinical features of HLH, including splenomegaly, elevated serum IFNγ, and anemia. Proteins mediating cell-cell contact, cytokine signaling or Toll-like receptor (TLR) signaling were analyzed. We show that neither the integrin CD18, which is involved in adhesion between antigen-presenting cells and effector T cells, nor tumor necrosis factor (TNF) made nonredundant contributions to the disease phenotype. Disruption of IFNγ signaling reduced immune cell activation in Unc13d(jinx/jinx) mice, but also resulted in uncontrolled viral proliferation and exaggerated release of inflammatory cytokines. Abrogating the function of myeloid differentiation primary response gene 88 (MyD88) in Unc13d(jinx/jinx) mice suppressed immune cell activation and controlled cytokine production in an IL-1 receptor 1 (IL-1R1)-independent way. Our findings implicate MyD88 as the key initiator of myeloid and lymphoid proliferation in HLH, and suggest that blockade of this signaling molecule may reduce immunopathology in patients.
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http://dx.doi.org/10.1182/blood-2011-01-329607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123024PMC
June 2011

Impact of β2 integrin deficiency on mouse natural killer cell development and function.

Blood 2011 Mar 14;117(10):2874-82. Epub 2011 Jan 14.

Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Marseille, France.

Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin-deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit(+) cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.
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http://dx.doi.org/10.1182/blood-2010-10-315457DOI Listing
March 2011

Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.

J Immunol 2010 Sep 26;185(5):2721-9. Epub 2010 Jul 26.

Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA.

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.
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http://dx.doi.org/10.4049/jimmunol.1001018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2989418PMC
September 2010

Fatty acid amide hydrolase shapes NKT cell responses by influencing the serum transport of lipid antigen in mice.

J Clin Invest 2010 Jun 17;120(6):1873-84. Epub 2010 May 17.

Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

The potent regulatory properties of NKT cells render this subset of lipid-specific T cells a promising target for immunotherapeutic interventions. The marine sponge glycolipid alpha-galactosylceramide (alphaGalCer) is the proto-typic NKT cell agonist, which elicits this function when bound to CD1d. However, our understanding of the in vivo properties of NKT cell agonists and the host factors that control their bioactivity remains very limited. In this report, we isolated the enzyme fatty acid amide hydrolase (FAAH) from mouse serum as an alphaGalCer-binding protein that modulates the induction of key effector functions of NKT cells in vivo. FAAH bound alphaGalCer in vivo and in vitro and was required for the efficient targeting of lipid antigens for CD1d presentation. Immunization of Faah-deficient mice with alphaGalCer resulted in a reduced systemic cytokine production, but enhanced expansion of splenic NKT cells. This distinct NKT response conferred a drastically increased adjuvant effect and strongly promoted protective CTL responses. Thus, our findings identify not only the presence of FAAH in normal mouse serum, but also its critical role in the tuning of immune responses to lipid antigens by orchestrating their transport and targeting for NKT cell activation. Our results suggest that the serum transport of lipid antigens directly shapes the quality of NKT cell responses, which could potentially be modulated in support of novel vaccination strategies.
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http://dx.doi.org/10.1172/JCI40451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877940PMC
June 2010

Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells.

Proc Natl Acad Sci U S A 2010 May 10;107(21):9759-64. Epub 2010 May 10.

Department of Genetics, The Scripps Research Institute, La Jolla, CA 92037, USA.

A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L --> Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised.
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http://dx.doi.org/10.1073/pnas.1005186107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906899PMC
May 2010

An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence.

Nat Immunol 2010 Apr 28;11(4):335-43. Epub 2010 Feb 28.

Department of Genetics, The Scripps Research Institute, La Jolla, California, USA.

Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea-induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes.
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http://dx.doi.org/10.1038/ni.1847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861894PMC
April 2010

Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5-deficient mice.

J Immunol 2010 Apr 26;184(7):3743-54. Epub 2010 Feb 26.

Department of Genetics, Scripps Research Institute, La Jolla, CA, USA.

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.
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http://dx.doi.org/10.4049/jimmunol.0903164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395861PMC
April 2010