bluuebunny/crossref_metadata_2025 huggingface dataset:
A NF-κB/Sp1 Region Is Essential for Chromatin Remodeling and Correct Transcription of a Human Granulocyte- Macrophage Colony-Stimulating Factor Transgene
Authors: Cakouros Dimitrios , Cockerill Peter N. , Bert Andrew G. , Mital Renu , Roberts Donna C. , Shannon M. Frances
Published: April 2014

The GM-CSF gene is expressed following activation of T cells. The proximal promoter and an upstream enhancer have previously been characterized using transfection and reporter assays in T cell lines in culture. A 10.5-kb transgene containing the entire human GM-CSF gene has also been shown to display inducible, position-independent, copy number-dependent transcription in mouse splenocytes. To determine the role of individual promoter elements in transgene function, mutations were introduced into the proximal promoter and activity assessed following the generation of transgenic mice. Of four mutations introduced into the transgene promoter, only one, in an NF-κB/Sp1 region, led to decreased induction of the transgene in splenocytes or bone marrow-derived macrophages. This mutation also affected the activity of reporter gene constructs stably transfected into T cell lines in culture, but not when transiently transfected into the same cell lines. The mutation alters the NF-κB family members that bind to the NF-κB site as well as reducing the binding of Sp1 to an adjacent element. A DNase I hypersensitive site that is normally generated at the promoter following T cell activation on the wild-type transgene does not appear in the mutant transgene. These results suggest that the NF-κB/Sp1 region plays a critical role in chromatin remodeling and transcription on the GM-CSF promoter in primary T cells.
Cytokine-Responsive Gene-2/IFN-Inducible Protein-10 Expression in Multiple Models of Liver and Bile Duct Injury Suggests a Role in Tissue Regeneration
Authors: Koniaris Leonidas G. , Zimmers-Koniaris Teresa , Hsiao Edward C. , Chavin Kenneth , Sitzmann James V. , Farber Joshua M.
Published: April 2014

IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl4 treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl4, d-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl4 injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.
Lacto-N-fucopentaose III Found on Schitosoma mansoni Egg Antigens Functions as Adjuvant for Proteins by Inducing Th2-Type Response
Authors: Okano Mitsuhiro , Satoskar Abhay R. , Nishizaki Kazunori , Harn Donald A.
Published: April 2014

We have recently demonstrated that induction of Th2 responses by Schistosoma mansoni egg Ag is largely due to carbohydrates on the Ag functioning as adjuvants. Lacto-N-fucopentaose III (LNFPIII), a polylactosamine sugar, is the predominant carbohydrate found in S. mansoni egg Ag. Therefore, using neoglycoprotein, we investigated whether LNFPIII induces in vivo Th2 response and functions as an adjuvant. Following intranasal immunization with LNFPIII linked to human serum albumin (HSA) (HSA-LNFPIII), BALB/c mice mounted a strong Th2 response and produced significantly higher levels of total IgE as well as HSA-specific IgG, IgG1, and IgE. HSA-LNFPIII was over 1000-fold more potent in inducing Ab production as compared with HSA alone. Although LNFPIII itself did not function as an epitope for either IgG or IgE, its conjugation with protein was essential for the adjuvant activity. Moreover, fucose residue on LNFPIII was crucial for induction of Ab production. Nasal lymphocytes from mice immunized with HSA-LNFPIII produced IL-4, IL-5, and IL-10, but not IFN-γ following in vitro stimulation with HSA or HSA-LNFPIII. In addition, these activated nasal lymphocytes also showed a significant increase of B7-2 expression on B220-positive cells. Furthermore, not only intranasal but also both i.p. and s.c. immunization with HSA-LNFPIII induced significant production of HSA-specific Abs compared with the immunization with HSA alone, suggesting that the activity of LNFPIII was not restricted on particular route of immunization. These results demonstrate that Lewis type carbohydrate LNFPIII can function as an adjuvant by their ability to induce a Th2 response.
Kaposi’s Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Constitutively Activates NF-κB and Induces Proinflammatory Cytokine and Chemokine Production Via a C-Terminal Signaling Determinant
Authors: Schwarz Mario , Murphy Philip M.
Published: April 2014

Kaposi’s sarcoma-associated herpesvirus (KSHV) is believed to be the causative agent of Kaposi’s sarcoma (KS), a multicentric growth factor-dependent tumor common in AIDS patients characterized histopathologically by spindle cell proliferation, angiogenesis, and leukocyte infiltration. Recently, open reading frame 74 of KSHV has been implicated as a major viral determinant of KS. Open reading frame 74 encodes KSHV G protein-coupled receptor (GPCR), a constitutively active chemokine receptor that directly transforms NIH 3T3 cells in vitro and induces multifocal KS-like lesions in KSHV-GPCR-transgenic mice. Interestingly, receptor-positive cells are very rare in lesions from these mice, implicating an indirect mechanism of tumorigenesis. In this regard, here we report that expression of KSHV-GPCR in transfected epithelial, monocytic, and T cell lines induced constitutive activation of the immunoregulatory transcription factors AP-1 and NF-κB. This was associated with constitutive induction of the proinflammatory NF-κB-dependent cytokines IL-1β, IL-6, and TNF-α, and chemokines monocyte chemoattractant protein-1 and IL-8, as well as the AP-1-dependent basic fibroblast growth factor. In addition, IL-2 and IL-4 production was induced in transfected Jurkat T cells. Truncation of the final five amino acids in the cytoplasmic tail of KSHV-GPCR caused complete loss of its transforming and NF-κB-inducing activities, without affecting receptor expression or ligand binding. These data suggest that KS results in part from KSHV-GPCR induction of proinflammatory cytokine and growth factor gene expression, mediated by a signaling determinant within the last five amino acids of the C terminus, a domain that is also critical for direct cell transformation.
A Retro-Inverso Peptide Mimic of CD28 Encompassing the MYPPPY Motif Adopts a Polyproline Type II Helix and Inhibits Encephalitogenic T Cells In Vitro
Authors: Srinivasan Mythily , Wardrop Richard M. , Gienapp Ingrid E. , Stuckman Scott S. , Whitacre Caroline C. , Kaumaya Pravin T. P.
Published: April 2014

Complete activation of T cells requires two signals: an Ag-specific signal delivered via the TCR by the peptide-MHC complex and a second costimulatory signal largely provided by B7:CD28/CTLA-4 interactions. Previous studies have shown that B7 blockade can either ameliorate experimental autoimmune encephalomyelitis by interfering with CD28 signaling or exacerbate the disease by concomitant blockade of CTLA-4 interaction. Therefore, we developed a functional CD28 mimic to selectively block B7:CD28 interactions. The design, synthesis, and structural and functional properties of the CD28 free peptide, the end group-blocked CD28 peptide, and its retro-inverso isomer are shown. The synthetic T cell-costimulatory receptor peptides fold into a polyproline type II helical structure commonly seen in regions of globular proteins involved in transient protein-protein interactions. The binding determinants of CD28 can be transferred onto a short peptide mimic of its ligand-binding region. The CD28 peptide mimics effectively block the expansion of encephalitogenic T cells in vitro suggesting the potential usefulness of the peptides for the treatment of autoimmune disease conditions requiring down-regulation of T cell responses.
Cutting Edge: Activation of NK Cell-Mediated Cytotoxicity by a SAP-Independent Receptor of the CD2 Family
Authors: Bouchon Axel , Cella Marina , Grierson Helen L. , Cohen Jeffrey I. , Colonna Marco
Published: April 2014

Some CD2 family receptors stimulate NK cell-mediated cytotoxicity through a signaling pathway, which is dependent on the recruitment of an adapter protein called SLAM-associated protein (SAP). In this work we identify a novel leukocyte cell surface receptor of the CD2 family called CD2-like receptor activating cytotoxic cells (CRACC). CRACC is expressed on cytotoxic lymphocytes, activated B cells, and mature dendritic cells, and activates NK cell-mediated cytotoxicity. Remarkably, although CRACC displays cytoplasmic motifs similar to those recruiting SAP, CRACC-mediated cytotoxicity occurs in the absence of SAP and requires activation of extracellular signal-regulated kinases-1/2. Thus, CRACC is a unique CD2-like receptor which mediates NK cell activation through a SAP-independent extracellular signal-regulated kinase-mediated pathway.
Cutting Edge: Antigen-Driven Lymphocyte Recruitment to the Lung Is Diminished in the Absence of Urokinase-Type Plasminogen Activator (uPA) Receptor, but Is Independent of uPA
Authors: Gyetko Margaret R. , Sud Sudha , Sonstein Joanne , Polak Timothy , Sud Anchal , Curtis Jeffrey L.
Published: April 2014

The requirement for urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in T lymphocyte migration is unknown. uPA−/− mice have fewer pulmonary lymphocytes in response to certain infections, but its unknown whether this is due to diminished recruitment. Primed, recipient mice were IT inoculated with Ag. Three days later, fluorescently labeled lymphoblasts from background-matched control wild-type (WT), uPA−/−, or uPAR−/− donor mice were injected i.v., and their recruitment was determined. Approximately twice the number of uPA−/− compared with WT lymphoblasts were recruited to the lungs of WT recipients. This difference was eliminated when uPA−/− and WT lymphoblasts were injected into uPA−/− recipients. Thus, the reduced number of lung lymphocytes in infected uPA−/− mice is not due to reduced recruitment. However, uPAR is critically involved in recruitment. Markedly fewer uPAR−/− compared with WT lymphoblasts were recruited to the lung. These findings suggest that uPAR may be a novel target for immune modulation in T lymphocyte-mediated disorders.
Inefficient ZAP-70 Phosphorylation and Decreased Thymic Selection In Vivo Result from Inhibition of NF-κB/Rel
Authors: Mora Ana L. , Stanley Sarah , Armistead Wade , Chan Andrew C. , Boothby Mark
Published: April 2014

Signaling from the TCR regulates T lymphoid survival, deletion by apoptosis, and selective clonal expansion. One set of signaling pathways activated during thymic selection leads to degradation of a cytosolic retention protein, the inhibitor of κB (IκB)α, followed by nuclear translocation of the NF-κB/Rel family of transcription factors. It has been found previously that NF-κB proteins mediate a pathway signaling the survival of mature T cells and protection of thymocytes against TNF-induced apoptosis. In contrast, we show in this study that a transgenic inhibitor of NF-κB/Rel signaling interferes with the negative selection of immature thymocytes by endogenous MHC ligands in vivo. Positive selection of the H-Y TCR also was diminished. This attenuation of thymic selection efficiency was associated with decreased ZAP-70 phosphorylation and TCR signaling of CD69 induction. These findings demonstrate that the NF-κB transcriptional pathway plays an important role in normal processes of clonal deletion and they indicate that the NF-κB/IκB axis can regulate the efficiency of TCR signaling.
Chronic Ethanol Inhibits NK Cell Cytolytic Activity: Role of Opioid Peptide β-Endorphin
Authors: Boyadjieva Nadka , Dokur Madhavi , Advis Juan P. , Meadows Gary G. , Sarkar Dipak K.
Published: April 2014

The role of β-endorphin (β-EP) in ethanol-altered NK cell cytolytic activity is studied using male Fischer-344 rats as an animal model. Ethanol was administered for 1, 2, 3, or 4 wk in a liquid diet containing 8.7% ethanol (v/v), which means that 37% of the total calories were derived from ethanol. Rats treated with ethanol for 1 wk showed an increase in hypothalamic and plasma levels of immunoreactive (IR)-β-EP, but displayed no significant effect on NK cell activity determined by 51Cr release assay, as compared with those in pair-fed and ad libitum-fed animals. However, animals treated with ethanol for 2, 3, or 4 wk showed decreased hypothalamic and plasma levels of IR-β-EP and decreased splenic NK cell activity. No significant decrease in the number of splenocytes and NK cells or in the percentage of NK cells was seen until after 3 and 4 wk of ethanol treatment. Exposure in vitro of splenic lymphocytes obtained from control animals to various concentrations of β-EP increased NK cell activity. The opiate antagonist naltrexone blocked the β-EP-stimulated effect. The in vitro NK cell response to β-EP was reduced in the splenocytes obtained from animals treated with ethanol for 2 wk, but not in those obtained from animals treated with ethanol for 1 wk as compared with those in control animals. Additionally, β-EP administration into the paraventricular nucleus of the hypothalamus stimulated NK cell cytolytic activity, whereas the opiate blocker administration reduced NK cell activity. The NK cell responses to paraventricular nucleus β-EP were reduced in the animals treated with ethanol for 2 wk. These data provide evidence for the first time that ethanol inhibits NK cell cytolytic activity, possibly by reducing β-EP-regulated splenic NK cell function.
Immunological Memory: Contribution of Memory B Cells Expressing Costimulatory Molecules in the Resting State
Authors: Bar-Or Amit , Oliveira Enedina M. L , Anderson David E. , Krieger Jeff I. , Duddy Martin , O’Connor Kevin C. , Hafler David A.
Published: April 2014

Traditionally, emphasis has been placed on the roles of Th cells in generating and amplifying both cellular and humoral memory responses. Little is known about the potential contributions of B cell subsets to immunological memory. Resting memory B cells have generally been regarded as poor APC, attributed in part to the relative paucity of costimulatory molecules identified on their surface. We describe a novel subpopulation of human memory B cells that express CD80 in their resting state, are poised to secrete particularly large amounts of class switched Igs, and can efficiently present Ag to and activate T cells. This functionally distinct B cell subset may represent an important mechanism by which quiescent human B cells can initiate and propagate rapid and vigorous immune memory responses. Finally, these studies extend recent observations in the murine system and highlight the phenotypic and functional diversity that exists within the human B cell memory compartment.
Superantigen-Induced T Cell:B Cell Conjugation Is Mediated by LFA-1 and Requires Signaling Through Lck, But Not ZAP-70
Authors: Morgan Margaret M. , Labno Christine M. , Van Seventer Gijs A. , Denny Michael F. , Straus David B. , Burkhardt Janis K.
Published: April 2014

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.
A Role for a RhoA/ROCK/LIM-Kinase Pathway in the Regulation of Cytotoxic Lymphocytes
Authors: Lou Zhenkun , Billadeau Daniel D. , Savoy Doris N. , Schoon Renee A. , Leibson Paul J.
Published: April 2014

Polarization of lipid rafts and granules to the site of target contact is required for the development of cell-mediated killing by cytotoxic lymphocytes. We have previously shown that these events require the activation of proximal protein tyrosine kinases. However, the downstream intracellular signaling molecules involved in the development of cell-mediated cytotoxicity remain poorly defined. We report here that a RhoA/ROCK/LIM-kinase axis couples the receptor-initiated protein tyrosine kinase activation to the reorganization of the actin cytoskeleton required for the polarization of lipid rafts and the subsequent generation of cell-mediated cytotoxicity. Pharmacologic and genetic interruption of any element of this RhoA/ROCK/LIM-kinase pathway inhibits both the accumulation of F-actin and lipid raft polarization to the site of target contact and the subsequent delivery of the lethal hit. These data define a specialized role for a RhoA→ROCK→LIM-kinase pathway in cytotoxic lymphocyte activation.
Rejection of Intraocular Tumors by CD4+ T Cells Without Induction of Phthisis
Authors: Schurmans Lucas R. H. M. , Diehl Linda , den Boer Annemieke T. , Sutmuller Roger P. M. , Boonman Zita F. H. M. , Medema Jan Paul , van der Voort Ellen I. H. , Laman Jon , Melief Cornelis J. M. , Jager Martine J. , Toes Rene E. M.
Published: April 2014

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4+ T cells. We now show that intraocular tumors can be eradicated by CD4+ Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8+ T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-α, Fas ligand, MHC class I, and CD8+ T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4+ Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4+ T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.
A New Generation of Melan-A/MART-1 Peptides That Fulfill Both Increased Immunogenicity and High Resistance to Biodegradation: Implication for Molecular Anti-Melanoma Immunotherapy
Authors: Blanchet Jean-Sébastien , Valmori Danila , Dufau Isabelle , Ayyoub Maha , Nguyen Christophe , Guillaume Philippe , Monsarrat Bernard , Cerottini Jean-Charles , Romero Pedro , Gairin Jean Edouard
Published: April 2014

Intense efforts of research are made for developing antitumor vaccines that stimulate T cell-mediated immunity. Tumor cells specifically express at their surfaces antigenic peptides presented by MHC class I and recognized by CTL. Tumor antigenic peptides hold promise for the development of novel cancer immunotherapies. However, peptide-based vaccines face two major limitations: the weak immunogenicity of tumor Ags and their low metabolic stability in biological fluids. These two hurdles, for which separate solutions exist, must, however, be solved simultaneously for developing improved vaccines. Unfortunately, attempts made to combine increased immunogenicity and stability of tumor Ags have failed until now. Here we report the successful design of synthetic derivatives of the human tumor Ag Melan-A/MART-1 that combine for the first time both higher immunogenicity and high peptidase resistance. A series of 36 nonnatural peptide derivatives was rationally designed on the basis of knowledge of the mechanism of degradation of Melan-A peptides in human serum and synthesized. Eight of them were efficiently protected against proteolysis and retained the antigenic properties of the parental peptide. Three of the eight analogs were twice as potent as the parental peptide in stimulating in vitro Melan-specific CTL responses in PBMC from normal donors. We isolated these CTL by tetramer-guided cell sorting and expanded them in vitro. The resulting CTL efficiently lysed tumor cells expressing Melan-A Ag. These Melan-A/MART-1 Ag derivatives should be considered as a new generation of potential immunogens in the development of molecular anti-melanoma vaccines.
Selective Activation of p38 Mitogen-Activated Protein Kinase Cascade in Human Neutrophils Stimulated by IL-1β
Authors: Suzuki Kenichi , Hino Masayuki , Kutsuna Haruo , Hato Fumihiko , Sakamoto Chikahiko , Takahashi Tatsuji , Tatsumi Noriyuki , Kitagawa Seiichi
Published: April 2014

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1β. IL-1β induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1β for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1β. IL-1β primed neutrophils for enhanced release of superoxide (O2−) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1β also induced O2− release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1β-induced O2− release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1β and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1β induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1β and activation of this cascade mediates IL-1β-induced O2− release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.
HLA-G Inhibits Rolling Adhesion of Activated Human NK Cells on Porcine Endothelial Cells
Authors: Forte Pietro , Pazmany Laszlo , Matter-Reissmann Ulrike B. , Stussi Georg , Schneider Mårten K. J. , Seebach Jörg D.
Published: April 2014

Human NK cells adhere to and lyse porcine endothelial cells (pEC) and therefore may contribute to the cell-mediated rejection of vascularized pig-to-human xenografts. Since MHC class I molecules inhibit the cytotoxic activity of NK cells, the expression of HLA genes in pEC has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity. HLA-G, a minimally polymorphic HLA class I molecule that can inhibit a wide range of NK cells, is an especially attractive candidate for this purpose. In this study we tested whether the expression of HLA-G on pEC inhibits the molecular mechanisms that lead to adhesion of human NK cells to pEC and subsequent xenogeneic NK cytotoxicity. To this end two immortalized pEC lines (2A2 and PED) were stably transfected with HLA-G1. Rolling adhesion of activated human NK cells to pEC monolayers and xenogeneic cytotoxicity against pEC mediated by polyclonal human NK lines as well as NK clones were inhibited by the expression of HLA-G. The adhesion was partially reversed by masking HLA-G on pEC with anti-HLA mAbs or by masking the HLA-G-specific inhibitory receptor ILT-2 on NK cells with the mAb HP-F1. The inhibition of NK cytotoxicity by HLA-G was only partially mediated by ILT-2, indicating a role for other unknown NK receptors. In conclusion, transgenic expression of HLA-G may be useful to prevent human NK cell responses to porcine xenografts, but is probably not sufficient on its own. Moreover, the blocking of rolling adhesion by HLA-G provides evidence for a novel biological function of HLA molecules.
Cutting Edge: IL-4 Receptor Expression by Non-Bone Marrow-Derived Cells Is Required to Expel Gastrointestinal Nematode Parasites
Authors: Urban Joseph F. , Noben-Trauth Nancy , Schopf Lisa , Madden Kathleen B. , Finkelman Fred D.
Published: April 2014

Expulsion of two gastrointestinal nematode parasites, Nippostrongylus brasiliensis and Trichinella spiralis, is similar in that both require IL-4Rα expression, but different in that T cells and mast cells are required for IL-4-induced expulsion of T. spiralis but not N. brasiliensis. To examine the role of IL-4Rα signaling in immunity to these parasites, we studied worm expulsion in chimeric mice that selectively expressed IL-4Rα on bone marrow-derived or non-bone marrow-derived cells. N. brasiliensis was expelled by mice that expressed IL-4Rα only on non-bone marrow-derived cells, but not by mice that expressed IL-4Rα only on bone marrow-derived cells. Although T. spiralis expulsion required IL-4Rα expression by both bone marrow- and non-bone marrow-derived cells, IL-4 stimulation eliminated the requirement for IL-4Rα expression by bone marrow-derived cells. Thus, direct IL-4Rα signaling of nonimmune gastrointestinal cells may be generally required to induce worm expulsion, even when mast cell and T cell responses are also required.
Cutting Edge: Interactions Through the IL-10 Receptor Regulate Autoimmune Diabetes
Authors: Phillips Jenny M. , Parish Nicole M. , Drage Martin , Cooke Anne
Published: April 2014

BDC2.5/nonobese diabetic (NOD) transgenic mice express a TCR from a diabetogenic T cell clone yet do not spontaneously develop diabetes at high incidence. Evidence exists showing that in the absence of endogenous TCR α-chain rearrangements this transgenic mouse spontaneously develops diabetes and that CTLA-4 negatively regulates diabetes onset. This strongly suggests that onset of diabetes in BDC2.5/NOD mice is governed by T cell regulation. We addressed the mechanism of immune regulation in BDC2.5/NOD mice. We find that activated spleen cells from young, but not old, BDC2.5/NOD mice are able to transfer diabetes to NOD-scid recipients. We have used anti-IL-10R to show that the failure of splenocytes from older mice to transfer diabetes is due to dominant regulation. We furthermore found that diabetes developed following anti-IL-10R treatment of 6-wk old BDC2.5/NOD mice indicating that endogenous IL-10 plays a key role in the regulation of diabetes onset in this transgenic mouse.
IL-3 Induces B7.2 (CD86) Expression and Costimulatory Activity in Human Eosinophils
Authors: Celestin Jocelyn , Rotschke Olaf , Falk Kirsten , Ramesh Narayanaswamy , Jabara Haifa , Strominger Jack , Geha Raif S.
Published: April 2014

Eosinophils in tissues are often present in intimate contact with T cells in allergic and parasitic diseases. Resting eosinophils do not express MHC class II proteins or costimulatory B7 molecules and fail to induce proliferation of T cells to Ags. IL-5 and GM-CSF induce MHC class II and B7 expression on eosinophils and have been reported in some studies to induce eosinophils to present Ag to T cells. The cytokine IL-3, like IL-5 and GM-CSF, is a survival and activating factor for eosinophils and the IL-3 receptor shares with the IL-5 and GM-CSF receptors a common signal transducing β-chain. IL-3-treated eosinophils expressed HLA-DR and B7.2, but not B7.1 on their surface and supported T cell proliferation in response to the superantigen toxic shock syndrome toxin 1, as well as the proliferation of HLA-DR-restricted tetanus toxoid (TT) and influenza hemagglutinin-specific T cell clones to antigenic peptides. This was inhibited by anti-B7.2 mAb. In contrast, IL-3-treated eosinophils were unable to present native TT Ag to either resting or TT-specific cloned T cells. In parallel experiments, eosinophils treated with IL-5 or GM-CSF were also found to present superantigen and antigenic peptides, but not native Ag, to T cells. These results suggest that eosinophils are deficient in Ag processing and that this deficiency is not overcome by cytokines that signal via the β-chain. Nevertheless, our findings suggest that eosinophils activated by IL-3 may contribute to T cell activation in allergic and parasitic diseases by presenting superantigens and peptides to T cells.
Tumor-Specific T Cell Activation by Recombinant Immunoreceptors: CD3ζ Signaling and CD28 Costimulation Are Simultaneously Required for Efficient IL-2 Secretion and Can Be Integrated Into One Combined CD28/CD3ζ Signaling Receptor Molecule
Authors: Hombach Andreas , Wieczarkowiecz Anja , Marquardt Thomas , Heuser Claudia , Usai Loretta , Pohl Christoph , Seliger Barbara , Abken Hinrich
Published: April 2014

Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA+ colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3ζ (BW431/26-scFv-Fc-CD3ζ), or FcεRIγ (BW431/26-scFv-Fc-γ). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the FcεRIγ or CD3ζ signaling receptor lysed specifically CEA+ tumor cells and secreted high amounts of IFN-γ upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with γ- or ζ-signaling receptors, but enhanced both IFN-γ secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-γ receptor-grafted T cells. Both purified CD4+ and CD8+ T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3ζ signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3ζ) with dual signaling properties. T cells grafted with the combined CD28/CD3ζ signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.
CD8+ T Lymphocytes in Double αβ TCR Transgenic Mice. I. TCR Expression and Thymus Selection in the Absence or in the Presence of Self-Antigen
Authors: Legrand Nicolas , Freitas Antonio A.
Published: April 2014

We derived Rag2-deficient mice bearing two rearranged αβ TCR transgenes, one specific for the HY male Ag and the second specific for the gp33-41 peptide of lymphocytic choriomeningitis virus, both restricted to the MHC H-2Db class I molecule. We found that, in female double transgenic (DTg) mice, most CD8 T cells express only the TCRβ chain from the aHY transgene. By comparing the mRNA species for both β-chains, we observed that in T cells from DTg mice the aHY TCRβ chain transcripts are abundant, whereas the anti-lymphocytic choriomeningitis virus TCRβ chain transcripts are rare. In contrast to TCRβ chain expression, most of the T cells from DTg mice express two TCRα chains. We examined the thymus selection of the dual-receptor CD8 T cells in the presence of self-Ag. We found that the presence of a second TCRα chain allows a significant number of CD8 T cells expressing a self-reactive receptor to escape central deletion and migrate to the peripheral pools of male mice. Differences in TCR and coreceptor expression between female and male MoaHY and DTg mice suggest that peripheral T cell survival requires an optimal level of signaling, which implies a process of “adaptation” of lymphocyte populations to the host environment.
Rhabdovirus Infection Induces Public and Private T Cell Responses in Teleost Fish
Authors: Boudinot Pierre , Boubekeur Samira , Benmansour Abdenour
Published: April 2014

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian αβ TCRs have been recently characterized in rainbow trout and in several teleost species, but the αβ T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRβ chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VβJβ combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vβ4Jβ1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.
T Cell Costimulation by the TNF Ligand BAFF
Authors: Huard Bertrand , Schneider Pascal , Mauri Davide , Tschopp Jürg , French Lars E.
Published: April 2014

The TNF ligand family member B cell-activating factor belonging to TNF family (BAFF, also called Blys, TALL-1, zTNF-4, or THANK) is an important survival factor for B cells. In this study, we show that BAFF is able to regulate T cell activation. rBAFF induced responses (thymidine incorporation and cytokine secretion) of T cells, suboptimally stimulated through their TCR. BAFF activity was observed on naive, as well as on effector/memory T cells (both CD4+ and CD8+ subsets), indicating that BAFF has a wide function on T cell responses. Analysis of the signal transduced by BAFF into T cells shows that BAFF has no obvious effect on T cell survival upon activation, but is able to deliver a complete costimulation signal into T cells. Indeed, BAFF is sufficient to induce IL-2 secretion and T cell division, when added to an anti-TCR stimulation. This highlights some differences in the BAFF signaling pathway in T and B cells. In conclusion, our results indicate that BAFF may play a role in the development of T cell responses, in addition to its role in B cell homeostasis.
Engagement of the FcεRI Stimulates the Production of IL-16 in Langerhans Cell-Like Dendritic Cells
Authors: Reich Kristian , Heine Andrea , Hugo Sabine , Blaschke Volker , Middel Peter , Kaser Arthur , Tilg Herbert , Blaschke Sabine , Gutgesell Carsten , Neumann Christine
Published: April 2014

Preferential uptake and presentation of IgE-bound allergens by epidermal Langerhans cells (LC) via the high affinity IgE receptor, FcεRI, is regarded as an important mechanism in the induction of cutaneous inflammation in atopic dermatitis. Here, we show that activation of monocyte-derived LC-like dendritic cells (LLDC) through engagement of FcεRI induces the expression of IL-16, a chemoattractant factor for dendritic cells, CD4+ T cells, and eosinophils. We found that ligation of FcεRI on LLDC derived from atopic dermatitis patients that express high levels of FcεRI increases IL-16 mRNA expression and storage of intracellular IL-16 protein and enhances the secretion of mature IL-16 in a biphasic manner. An early release of IL-16 (peak at 4 h) is independent of protein synthesis, while a more delayed release (peak at 12 h) requires protein synthesis and occurs subsequent to the induction of IL-16 mRNA and intracellular accumulation of pro-IL-16. There was evidence that LLDC use caspase-1 to process IL-16, as inhibition of caspase-1, but not of caspase-3, partially prevented the release of IL-16 in response to ligation of FcεRI. In an in vivo model of IgE-dependent LC activation, the atopy patch test, positive skin reactions were also associated with the induction of IL-16 in epidermal dendritic cells. These data indicate that IL-16 released from LC after allergen-mediated activation through FcεRI may link IgE-driven and cellular inflammatory responses in diseases such as atopic dermatitis.
β-Amyloid Fibrils Activate the C1 Complex of Complement Under Physiological Conditions: Evidence for a Binding Site for Aβ on the C1q Globular Regions
Authors: Tacnet-Delorme Pascale , Chevallier Sylvie , Arlaud Gérard J.
Published: April 2014

Previous studies based on the use of serum as a source of C have shown that fibrils of β-amyloid peptides that accumulate in the brain of patients with Alzheimer’s disease have the ability to bind C1q and activate the classical C pathway. The objective of the present work was to test the ability of fibrils of peptide Aβ1–42 to trigger direct activation of the C1 complex and to carry out further investigations on the site(s) of C1q involved in the interaction with Aβ1–42. Using C1 reconstituted from purified C1q, C1r, and C1s, it was shown that Aβ1–42 fibrils trigger direct C1 activation both in the absence of C1 inhibitor and at C1 inhibitor:C1 ratios up to 8:0, i.e., under conditions consistent with the physiological context in serum. The truncated peptide Aβ12–42 and the double mutant (D7N, E11Q) of Aβ1–42 did not yield C1 activation, providing further evidence that the C1 binding site of β-amyloid fibrils is located in the acidic N-terminal 1–11 region of the Aβ1–42 peptide. Binding studies performed using a solid phase assay provided strong evidence that C1q interacts with Aβ1–42 fibrils through its C-terminal globular regions. In contrast to previous studies based on a different experimental design, no significant involvement of the C1q collagen-like domain was detected. These findings were confirmed by additional experiments based on C1 activation and C4 consumption assays. These observations provide direct evidence of the ability of β-amyloid fibrils to trigger activation of the classical C pathway and further support the hypothesis that C activation may be a component of the pathogenesis of Alzheimer’s disease.
The Alternatively Spliced Domain TnFnIII A1A2 of the Extracellular Matrix Protein Tenascin-C Suppresses Activation-Induced T Lymphocyte Proliferation and Cytokine Production
Authors: Puente Navazo Marta D. , Valmori Danila , Rüegg Curzio
Published: April 2014

Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A–D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.
Intramembrane Proteolysis of Signal Peptides: An Essential Step in the Generation of HLA-E Epitopes
Authors: Lemberg Marius K. , Bland Felicity A. , Weihofen Andreas , Braud Veronique M. , Martoglio Bruno
Published: April 2014

Signal sequences of human MHC class I molecules are a unique source of epitopes for newly synthesized nonclassical HLA-E molecules. Binding of such conserved peptides to HLA-E induces its cell surface expression and protects cells from NK cell attack. After cleavage from the pre-protein, we show that the liberated MHC class I signal peptide is further processed by signal peptide peptidase in the hydrophobic, membrane-spanning region. This cut is essential for the release of the HLA-E epitope-containing fragment from the lipid bilayer and its subsequent transport into the lumen of the endoplasmic reticulum via the TAP.
Trypsin Induces Activation and Inflammatory Mediator Release from Human Eosinophils Through Protease-Activated Receptor-2
Authors: Miike Satoshi , McWilliam Andrew S. , Kita Hirohito
Published: April 2014

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50∼70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.
A Ligand for the Chemokine Receptor CCR7 Can Influence the Homeostatic Proliferation of CD4 T Cells and Progression of Autoimmunity
Authors: Ploix Corinne , Lo David , Carson Monica J.
Published: April 2014

Homeostasis of T cell numbers in the periphery implies an ability of lymphocytes to sense cell numbers. Although the mechanisms are unknown, we find that the chemokine CCL21 (also known as TCA4, SLC, 6Ckine), a ligand for the chemokine receptor CCR7, can regulate homeostasis of CD4 (but not CD8) T cells. In the absence of CCR7 ligands, transferred CD4 T cells failed to expand in lymphopenic hosts, whereas in the presence of CCL21 overexpression, homeostatic CD4 T cell proliferation occurred even in nonlymphopenic recipients. Ag-specific CD4 T cells transferred into Ag-expressing mice proliferated and induced autoimmunity only in lymphopenic recipients. Pancreatic expression of CCL21 was sufficient to replace the requirement for lymphopenia in the progression of autoimmune disease. These results suggest that CD4 T cells use local concentrations of CCR7 ligands as an index of T cell steady state numbers and that homeostatic expansion of the T cell population may be a contributing factor in the development of autoimmune disease.
Cell Surface Targeting of Heat Shock Protein gp96 Induces Dendritic Cell Maturation and Antitumor Immunity
Authors: Zheng Hong , Dai Jie , Stoilova Diliana , Li Zihai
Published: April 2014

gp96 is a residential heat shock protein of the endoplasmic reticulum that has been implicated in the activation of dendritic cells (DCs) for the initiation of adaptive immunity. By genetic targeting of gp96 onto the cell surface, we demonstrate that direct access of gp96 to DCs induces their maturation, resulting in secretion of proinflammatory cytokines IL-1β, IL-12, and chemokine monocyte chemoattractant protein-1 and up-regulation of the expression of MHC class I, MHC class II, CD80, CD86, and CD40. Furthermore, surface expression of gp96 on tumor cells renders them regressive via a T lymphocyte-dependent mechanism. This work reinforces the notion that gp96 is an endogenous DC activator and unveils that the context in which Ag is delivered to the immune system, in this case surface expression of gp96, has profound influence on immunity. It also establishes a principle of bridging innate and adaptive immunity for cancer immunotherapy by surface targeting of an intracellular heat shock protein.