bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of CD70 on Dendritic Cells through CD40 or TLR Stimulation Contributes to the Development of CD8+ T Cell Responses in the Absence of CD4+ T Cells
Authors: Bullock Timothy N. J. , Yagita Hideo
Published: April 2014

The expansion of CD8+ T cells in response to Ag can be characterized as either dependent or independent of CD4+ T cells. The factors that influence this dichotomy are poorly understood but may be dependent upon the degree of inflammation associated with the Ag. Using dendritic cells derived from MHC class II-deficient mice to avoid interaction with CD4+ T cells in vivo, we have compared the immunogenicity of peptide-pulsed dendritic cells stimulated with molecules associated with infection to those stimulated via CD40. In the absence of CD4+ T cell help, the expansion of primary CD8+ T cells after immunization with TNF-α- or poly(I:C)-stimulated dendritic cells was minimal. In comparison, LPS- or CpG-stimulated dendritic cells elicited substantial primary CD8+ T cell responses, though not to the same magnitude generated by immunization with CD40L-stimulated dendritic cells. Remarkably, mice immunized with any stimulated dendritic cell population generated fully functional recall CD8+ T cells without the aid of CD4+ T cell help. The observed hierarchy of immunogenicity was closely correlated with the expression of CD70 (CD27L) on the stimulated dendritic cells, and Ab-mediated blockade of CD70 substantially prevented the CD4+ T cell-independent expansion of primary CD8+ T cells. These results indicate that the expression of CD70 on dendritic cells is an important determinant for helper-dependence of primary CD8+ T cell expansion and provide an explanation for the ability of a variety of pathogens to stimulate primary CD8+ T cell responses in the absence of CD4+ T cells.
The Reciprocal Interaction of NK Cells with Plasmacytoid or Myeloid Dendritic Cells Profoundly Affects Innate Resistance Functions
Authors: Gerosa Franca , Gobbi Andrea , Zorzi Penelope , Burg Stephanie , Briere Francine , Carra Giuseppe , Trinchieri Giorgio
Published: April 2014

A reciprocal activating interaction between NK cells and dendritic cells (DC) has been suggested to play a role in the functional regulation of these cells in immunity, but it has been studied only using in vitro generated bone marrow- or monocyte-derived DC. We report that human peripheral blood plasmacytoid DC (pDC) and myeloid DC are necessary to induce NK cell function depending on the type of microbial stimulus. pDC and myeloid DC are required for strongly increased NK cytolytic activity and CD69 expression, in response to inactivated influenza virus or CpG-containing oligonucleotides and poly(I:C), respectively. Secreted type I IFN is required and sufficient for the augmentation of NK cell cytolytic activity in the coculture with pDC or myeloid DC, whereas CD69 expression is dependent on both type I IFN and TNF. In addition, in response to poly(I:C), myeloid DC induce NK cells to produce IFN-γ through a mechanism dependent on both IL-12 secretion and cell contact between NK cells and myeloid DC, but independent of type I IFN. IL-2-activated NK cells have little to no cytolytic activity for immature myeloid DC and pDC, but are able to induce maturation of these cells. Moreover, IL-2-activated NK cells induce, in the presence of a suboptimal concentration of CpG-containing oligonucleotides, a strong IFN-α and TNF production. These data suggest that the reciprocal functional interaction between NK cells and either pDC or myeloid DC may play an important physiological role in the regulation of both innate resistance and adaptive immunity to infections.
Dendritic Cells and NK Cells Stimulate Bystander T Cell Activation in Response to TLR Agonists through Secretion of IFN-αβ and IFN-γ
Authors: Kamath Arun T. , Sheasby Christopher E. , Tough David F.
Published: April 2014

Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-αβ, secreted by dendritic cells (DCs), and IFN-γ, secreted by NK cells; notably, IFN-γ-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-γ by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-γ, maximal IFN-γ production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-αβ, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-γ, whereas IFN-αβ was shown to be important for induction of T cell bystander activation and NK cell IFN-γ production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells.
Importance of Cellular Microenvironment and Circulatory Dynamics in B Cell Immunotherapy
Authors: Gong Qian , Ou Qinglin , Ye Shiming , Lee Wyne P. , Cornelius Jennine , Diehl Lauri , Lin Wei Yu , Hu Zhilan , Lu Yanmei , Chen Yongmei , Wu Yan , Meng Y. Gloria , Gribling Peter , Lin Zhonghua , Nguyen Kathy , Tran Thanhvien , Zhang Yifan , Rosen Hugh , Martin Flavius , Chan Andrew C.
Published: April 2014

B cell immunotherapy has emerged as a mainstay in the treatment of lymphomas and autoimmune diseases. Although the microenvironment has recently been demonstrated to play critical roles in B cell homeostasis, its contribution to immunotherapy is unknown. To analyze the in vivo factors that regulate mechanisms involved in B cell immunotherapy, we used a murine model for human CD20 (hCD20) expression in which treatment of hCD20+ mice with anti-hCD20 mAbs mimics B cell depletion observed in humans. We demonstrate in this study that factors derived from the microenvironment, including signals from the B cell-activating factor belonging to the TNF family/BLyS survival factor, integrin-regulated homeostasis, and circulatory dynamics of B cells define distinct in vivo mechanism(s) and sensitivities of cells in anti-hCD20 mAb-directed therapies. These findings provide new insights into the mechanisms of immunotherapy and define new opportunities in the treatment of cancers and autoimmune diseases.
Matrix Metalloproteinase-9 in Macrophages Induces Thymic Neovascularization following Thymocyte Apoptosis
Authors: Odaka Chikako , Tanioka Masatoshi , Itoh Takeshi
Published: April 2014

Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.
p53 Potentiation of Tumor Cell Susceptibility to CTL Involves Fas and Mitochondrial Pathways
Authors: Thiery Jérôme , Abouzahr Soraya , Dorothee Guillaume , Jalil Abdelali , Richon Catherine , Vergnon Isabelle , Mami-Chouaib Fathia , Chouaib Salem
Published: April 2014

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.
Different MHC Class I Heavy Chains Compete with Each Other for Folding Independently of β2-Microglobulin and Peptide
Authors: Tourdot Sophie , Nejmeddine Mohamed , Powis Simon J. , Gould Keith G.
Published: April 2014

We reported previously that different MHC class I molecules can compete with each other for cell surface expression in F1 hybrid and MHC class I transgenic mice. In this study, we show that the competition also occurs in transfected cell lines, and investigate the mechanism. Cell surface expression of an endogenous class I molecule in Chinese hamster ovary (CHO) cells was strongly down-regulated when the mouse Kd class I H chain was introduced by transfection. The competition occurred only after Kd protein translation, not at the level of RNA, and localization studies of a CHO class I-GFP fusion showed that the presence of Kd caused retention of the hamster class I molecule in the endoplasmic reticulum. The competition was not for β2-microglobulin, because a single chain version of Kd that included mouse β2-microglobulin also had a similar effect. The competition was not for association with TAP and loading with peptide, because a mutant form of the Kd class I H chain, not able to associate with TAP, caused the same down-regulation of hamster class I expression. Moreover, Kd expression led to a similar level of competition in TAP2-negative CHO cells. Competition for cell surface expression was also found between different mouse class I H chains in transfected mouse cells, and this competition prevented association of the H chain with β2-microglobulin. These unexpected new findings show that different class I H chains compete with each other at an early stage of the intracellular assembly pathway, independently of β2-microglobulin and peptide.
The Rat Expresses Two Complement Factor C4 Proteins, but Only One Isotype Is Expressed in the Liver
Authors: Roos Christian , Dressel Ralf , Schmidt Bernhard , Günther Eberhard , Walter Lutz
Published: April 2014

The complement component C4 is well known for its complex genetics in human and mouse where it is part of a tandemly duplicated module. For the rat, no such information had been available until recently. A C4 gene duplication could be identified also in the rat, but the duplicated module maps ∼200 kb centromerically from the canonical C4-1 gene. In this study, we present the genomic organization of the two C4 gene-containing modules and the expression of the two C4 genes in the rat (Rattus norvegicus). The duplicated module contains an intact C4 gene as well as Cyp21 and Stk19 pseudogenes. Quantitative mRNA expression analyses revealed that both C4 genes are transcribed in various organs and tissues, but displaying ample differences of C4-1 and C4-2 expression. Most notably, C4-2 is not expressed in the liver. At variance to the mouse, the expression of the rat C4 genes does not exhibit any sex dependency. By using two-dimensional gel electrophoresis and mass spectrometry, products of both C4 genes could be identified in rat serum samples. These two rat C4 isotypes are nearly identical, but differ in a functionally important amino acid residue that is known to influence the functional properties of the C4 isotypes in human.
Vaccination with a Human High Molecular Weight Melanoma-Associated Antigen Mimotope Induces a Humoral Response Inhibiting Melanoma Cell Growth In Vitro
Authors: Wagner Stefan , Hafner Christine , Allwardt Dorothee , Jasinska Joanna , Ferrone Soldano , Zielinski Christoph C. , Scheiner Otto , Wiedermann Ursula , Pehamberger Hubert , Breiteneder Heimo
Published: April 2014

Peptide mimics of a conformational epitope that is recognized by a mAb with antitumor activity are promising candidates for formulations of anticancer vaccines. These mimotope vaccines are able to induce a polyclonal Ab response focused to the determinant of the mAb. Such attempts at cancer immunotherapy are of special interest for malignant melanoma that is highly resistant to chemotherapy and radiotherapy. In this study, we describe for the first time the design and immunogenicity of a vaccine containing a mimotope of the human high m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then generated by coupling the most suitable candidate mimotope to tetanus toxoid as an immunogenic carrier. Immunization of rabbits with this vaccine induced a specific humoral immune response directed toward the epitope recognized by the mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26% lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate a possible application of this mimotope vaccine as a novel immunotherapeutic agent for the treatment of malignant melanoma.
Enhanced Production of IL-10 by Dendritic Cells Deficient in CIITA
Authors: Yee Christina S. K. , Yao Yongxue , Xu Qi , McCarthy Brian , Sun-Lin Deqin , Tone Masahide , Waldmann Herman , Chang Cheong-Hee
Published: April 2014

Dendritic cells (DC) are professional APCs that play a critical role in regulating immunity. In DC, maturation-induced changes in MHC class II expression and Ag presentation require transcriptional regulation by CIITA. To study the role of CIITA in DC, we evaluated key cell functions in DC from CIITA-deficient (CIITA−/−) mice. The ability to take up Ag, measured by fluid phase endocytosis, was comparable between CIITA−/− and control DC. Although CIITA−/− DC lack MHC class II, they maintained normal expression of costimulatory molecules CD80, CD86, and CD40. In contrast, CIITA−/− DC activated with LPS or CpG expressed increased IL-10 levels, but normal levels of TNF-α and IL-12 relative to control. Enhanced IL-10 was due to greater IL-10 mRNA in CIITA−/− DC. Aβ−/− DC, which lack MHC class II but express CIITA normally, had exhibited no difference in IL-10 compared with control. When CIITA was cotransfected with an IL-10 promoter-reporter into a mouse monocyte cell line, RAW 264.7, IL-10 promoter activity was decreased. In addition, reintroducing CIITA into CIITA−/− DC reduced production of IL-10. In all, these data suggest that CIITA negatively regulates expression of IL-10, and that CIITA may direct DC function in ways that extend beyond control of MHC class II.
The Role of p53 and Fas in a Model of Acute Murine Graft-versus-Host Disease
Authors: Yada Shinichiro , Takamura Noriaki , Inagaki-Ohara Kyoko , O’Leary Melissa K. , Wasem Christoph , Brunner Thomas , Green Douglas R. , Lin Tesu , Pinkoski Michael J.
Published: April 2014

Graft-vs-host disease (GVHD) is a devastating, frequently fatal, pathological condition associated with lesions in specific target organs, including the intestine, liver, lung, and skin, as well as pancytopenia and alopecia. Bone marrow (BM) atrophy is observed in acutely diseased animals, but the underlying mechanisms of hemopoietic stem cell depletion remained to be established. We used an experimental mouse model of acute GVHD in which parental cells were injected into F1 hosts preconditioned by sublethal irradiation. The resulting graft-vs-host response was kinetically consistent, resulting in lethality within 3 wk. We observed disease pathology in the liver and small intestine, and consistent with previous observations, we found BM atrophy to be a factor in the onset of acute disease. The product of the protooncogene, p53, is known to be a key player in many physiological examples of apoptosis. We investigated the role of p53 in the apoptosis of BM cells (BMC) during the development of acute disease and found that at least one copy of the p53 gene is necessary for depletion of BM and subsequent lethality in host animals. BM depletion was preceded by induction of the death receptor, Fas, on the surface of host stem cells, and induction of Fas was coincidental with the sensitization of BMC to Fas-mediated apoptosis. Our data indicate that BM depletion in acute GVHD is mediated by p53-dependent up-regulation of Fas on BMC, which leads to Fas-dependent depletion and subsequent disease.
The Pathway of Antigen Uptake and Processing Dictates MHC Class II-Mediated B Cell Survival and Activation
Authors: Nashar Toufic O. , Drake James R.
Published: April 2014

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50–100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4+ T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.
Phosphostim-Activated γδ T Cells Kill Autologous Metastatic Renal Cell Carcinoma
Authors: Viey Emilie , Fromont Gaëlle , Escudier Bernard , Morel Yannis , Da Rocha Sylvie , Chouaib Salem , Caignard Anne
Published: April 2014

Metastatic renal cell carcinoma, inherently resistant to conventional treatments, is considered immunogenic. Indeed, partial responses are obtained after treatment with cytokines such as IL-2 or IFN-α, suggesting that the immune system may control the tumor growth. In this study, we have investigated the ability of the main subset of peripheral γδ lymphocytes, the Vγ9Vδ2-TCR T lymphocytes, to induce an effective cytotoxic response against autologous primary renal cell carcinoma lines. These γδ T cells were expanded ex vivo using a Vγ9Vδ2 agonist, a synthetic phosphoantigen called Phosphostim. From 11 of 15 patients, the peripheral Vγ9Vδ2 T cells were amplified in vitro by stimulating PBMCs with IL-2 and Phosphostim molecule. These expanded Vγ9Vδ2 T cells express activation markers and exhibit an effector/memory phenotype. They display a selective lytic potential toward autologous primary renal tumor cells and not against renal NC. The lytic activity involves the perforin-granzyme pathway and is mainly TCR and NKG2D receptor dependent. Furthermore, an increased expression of MHC class I-related molecule A or B proteins, known ligands of NKG2D, are detected on primary renal tumor cells. Interestingly, from 2 of the 11 positive cultures in response to Phosphostim, expanded-Vγ9Vδ2 T cells present an expression of killer cell Ig-like receptors, suggesting their prior recruitment in vivo. Unexpectedly, on serial frozen sections from three tumors, we observe a γδ lymphocyte infiltrate that was mainly composed of Vγ9Vδ2 T cells. These results outline that Vγ9Vδ2-TCR effectors may represent a promising approach for the treatment of metastatic renal cell carcinoma.
The CD200 Receptor Is a Novel and Potent Regulator of Murine and Human Mast Cell Function
Authors: Cherwinski Holly M. , Murphy Craig A. , Joyce Barbara L. , Bigler Mike E. , Song Yaoli S. , Zurawski Sandra M. , Moshrefi Mehrdad M. , Gorman Daniel M. , Miller Kathy L. , Zhang Shuli , Sedgwick Jonathan D. , Phillips Joseph H.
Published: April 2014

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcεRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcεRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.
Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo
Authors: Hoffmann Peter R. , Kench Jennifer A. , Vondracek Andrea , Kruk Ellen , Daleke David L. , Jordan Michael , Marrack Philippa , Henson Peter M. , Fadok Valerie A.
Published: April 2014

Phosphatidylserine (PS) on apoptotic cells promotes their uptake and induces anti-inflammatory responses in phagocytes, including TGF-β release. Little is known regarding the effects of PS on adaptive immune responses. We therefore investigated the effects of PS-containing liposomes on immune responses in mice in vivo. PS liposomes specifically inhibited responses to Ags as determined by decreased draining lymph node tissue mass, with reduced numbers of total leukocytes and Ag-specific CD4+ T cells. There was also a decrease in formation and size of germinal centers in spleen and lymph nodes, accompanied by decreased levels of Ag-specific IgG in blood. Many of these effects were mimicked by an agonistic Ab-specific for the PS receptor. TGF-β appears to play a critical role in this inhibition, as the inhibitory effects of PS were reversed by in vivo administration of anti-TGF-β Ab. PS-containing liposomes did not appear to directly inhibit dendritic cell maturation in vitro in response to a variety of stimuli, nor did it prevent their migration to regional lymph nodes in vivo, suggesting that the inhibitory effects may have resulted from complicated interactions between tissue cells and dendritic cells, subsequently inhibiting their ability to productively activate T lymphocytes.
Tim-3+ T-bet+ Tumor-Specific Th1 Cells Colocalize with and Inhibit Development and Growth of Murine Neoplasms
Authors: Simmons William J. , Koneru Mythili , Mohindru Mani , Thomas Rajan , Cutro Scott , Singh Parul , DeKruyff Rosemarie H. , Inghirami Giorgio , Coyle Anthony J. , Kim Byung S. , Ponzio Nicholas M.
Published: April 2014

Although T cells infiltrate many types of murine and human neoplasms, in many instances tumor-specific cytotoxicity is not observed. Strategies to stimulate CTL-mediated antitumor immunity have included in vitro stimulation and/or genetic engineering of T cells, followed by adoptive transfer into tumor-bearing hosts. In this model of B cell lymphoma in SJL/J mice, we used Tim-3+ T-bet+ Th1 cells to facilitate the development of tumor-specific CTL. Tumor-specific Th1 cell lines were polarized with IL-12 during in vitro stimulation and long term maintenance. As few as 5 million Tim-3+ T-bet+ Th1 cells enabled recipients to resist growth of malignant transplantable cells. In addition, similar numbers of Th1 cells injected into 2- to 3-mo-old mice inhibited development of the spontaneous primary lymphomas, which normally arise in 90% of aging mice. CFSE+ Th1 cells colocalized with injected tumor cells in vivo and formed conjugates with the tumor cells within follicles, whereas in nontumor-challenged recipients the CFSE+ Th1 cells localized only within the T cell zones of the spleen. These results provide evidence that adoptive immunotherapy with Tim-3+ T-bet+ tumor-specific Th1 cells can be used to induce host cytotoxic responses that inhibit the development and growth of neoplastic cells.
IFN-γ Determines Distinct Clinical Outcomes in Autoimmune Encephalomyelitis
Authors: Wensky Allen K. , Furtado Glaucia C. , Garibaldi Marcondes Maria Cecilia , Chen Shaohua , Manfra Denise , Lira Sergio A. , Zagzag David , Lafaille Juan J.
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS initiated by autoreactive CD4+ T cells. EAE classically presents with a progressive ascending paralysis and is a model of multiple sclerosis that recapitulates some aspects of the disease. In this report we describe a mouse strain that spontaneously develops a severe, nonclassical form of EAE with 100% incidence. The distinct clinical phenotype is marked initially by a slight head tilt, progressing to a severe head tilt, spinning, or a rotatory motion. Classical EAE spontaneously occurs in myelin basic protein (MBP)-specific TCR transgenic RAG-1−/− mice (referred to as T/R−), whereas nonclassical EAE spontaneously occurs in T/R− IFN-γ−/− mice (T/R−γ−). Thus, the TCR recognizes the same Ag (MBP) and uses identical TCR in both cases. The cellular infiltrate in nonclassical EAE is predominantly found in the brainstem and cerebellum, with very little inflammation in the spinal cord, which is primarily affected in classical disease. Importantly, depending on the genetic makeup and priming conditions of the MBP-specific T cells, nonclassical disease can occur in the presence of an inflammatory infiltrate with eosinophilic, neutrophilic, or monocytic characteristics. Finally, we believe that nonclassical spontaneous EAE could be a useful model for the study of some characteristics of multiple sclerosis not observed in classical EAE, such as the inflammatory responses in the brainstem and cerebellum that can cause vertigo.
Rolling of Th1 Cells via P-Selectin Glycoprotein Ligand-1 Stimulates LFA-1-Mediated Cell Binding to ICAM-1
Authors: Atarashi Kazuyuki , Hirata Takako , Matsumoto Masanori , Kanemitsu Naotoshi , Miyasaka Masayuki
Published: April 2014

Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 or CCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.
OX40 Signals during Priming on Dendritic Cells Inhibit CD4 T Cell Proliferation: IL-4 Switches off OX40 Signals Enabling Rapid Proliferation of Th2 Effectors
Authors: Kim Mi-Yeon , Bekiaris Vasilios , McConnell Fiona M. , Gaspal Fabrina M. C. , Raykundalia Chandra , Lane Peter J. L.
Published: April 2014

In this study we examined the role and regulation of OX40 signals during CD4 T cell priming on dendritic cells (DCs). Contrary to expectation, OX40-deficient cells proliferated more rapidly than their normal counterparts, particularly when stimulated with peptide in the absence of added cytokines. This proliferative advantage was not apparent for Th2-differentiated cells. When the reasons for this were investigated, we found that the cytokine IL-4 specifically down-regulated expression of OX40 ligand on T, B, and DCs, but not on the CD4+CD3− cells linked with selection of Th2 cells into the memory compartment. OX40 ligand expression was also down-regulated on rapidly proliferating Th1 effectors. These data are compatible with OX40 signals acting during priming as a check on naive T cell proliferation while T cells integrate additional DC signals. This would serve to limit inappropriate T cell responses. In contrast, OX40 signals from CD4+CD3− cells located in the outer T zone select proliferating Th2 effectors into the memory T cell pool.
Sonic Hedgehog Is Produced by Follicular Dendritic Cells and Protects Germinal Center B Cells from Apoptosis
Authors: Sacedón Rosa , Díez Blanca , Nuñez Vanesa , Hernández-López Carmen , Gutierrez-Frías Cruz , Cejalvo Teresa , Outram Susan V. , Crompton Tessa , Zapata Agustín G. , Vicente Angeles , Varas Alberto
Published: April 2014

The Hedgehog (Hh) signaling pathway is involved in the development of many tissues during embryogenesis, but has also been described to function in adult self-renewing tissues. In the immune system, Sonic Hedgehog (Shh) regulates intrathymic T cell development and modulates the effector functions of peripheral CD4+ T cells. In this study we investigate whether Shh signaling is involved in peripheral B cell differentiation in mice. Shh is produced by follicular dendritic cells, mainly in germinal centers (GCs), and GC B cells express both components of the Hh receptor, Patched and Smoothened. Blockade of the Hh signaling pathway reduces the survival, and consequently the proliferation and Ab secretion, of GC B cells. Furthermore, Shh rescues GC B cells from apoptosis induced by Fas ligation. Taken together, our data suggest that Shh is one of the survival signals provided by follicular dendritic cells to prevent apoptosis in GC B cells.
CD45 Signals outside of Lipid Rafts to Promote ERK Activation, Synaptic Raft Clustering, and IL-2 Production
Authors: Zhang Min , Moran Miriana , Round June , Low Teresa A. , Patel Viresh P. , Tomassian Tamar , Hernandez Joseph D. , Miceli M. Carrie
Published: April 2014

CD45 is dynamically repositioned within lipid rafts and the immune synapse during T cell activation, although the molecular consequences of CD45 repositioning remain unclear. In this study we examine the role of CD45 membrane compartmentalization in regulating murine T cell activation. We find that raft-localized CD45 antagonizes IL-2 production by opposing processive TCR signals, whereas raft-excluded CD45 promotes ERK-dependent polarized synaptic lipid raft clustering and IL-2 production. We propose that these dual CD45 activities ensure that only robust TCR signals proceed, whereas signals meeting threshold requirements are potentiated. Our findings highlight membrane compartmentalization as a key regulator of CD45 function and elucidate a novel signal transduction pathway by which raft-excluded CD45 positively regulates T cell activation.
Functional and Molecular Analysis of the Double-Positive Stage-Specific CD8 Enhancer E8III during Thymocyte Development
Authors: Feik Nicholas , Bilic Ivan , Tinhofer Johanna , Unger Bernd , Littman Dan R. , Ellmeier Wilfried
Published: April 2014

Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8III enhancer, which directs expression in double-positive (DP) thymocytes, and E8II, which is active in DP cells and CD8+ T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8III enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8III in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8II is also deleted. These results indicate that E8III is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development.
A Single Nucleotide Polymorphism on the Promoter of eotaxin1 Associates with Its mRNA Expression and Asthma Phenotypes
Authors: Chang Hun Soo , Kim Jung Sun , Lee June Hyuk , Cho Jung Il , Rhim Tai Youn , Uh Soo-Taek , Park Byung Lae , Chung Il Yup , Park Choon-Sik , Shin Hyoung Doo
Published: April 2014

Eotaxin1 plays a pivotal role in eosinophil-associated inflammation. Previously, we demonstrated 14 single-nucleotide polymorphisms (SNPs) in the human eotaxin1 gene and the association between the EOT+67G>A allele and the level of IgE. In this study, we investigated the association between the SNPs and plasma eotaxin1 levels, peripheral blood eosinophil counts, and PC20 methacholine values in normal and asthmatic subjects, and the effects of SNPs on the process of eotaxin1 production. The EOT−576C>T and EOT−384A>G polymorphisms and haplotypes (ht1 and ht4) were significantly associated with plasma eotaxin1 levels in the asthmatics (p < 0.001–0.040). The log [plasma eotaxin1] values correlated with the log [serum total IgE] values in the asthmatics and the normal controls (p = 0.012 and p = 0.004, respectively), and with the log [PC20 methacholine] values in the asthmatics (p = 0.014). A DNA-protein complex was formed with EOT−384A>G, but not with the other SNPs of the promoter. The interaction was stronger with the minor allele than with the common allele, and was reduced upon TNF-α exposure. TNF-α-stimulated PBMCs from the asthmatics with the minor allele homozygote expressed significantly lower levels of eotaxin1 mRNA than those from individuals with the common allele. The EOT+67G>A polymorphism, which substitutes alanine with threonine, did not affect eotaxin1 production or activity. Our data suggest that the EOT−384A>G SNP participates in the regulation of eotaxin1 expression by providing a potential binding site for a repressor, and that the ANOVA of EOT-384A>G may predict asthma phenotypes.
Monocyte Chemoattractant Protein-1 and CCR2 Interactions Are Required for IFN-α/β-Induced Inflammatory Responses and Antiviral Defense in Liver
Authors: Hokeness Kirsten L. , Kuziel William A. , Biron Christine A. , Salazar-Mather Thais P.
Published: April 2014

IFN-α/β-mediated functions promote production of MIP-1α (or CCL3) by mediating the recruitment of MIP-1α-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-γ delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-α/β and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1α during infection and was dependent on IFN-α/β effects for induction. Resident F4/80+ liver leukocytes were identified as primary IFN-α/β responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1α and IFN-γ in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.
Myeloperoxidase Plays Critical Roles in Killing Klebsiella pneumoniae and Inactivating Neutrophil Elastase: Effects on Host Defense
Authors: Hirche Tim O. , Gaut Joseph P. , Heinecke Jay W. , Belaaouaj Azzaq
Published: April 2014

Activated neutrophils use myeloperoxidase (MPO) to generate an array of potent toxic oxidants. In the current studies we used genetically altered mice deficient in MPO to investigate the role of the enzyme in host defense against the Gram-negative bacterium Klebsiella pneumoniae, an important human pathogen. For comparison, we used mice deficient in the antimicrobial molecule, neutrophil elastase (NE). When challenged i.p., mice deficient in either MPO or NE were markedly more susceptible to bacterial infection and death. In vitro studies suggested that MPO impairs the morphology of bacteria in a distinctive way. Of importance, our in vitro studies found that MPO mediated oxidative inactivation of NE, an enzyme that has been widely implicated in the pathogenesis of various tissue-destructive diseases. This pathway of oxidative inactivation may be physiologically relevant, because activated neutrophils isolated from MPO-deficient mice exhibited increased elastase activity. Our observations provide strong evidence that MPO, like NE, is a key player in the killing of K. pneumoniae bacteria. They also suggest that MPO may modulate NE to protect the host from the tissue-degrading activity of this proteinase.
Surface-Expressed TLR6 Participates in the Recognition of Diacylated Lipopeptide and Peptidoglycan in Human Cells
Authors: Nakao Yoshiya , Funami Kenji , Kikkawa Satomi , Taniguchi Mitsue , Nishiguchi Miyuki , Fukumori Yasuhiro , Seya Tsukasa , Matsumoto Misako
Published: April 2014

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-κB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys30 and Cys36 in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-κB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.
Prototype Alzheimer’s Disease Vaccine Using the Immunodominant B Cell Epitope from β-Amyloid and Promiscuous T Cell Epitope Pan HLA DR-Binding Peptide
Authors: Agadjanyan Michael G. , Ghochikyan Anahit , Petrushina Irina , Vasilevko Vitaly , Movsesyan Nina , Mkrtichyan Mikayel , Saing Tommy , Cribbs David H.
Published: April 2014

Immunization of amyloid precursor protein transgenic mice with fibrillar β-amyloid (Aβ) prevents Alzheimer’s disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Aβ, containing the B and T cell self epitopes of Aβ, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Aβ Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Aβ immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Aβ in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Aβ1–15 sequence lacks the T cell epitope of Aβ. Immunization of BALB/c mice with the PADRE-Aβ1–15 epitope vaccine produced high titers of anti-Aβ Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Aβ peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.
Immune Activation of Type I IFNs by Listeria monocytogenes Occurs Independently of TLR4, TLR2, and Receptor Interacting Protein 2 but Involves TANK-Binding Kinase 1
Authors: O’Connell Ryan M. , Vaidya Sagar A. , Perry Andrea K. , Saha Supriya K. , Dempsey Paul W. , Cheng Genhong
Published: April 2014

Type I IFNs are well established antiviral cytokines that have also been shown to be induced by bacteria. However, the signaling mechanisms regulating the activation of these cytokines during bacterial infections remain poorly defined. We show that although Gram-negative bacteria can activate the type I IFN pathway through TLR4, the intracellular Gram-positive bacterium Listeria monocytogenes (LM) can do so independently of TLR4 and TLR2. Furthermore, experiments using genetic mutants and chemical inhibitors suggest that LM-induced type I IFN activation occurs by an intracellular pathway involving the serine-threonine kinase TNFR-associated NF-κB kinase (TANK)-binding kinase 1 (TBK1). Interestingly, receptor-interacting protein 2, a component of the recently discovered nucleotide-binding oligomerization domain-dependent intracellular detection pathway, was not involved. Taken together, our data describe a novel signal transduction pathway involving TBK1 that is used by LM to activate type I IFNs. Additionally, we provide evidence that both the LM- and TLR-dependent pathways converge at TBK1 to activate type I IFNs, highlighting the central role of this molecule in modulating type I IFNs in host defense and disease.
Opposing and Hierarchical Roles of Leukotrienes in Local Innate Immune versus Vascular Responses in a Model of Sepsis
Authors: Benjamim Claudia F. , Canetti Claudio , Cunha Fernando Q. , Kunkel Steven L. , Peters-Golden Marc
Published: April 2014

The 5-lipoxygenase (5-LO)-derived leukotrienes (LTs) influence both local innate immunity and vascular responses, but the relative importance of effects on these two processes in sepsis is unknown. In a cecal ligation and puncture model of peritonitis with severe sepsis, 5-LO−/− mice showed a reduction in peritoneal neutrophil accumulation and an increase in the number of bacteria in the peritoneal cavity. Despite this impairment of local innate immunity, the null mice exhibited a marked improvement in survival, and this protection was also seen in wild-type animals treated with the LT synthesis inhibitor MK 886. A survival advantage in severe sepsis was also observed in mice treated with the cysteinyl-LT receptor antagonist MK 571, but not with the LTB4 receptor antagonist CP 105, 696. Protection in the 5-LO−/− mice was associated with reduced vascular leak and serum lactate levels. Moreover, wild-type mice treated with MK 571 exhibited less sepsis-induced hypotension. These data demonstrate opposing effects of cysteinyl-LTs on innate immune vs hemodynamic responses, demonstrating protective effects on local immunity and deleterious effects on the vasculature. They also suggest the possible therapeutic utility of targeting vascular events in sepsis with cysteinyl-LT blockade.
Tissue Transglutaminase-Mediated Formation and Cleavage of Histamine-Gliadin Complexes: Biological Effects and Implications for Celiac Disease
Authors: Qiao Shuo-Wang , Piper Justin , Haraldsen Guttorm , Øynebråten Inger , Fleckenstein Burkhard , Molberg Øyvind , Khosla Chaitan , Sollid Ludvig M.
Published: April 2014

Celiac disease is an HLA-DQ2-associated disorder characterized by an intestinal T cell response. The disease-relevant T cells secrete IFN-γ upon recognition of gluten peptides that have been deamidated in vivo by the enzyme tissue transglutaminase (transglutaminase 2 (TG2)). The celiac intestinal mucosa contains elevated numbers of mast cells, and increased histamine secretion has been reported in celiac patients. This appears paradoxical because histamine typically biases T cell responses in the direction of Th2 instead of the Th1 pattern seen in the celiac lesions. We report that histamine is an excellent substrate for TG2, and it can be efficiently conjugated to gluten peptides through TG2-mediated transamidation. Histamine-peptide conjugates do not exert agonistic effects on histamine receptors, and scavenging of biologically active histamine by gluten peptide conjugation can have physiological implications and may contribute to the mucosal IFN-γ response in active disease. Interestingly, TG2 is able to hydrolyze the peptide-histamine conjugates when the concentrations of substrates are lowered, thereby releasing deamidated gluten peptides that are stimulatory to T cells.