bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4+ Th2 Cell Recognition of HLA-DR-Restricted Epitopes Derived from CAMEL: A Tumor Antigen Translated in an Alternative Open Reading Frame
Authors: Slager Elisabeth H. , Borghi Martina , van der Minne Carolien E. , Aarnoudse Corlien A. , Havenga Menzo J. E. , Schrier Peter I. , Osanto Susanne , Griffioen Marieke
Published: April 2014

Tumor Ag NY-ESO-1 is an attractive target for immunotherapy of cancer, since both CD8+ CTL and CD4+ Th cells against NY-ESO-1 have been described. Moreover, NY-ESO-1 as well as the highly homologous tumor Ag LAGE-1 are broadly expressed in various tumor types. Interestingly, the NY-ESO-1 and LAGE-1 genes also encode for proteins translated in an alternative open reading frame. These alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes, respectively, have been demonstrated to be immunogenic, since CTL specific for these proteins have been isolated from melanoma patients. In this study a panel of advanced melanoma patients was screened for the presence of Th cells specific for the alternatively translated tumor Ags NY-ESO-ORF2 and CAMEL. PBMC of melanoma patients were stimulated for 4 days with mixes of overlapping peptides covering the entire NY-ESO-ORF2 and CAMEL protein sequences and were tested for the release of type 1 (IFN-γ) and type 2 (IL-13) cytokines in ELISPOT assays. In three of 15 patients, T cells specific for two CAMEL peptides (CAMEL71–92 and CAMEL81–102) could be detected. From one of these patients, CD4+ T cell clones specific for CAMEL81–102 could be generated. These clones recognized a naturally processed epitope presented in both HLA-DR11 and HLA-DR12 and produced high levels of IL-4, IL-5, and IL-13. In conclusion, this study shows the presence of Th cells specific for the alternatively translated tumor Ag CAMEL in melanoma patients and is the first report that describes the isolation of tumor Ag-specific CD4+ Th 2 clones.
A Novel Anti-Inflammatory Role for Simvastatin in Inflammatory Arthritis
Authors: Leung Bernard P. , Sattar Naveed , Crilly Anne , Prach Morag , McCarey David W. , Payne Helen , Madhok Rajan , Campbell Carol , Gracie J. Alastair , Liew Foo Y. , McInnes Iain B.
Published: April 2014

3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exert favorable effects on lipoprotein metabolism, but may also possess anti-inflammatory properties. Therefore, we explored the activities of simvastatin, a lipophilic statin, in a Th1-driven model of murine inflammatory arthritis. We report in this study that simvastatin markedly inhibited not only developing but also clinically evident collagen-induced arthritis in doses that were unable to significantly alter cholesterol concentrations in vivo. Ex vivo analysis demonstrated significant suppression of collagen-specific Th1 humoral and cellular immune responses. Moreover, simvastatin reduced anti-CD3/anti-CD28 proliferation and IFN-γ release from mononuclear cells derived from peripheral blood and synovial fluid. Proinflammatory cytokine production in vitro by T cell contact-activated macrophages was suppressed by simvastatin, suggesting that such observations have direct clinical relevance. These data clearly illustrate the therapeutic potential of statin-sensitive pathways in inflammatory arthritis.
The In Vitro and In Vivo Effects of Anti-Galactose Antibodies on Endothelial Cell Activation and Xenograft Rejection
Authors: Xu Hui , Yin Dengping , Naziruddin Bashoo , Chen Libing , Stark Aileen , Wei Yuanyuan , Lei Ying , Shen JiKun , Logan John S. , Byrne Guerard W. , Chong Anita S.-F.
Published: April 2014

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the α-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal−/− mice show a range of affinity for the α-Gal epitope, and that affinity was generally increased as the VH gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced α-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.
Increased Bone Mass Is a Part of the Generalized Lymphoproliferative Disorder Phenotype in the Mouse
Authors: Katavić Vedran , Lukić Ivan Krešimir , Kovačić Nataša , Grčević Danka , Lorenzo Joseph A. , Marušić Ana
Published: April 2014

We investigated the bone phenotype of mice with generalized lymphoproliferative disorder (gld) due to a defect in the Fas ligand-mediated apoptotic pathway. C57BL/6-gld mice had greater whole body bone mineral density and greater trabecular bone volume than their wild-type controls. gld mice lost 5-fold less trabecular bone and had less osteoclasts on bone surfaces after ovariectomy-induced bone resorption. They also formed more bone in a model of osteogenic regeneration after bone marrow ablation, had less osteoclasts on bone surfaces and less apoptotic osteoblasts. gld and wild-type mice had similar numbers of osteoclasts in bone marrow cultures, but marrow stromal fibroblasts from gld mice formed more alkaline phosphatase-positive colonies. Bone diaphyseal shafts and bone marrow stromal fibroblasts produced more osteoprotegerin mRNA and protein than wild-type mice. These findings provide evidence that the disturbance of the bone system is a part of generalized lymphoproliferative syndrome and indicates the possible role of osteoprotegerin as a regulatory link between the bone and immune system.
Peptide Nucleic Acid Antisense Prolongs Skin Allograft Survival by Means of Blockade of CXCR3 Expression Directing T Cells into Graft
Authors: Jiankuo Ming , Xingbing Wang , Baojun Huang , Xiongwin Wu , Zhuoya Li , Ping Xiong , Yong Xu , Anting Liu , Chunsong Hu , Feili Gong , Jinquan Tan
Published: April 2014

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-γ-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-γ/CXCL9. It was reported that CXC chemokines IFN-γ-inducible protein 10/CXCL10 and monokine induced by IFN-γ/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 ± 2.4 days) compared with physiological saline treatment (7.5 ± 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 ± 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.
Normal Tissue Depresses While Tumor Tissue Enhances Human T Cell Responses In Vivo to a Novel Self/Tumor Melanoma Antigen, OA1
Authors: Touloukian Christopher E. , Leitner Wolfgang W. , Schnur Rhonda E. , Robbins Paul F. , Li Yong , Southwood Scott , Sette Alessandro , Rosenberg Steven A. , Restifo Nicholas P.
Published: April 2014

Antitumor T cells often recognize targets that are nonmutated “self” tissue differentiation Ags, but the relative impact of Ag expression by normal and transformed tissue for a human self/tumor Ag has not been studied. To examine the influence of self-tolerance mechanisms on the function of self/tumor-specific T cell responses in humans, we sought to identify an Ag that was expressed, processed, and presented in an MHC-restricted fashion by tumor cells, but for which there was the human equivalent of a “knockout.” In this study, we report the first immunological characterization of a melanoma/melanocyte differentiation Ag, called OA1, which meets these criteria. This Ag, an X chromosome-encoded melanoma/melanocyte differentiation Ag, was completely deleted in a male patient. Using a newly identified HLA-A*2402-restricted epitope (LYSACFWWL) to study T cell tolerance, we found that OA1-specific T cell reactivity was more than five SD higher in the knockout patient that in normal controls. These data provide compelling evidence for T cell tolerance to OA1 in humans. Most surprisingly, we found elevated levels of OA1-specific T cells in patients with metastatic malignant melanoma, indicating that the tumor-bearing state partially reversed tolerance observed in normal (non-“knockout”) individuals. Taken together, these findings indicated that tolerance can exist for self/tumor Ags in humans, and that this tolerance could be partially abrogated by the growth of the tumor, increasing the reactivity of tumor Ag-specific T cells. Thus, the tumor-bearing state reverses, in part, the tolerance of T cells that results from the normal expression of tissue differentiation Ags.
Influence of Coinfecting Pathogens on HIV Expression: Evidence for a Role of Toll-Like Receptors
Authors: Báfica André , Scanga Charles A. , Schito Marco , Chaussabel Damien , Sher Alan
Published: April 2014

Immune activation of HIV gene expression as a consequence of the host response to coinfecting pathogens has been implicated as an important factor in AIDS progression. Immune responsiveness to many of the infectious agents associated with HIV has been demonstrated to depend on a family of innate recognition molecules, known as Toll-like receptors (TLR). Therefore, TLR-pathogen interactions could play an indirect role in regulating HIV-associated disease. In this review, we summarize emerging evidence for the influence of TLR recognition on HIV gene activation and AIDS progression.
Expression Characteristics and Stimulatory Functions of CD43 in Human CD4+ Memory T Cells: Analysis Using a Monoclonal Antibody to CD43 That Has a Novel Lineage Specificity
Authors: Kyoizumi Seishi , Ohara Takaaki , Kusunoki Yoichiro , Hayashi Tomonori , Koyama Kazuaki , Tsuyama Naohiro
Published: April 2014

We have used HSCA-2, an mAb that recognizes a sialic acid-dependent epitope on the low molecular mass (∼115-kDa) glycoform of CD43 that is expressed in resting T and NK cells, to examine the expression characteristics and stimulatory functions of CD43 in human CD4+ memory T cells. Having previously reported that the memory cells that respond to recall Ags in a CD4+CD45RO+ T cell population almost all belong to a subset whose surface CD43 expression levels are elevated, we now find that exposing these same memory T cells to HSCA-2 mAb markedly increases their proliferative responsiveness to recall Ags. We think it unlikely that this increase in responsiveness is a result of CD43-mediated monocyte activation, especially given that the HSCA-2 mAb differs from all previously used CD43 mAbs in having no obvious binding specificity for monocyte CD43. Predictably, treatment with HSCA-2 mAb did not lead to significant recall responses in CD4+CD45RO+ T cells, whose CD43 expression levels were similar to or lower than those of naive cells. Other experiments indicated that the HSCA-2 mAb was capable of enhancing the proliferative responsiveness of CD4+ memory T cells that had been exposed to polyclonal stimulation by monocyte-bound CD3 mAb and could also act in synergy with CD28 mAb to enhance the responsiveness of CD4+ T cells to CD3 stimulation. Taken together, these findings suggest that the CD43 molecules expressed on CD4+ memory T cells may be capable of enhancing the costimulatory signaling and hence providing accessory functions to TCR-mediated activation processes.
c-Maf and JunB Mediation of Th2 Differentiation Induced by the Type 2 G Protein-Coupled Receptor (VPAC2) for Vasoactive Intestinal Peptide
Authors: Voice Julia , Donnelly Samantha , Dorsam Glenn , Dolganov Gregory , Paul Sudhir , Goetzl Edward J.
Published: April 2014

Vasoactive intestinal peptide and its G protein-coupled receptors, VPAC1 and VPAC2, regulate critical aspects of innate and adaptive immunity. T cell VPAC2Rs mediate changes in cytokine generation, which potently increase the Th2/Th1 ratio and consequently shift the effector responses toward allergy and inflammation. To examine mechanisms of VPAC2 promotion of the Th2 phenotype, we analyzed controls of IL-4 transcription in CD4 T cells from T cell-targeted VPAC2 transgenic (Tg), VPAC2 knockout, and wild-type (WT) mice. c-maf and junB mRNA, protein, and activity were significantly up-regulated to a higher level in TCR-stimulated CD4 T cells from Tg mice compared with those from knockout and WT C57BL/6 mice. In contrast, GATA3, T-bet, and NFATc levels were identical in WT and Tg CD4 T cells. Vasoactive intestinal peptide binding to VPAC2 on CD4 T cells specifically induces an up-regulation of the Th2-type transcription factors c-Maf and JunB, which consequently enhances IL-4 and IL-5 production, leading to a Th2-type phenotype.
Expression of Human CD1d Molecules Protects Target Cells from NK Cell-Mediated Cytolysis
Authors: Campos-Martín Yolanda , Gómez del Moral Manuel , Gozalbo-López Beatriz , Suela Javier , Martínez-Naves Eduardo
Published: April 2014

The cytotoxic activity of NK cells can be inhibited by classical and nonclassical MHC molecules. The CD1 system is formed by a family of glycoproteins that are related to classical MHC. CD1a, b, and c molecules present lipids or glycolipids to T cells and are involved in defense against microbial infections, especially mycobacteria. It has been shown recently that these molecules can inhibit target cell lysis by human NK cells. It has also been shown that mouse CD1d molecules can protect cells from NK cell-mediated cytotoxicity. In the present study, we describe how human CD1d, orthologous to murine CD1 molecules, can inhibit NK cell-mediated cytolysis. We have expressed CD1d in the HLA class I-deficient cell lines L721.221 and C1R. The inhibitory effect is observed when effector NK cells from different donors are used, as well as in different cell lines with NK activity. The inhibitory effect was reversed by incubating the target cells with a mAb specific for human CD1d. Incubation of target cells with the ligands for CD1d, α-galactosylceramide (α-GalCer), and β-GalCer abolishes the protective effect of CD1d in our in vitro killing assays. Staining the effector cells using CD1d tetramers loaded with α-GalCer was negative, suggesting that the putative inhibitory receptor does not recognize CD1d molecules loaded with α-GalCer.
Costimulation via Glucocorticoid-Induced TNF Receptor in Both Conventional and CD25+ Regulatory CD4+ T Cells
Authors: Kanamaru Fumiko , Youngnak Pornpan , Hashiguchi Masaaki , Nishioka Tomohisa , Takahashi Takeshi , Sakaguchi Shimon , Ishikawa Isao , Azuma Miyuki
Published: April 2014

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25− CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25−CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-γ, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-κB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.
Elevated TGF-β1 Secretion and Down-Modulation of NKG2D Underlies Impaired NK Cytotoxicity in Cancer Patients
Authors: Lee June-Chul , Lee Kyung-Mi , Kim Dong-Wan , Heo Dae Seog
Published: April 2014

NK cell function in cancer patients is severely impaired, but the mechanism underlying this impairment is not clearly understood. In this study we show evidence that TGF-β1 secreted by tumors is responsible for the poor NK lytic activity via down-regulating an NK-activating receptor, NKG2D. The plasma level of TGF-β1 in human lung cancer or colorectal cancer patients was elevated compared with that in normal volunteers, and this elevation was inversely correlated with surface expression of NKG2D on NK cells in these patients. Incubation of NK cells with plasma obtained from cancer patients specifically down-modulated surface NKG2D expression, whereas addition of neutralizing anti-TGF-β1 mAbs completely restored surface NKG2D expression. Likewise, incubation of NK cells and lymphokine-activated killer cells with TGF-β1 resulted in dramatic reduction of surface NKG2D expression associated with impaired NK cytotoxicity. Modulation of NKG2D by TGF-β1 was specific, as expression of other NK receptors, CD94/NKG2A, CD44, CD16, 2B4, or CD56, was not affected by TGF-β1. Impaired NK cytotoxicity by TGF-β1 was not due to alteration of lytic moieties, such as perforin or Fas, or apoptotic pathway, but, rather, appeared to be due to lack of NKG2D expression. Taken together, our data suggest that impaired NK function in cancer patients can be attributed to down-modulation of activating receptors, such as NKG2D, via secretion of TGF-β1.
Peptides Identified through Phage Display Direct Immunogenic Antigen to Dendritic Cells
Authors: Curiel Tyler J. , Morris Cindy , Brumlik Michael , Landry Samuel J. , Finstad Kristiaan , Nelson Anne , Joshi Virendra , Hawkins Christopher , Alarez Xavier , Lackner Andrew , Mohamadzadeh Mansour
Published: April 2014

Dendritic cells (DC) play a critical role in adaptive immunity by presenting Ag, thereby priming naive T cells. Specific DC-binding peptides were identified using a phage display peptide library. DC-peptides were fused to hepatitis C virus nonstructural protein 3 (NS3) while preserving DC targeting selectivity and Ag immunogenicity. The NS3-DC-peptide fusion protein was efficiently presented to CD4+ and CD8+ T cells derived from hepatitis C virus-positive blood cells, inducing their activation and proliferation. This immunogenic fusion protein was significantly more potent than NS3 control fusion protein or NS3 alone. In chimeric NOD-SCID mice transplanted with human cells, DC-targeted NS3 primed naive CD4+ and CD8+ T cells for potent NS3-specific proliferation and cytokine secretion. The capacity of peptides to specifically target immunogenic Ags to DC may establish a novel strategy for vaccine development.
CD27 Is Acquired by Primed B Cells at the Centroblast Stage and Promotes Germinal Center Formation
Authors: Xiao Yanling , Hendriks Jenny , Langerak Petra , Jacobs Heinz , Borst Jannie
Published: April 2014

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B cells. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28−/− mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by CD28+ Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation.
Lymphotoxin-β Receptor Activation by Activated T Cells Induces Cytokine Release from Mouse Bone Marrow-Derived Mast Cells
Authors: Stopfer Peter , Männel Daniela N. , Hehlgans Thomas
Published: April 2014

Lymphotoxin-β receptor (LTβR) signaling is known to play a key role in embryonic lymphoid organ formation as well as maintenance of lymphoid architecture. Activation of the LTβR is induced by either the heterotrimeric lymphotoxin-α1β2 (LTα1β2) or the homotrimeric LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV gpD for herpes virus entry mediator, a receptor expressed by T lymphocyte). Both ligands are expressed on activated lymphocytes. As mast cells reside in close proximity to activated T cells in some inflammatory tissues, we examined the expression of LTβR on bone marrow-derived mast cells and asked whether the LTβR-ligand interaction would allow communication between mast cells and activated T cells. We found that mast cells express LTβR at the mRNA as well as at the protein level. To investigate LTβR-specific mast cell activation, the LTβR on BMMC from either wild-type or LTβR-deficient mice was stimulated with recombinant mouse LIGHT or agonistic mAbs in the presence of ionomycin. LTβR-specific release of the cytokines IL-4, IL-6, TNF, and the chemokines macrophage inflammatory protein 2 and RANTES was detected. Moreover, coculture of mast cells with T cells expressing the LTβR ligands also entailed the release of these cytokines. Interference with a specific LTβR inhibitor resulted in significant suppression of mast cell cytokine release. These data clearly show that LTβR expressed on mast cells can transduce a costimulatory signal in T cell-dependent mast cell activation.
MHC-Restricted Ig V Region-Driven T-B Lymphocyte Collaboration: B Cell Receptor Ligation Facilitates Switch to IgG Production
Authors: Munthe Ludvig A. , Os Audun , Zangani Michael , Bogen Bjarne
Published: April 2014

B cells spontaneously process their endogenous Ig and present V region peptides on their MHC class II molecules. We have here investigated whether B cells collaborate with V region-specific CD4+ T cells in vivo. By use of paired Ig L chain-transgenic and TCR-transgenic mice and cell transfer into normal hosts, we demonstrate that B cell presentation of a VL region peptide to CD4+ T cells results in germinal centers, plasma cells, and Ab secretion. Because the transgenic B cells have a fixed L chain but polyclonal H chains, their B cell receptor (BCR) repertoire is diverse and may bind a multitude of ligands. In a hapten-based system, BCR ligation concomitant with V region-driven T-B collaboration induced germinal center formation and an IgM → IgG isotype switch. In the absence of BCR ligation, mainly IgM was produced. Consistent with this, prolonged V region-driven T-B collaboration resulted in high titers of IgG autoantibodies against ubiquitous self-Ags, while natural-type Abs against exotic bacteria remained IgM. Taken together, V region-driven T-B collaboration may explain induction of natural IgM Abs (absence of BCR ligation) and IgG autoantibodies (BCR ligation by autoantigen) and may be involved in the development of autoimmunity.
Rat Plasmacytoid Dendritic Cells Are an Abundant Subset of MHC Class II+ CD4+CD11b−OX62− and Type I IFN-Producing Cells That Exhibit Selective Expression of Toll-Like Receptors 7 and 9 and Strong Responsiveness to CpG
Authors: Hubert Francois-Xavier , Voisine Cécile , Louvet Cédric , Heslan Michèle , Josien Régis
Published: April 2014

We have identified in the rat a new subset of MHC class II+ CD4+CD3−CD11b− leukocytes that produce high amounts of type I IFN upon viral stimulation and that appeared homologous to plasmacytoid DC (pDC) previously described in humans and mice. These cells exhibited the following phenotype: CD5+,CD90+,CD45R+,CD45RC+,CD11c−,CD161a+,CD200+,CD172a+,CD32+,CD86+. Rat pDC did not express the DC-specific marker OX62 and were more abundant in the spleen than the classical CD4+ and CD4− subsets of OX62+CD11b+ DC we previously described that produced very little, if any, type I IFN. Spleen pDC exhibited an undifferentiated morphology and rapidly died in vitro, but showed extensive dendrite formation, survival, maturation, and moderate type I IFN production upon stimulation by oligonucleotides containing type B CpG motifs (CpG ODN). Type A CpG ODN and CD40 ligand induced pDC to produce large amounts of type I IFN, but did not promote maturation. CpG ODN and CD40 ligand, but not influenza virus, induced IL-12p40 and IL-6 secretion. Spleen pDC did not produce IL-12p70, TNF-α, IL-1β, or IL-10 using these stimulation conditions. Correlating with their strong responsiveness to virus and CpG ODN, rat pDC specifically expressed Toll-like receptor 7 and 9 mRNA. Fresh spleen pDC were poor stimulators of allogenic CD4+ and CD8+ T cells, but became potent inducers of allogenic T cell proliferation as well as Th1 differentiation after stimulation by type B CpG. Therefore, rat pDC appear very similar to human pDC, indicating that the specific phenotype and functions of pDC have been highly conserved between species.
Repression of IFN-γ Expression by Peroxisome Proliferator-Activated Receptor γ
Authors: Cunard Robyn , Eto Yoko , Muljadi Julie T. , Glass Christopher K. , Kelly Carolyn J. , Ricote Mercedes
Published: April 2014

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in a wide variety of cells. Our studies and others have demonstrated that both human and murine T cells express PPARγ and that expression can be augmented over time in mitogen-activated splenocytes. PPARγ ligands decrease proliferation and IL-2 production, and induce apoptosis in both B and T cells. PPARγ ligands have also been shown to be anti-inflammatory in multiple models of inflammatory disease. In the following study, we demonstrate for the first time that PPARγ is expressed in both murine CD4 and CD8 cells and that PPARγ ligands directly decrease IFN-γ expression by murine and transformed T cell lines. Unexpectedly, GW9662, a PPARγ antagonist, increases lymphocyte IFN-γ expression. Transient transfection studies reveal that PPARγ ligands, in a PPARγ-dependent manner, potently repress an IFN-γ promoter construct. Repression localizes to the distal conserved sequence of the IFN-γ promoter. Our studies also demonstrate that PPARγ acts on the IFN-γ promoter by interfering with c-Jun activation. These studies suggest that many of the observed anti-inflammatory effects of PPARγ ligands may be related to direct inhibition of IFN-γ by PPARγ.
Bap29/31 Influences the Intracellular Traffic of MHC Class I Molecules
Authors: Paquet Marie-Eve , Cohen-Doyle Myrna , Shore Gordon C. , Williams David B.
Published: April 2014

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with β2-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.
Binding of Cytoplasmic Proteins to the CD19 Intracellular Domain Is High Affinity, Competitive, and Multimeric
Authors: Brooks Stephen R. , Kirkham Perry M. , Freeberg Larry , Carter Robert H.
Published: April 2014

CD19 is required for the development of B1 and marginal zone B cells, for Ab responses, and for B cell memory. CD19 immunoprecipitates contain a complex of cytoplasmic proteins, including Lyn, Vav, phospholipase Cγ2 (PLCγ2), Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Which of these bind directly to CD19 and the strengths of the interactions are unknown. These issues are important in understanding the signaling functions of CD19, which are crucial for normal B cell physiology. Using purified, recombinant proteins, we now show that each of these signaling proteins contains at least one Src homology 2 (SH2) domain that interacts directly with the phosphorylated CD19 cytoplasmic domain. The affinities of binding of the SH2 domains of Vav, p85, and Grb2 to CD19 are each in the nanomolar range by surface plasmon resonance (Biacore) analysis. Binding of Lyn and PLCγ2 do not fit 1:1 modeling. However, analyses of binding data (Lyn) and competition experiments (PLCγ2) suggest that these bind with comparable affinity. Competition experiments demonstrate that SH2 domains whose binding is dependent on the same CD19 tyrosine(s) compete for binding, but these SH2 domains do not impede binding of different SH2 domains to other CD19 tyrosines. We conclude that binding to the CD19 cytoplasmic domain is multimeric, high affinity, and competitive. The high affinity of the interactions also suggests that tyrosines that were nonessential in vivo are nevertheless functional. A preliminary structural model suggests that CD19 forms a signaling complex containing multiple cytoplasmic proteins in close proximity to each other and to the plasma membrane.
Protection against Progressive Leishmaniasis by IFN-β
Authors: Mattner Jochen , Wandersee-Steinhäuser Alexandra , Pahl Andreas , Röllinghoff Martin , Majeau Gerard R. , Hochman Paula S. , Bogdan Christian
Published: April 2014

Type I IFNs (IFN-αβ) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-β protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (106) or low (104) number of Leishmania major parasites, respectively. IFN-β treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-γ and IL-12 in the draining lymph node. Low, but not high doses of IFN-β caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-β-induced increase of IFN-γ production was dependent on STAT4. Protection by IFN-β strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-β led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-β as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.
Neuroregulatory Events Follow Adaptive Immune-Mediated Elimination of HIV-1-Infected Macrophages: Studies in a Murine Model of Viral Encephalitis
Authors: Poluektova Larisa , Gorantla Santhi , Faraci Jill , Birusingh Kevin , Dou Huanyu , Gendelman Howard E.
Published: April 2014

HIV-1-specific cellular immunity serves to eliminate infected cells and disease. However, how this process specifically affects the CNS is poorly understood. To mirror the regulatory events that occur in human brain after HIV-1 infection, a murine model of viral encephalitis was used to study relationships, over time, among lymphocyte-mediated infected cell elimination, innate immune responses, and neuropathology. Nonobese diabetic SCID mice were reconstituted with human PBL and a focal encephalitis induced by intracranial injection of autologous HIV-1-infected, monocyte-derived macrophages (MDM). On days 7, 14, and 21 after MDM injection into the basal ganglia, the numbers of human lymphocytes and mouse monocytes, virus-infected MDM, glial (astrocyte and microglial) responses, cytokines, inducible NO (iNOS), neurotrophic factors, and neuronal Ags were determined in brain by immunohistochemistry, real-time PCR, and Western blot assays. Microglia activation, astrocytosis, proinflammatory cytokines, and iNOS expression accompanied the loss of neuronal Ags. This followed entry of human lymphocytes and mouse monocytes into the brain on days 7 and 14. Elimination of virus-infected human MDM, expression of IL-10, neurotropins, and a down-regulation of iNOS coincided with brain tissue restoration. Our results demonstrate that the degree of tissue damage and repair parallels the presence of infected macrophages and effectors of innate and adaptive immunity. This murine model of HIV-1 encephalitis can be useful in elucidating the role played by innate and adaptive immunity in disease progression and resolution.
Differential Immune Responses and Protective Efficacy Induced by Components of a Tuberculosis Polyprotein Vaccine, Mtb72F, Delivered as Naked DNA or Recombinant Protein
Authors: Skeiky Yasir A. W. , Alderson Mark R. , Ovendale Pamela J. , Guderian Jeffrey A. , Brandt Lise , Dillon Davin C. , Campos-Neto Antonio , Lobet Yves , Dalemans Wilfried , Orme Ian M. , Reed Steven G.
Published: April 2014

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32C-Mtb39-Mtb32N. Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-γ responses directed against the first two components of the polyprotein and a strong CD8+ T cell response directed exclusively against Mtb32C. In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-γ response and a weak CD8+ T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-γ and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8+ response directed against the same Mtb32C epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.
WSX-1: A Key Role in Induction of Chronic Intestinal Nematode Infection
Authors: Bancroft Allison J. , Humphreys Neil E. , Worthington John J. , Yoshida Hiroki , Grencis Richard K.
Published: April 2014

Chronic infection by the gastrointestinal nematode Trichuris muris in susceptible AKR mice, which mount a Th1 response, is associated with IL-27p28 expression in the cecum. In contrast to wild-type mice, mice that lack the WSX-1/IL-27R gene fail to harbor a chronic infection, having significantly lower Th1 responses. The lower level of Ag-specific IFN-γ-positive cells in WSX-1 knockout (KO) mice was found to be CD4+ T cell specific, and the KO mice also had increased levels of IL-4-positive CD4+ T cells. Polyclonal activation of mesenteric lymph node cells from naive WSX-1 KO or wild-type mice demonstrated that there was no inherent defect in the production of IFN-γ by CD4+ T cells, suggesting the decrease in these cells seen in infected WSX-1 KO mice is an in vivo Ag-driven effect. IL-12 treatment of WSX-1 KO mice failed to rescue the type 1 response, resulting in unaltered type-2-driven resistance. Infection of WSX-1 KO mice was also associated with a reduction of IL-27/WSX-1 downstream signaling gene expression within the cecum. These studies demonstrate an important role for WSX-1 signaling in the promotion of type 1 responses and chronic gastrointestinal nematode infection.
Rac1 and Toll-IL-1 Receptor Domain-Containing Adapter Protein Mediate Toll-Like Receptor 4 Induction of HIV-Long Terminal Repeat
Authors: Equils Ozlem , Madak Zeynep , Liu Chunrin , Michelsen Kathrin S. , Bulut Yonca , Lu Daning
Published: April 2014

Opportunistic infections, common in HIV-1-infected patients, increase HIV replication; however, the intracellular signaling mechanisms involved are not clearly known. We have shown that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate microbial Ag-induced HIV-long terminal repeat (HIV-LTR) trans-activation and HIV-1 replication, and that LPS-induced HIV-LTR trans-activation is mediated through myeloid differentiation adapter protein. Recently, Toll-IL-1R domain-containing adapter protein (TIRAP) has been identified as an adapter molecule that mediates responses to TLR2 and TLR4 ligands, and TIRAP was suggested to provide signaling specificity for different TLRs. Rac1, a small GTP-binding protein that is activated upon LPS stimulation of macrophages, activates phosphatidylinositol 3-kinase and Akt and leads to NF-κB activation. The roles of Rac1 and TIRAP in LPS activation of HIV replication is not known. In the present study we show that LPS stimulation of human microvessel endothelial cells leads to Rac1 activation. Constitutively active Rac1 (Rac1V12) simulated the effect of LPS to activate HIV-LTR, whereas the expression of dominant negative Rac1 (Rac1N17) partially blocked LPS-induced HIV-LTR trans-activation. Rac1V12-induced HIV-LTR activation was independent of myeloid differentiation adapter protein, and dominant negative TIRAP blocked Rac1V12-induced HIV-LTR trans-activation. In this study we show for the first time that activation of Rac1 leads to HIV-LTR trans-activation, and this is mediated through TIRAP. Together these results underscore the importance of Rac1 and TIRAP in TLR4 activation of HIV replication and help delineate the signaling pathways induced by TLRs to mediate microbial Ag-induced HIV replication and HIV pathogenesis.
CCR2-Dependent Trafficking of F4/80dim Macrophages and CD11cdim/intermediate Dendritic Cells Is Crucial for T Cell Recruitment to Lungs Infected with Mycobacterium tuberculosis
Authors: Peters Wendy , Cyster Jason G. , Mack Matthias , Schlöndorff Detlef , Wolf Andrea J. , Ernst Joel D. , Charo Israel F.
Published: April 2014

We previously reported that CCR2−/− mice are susceptible to Mycobacterium tuberculosis infection. Susceptibility was associated with an early and sustained macrophage trafficking defect, followed by delayed recruitment of dendritic cells (DCs) and T cells to the lungs. However, the relative importance of the lack of CCR2 expression by macrophages and DCs vs T cells in susceptibility to infection was unclear. In this study, we used mixed bone marrow transplantation to create mice in which the genotype of the T cells was either CCR2+/+ or CCR2−/− while maintaining the genotype of the myeloid cells as CCR2+/+. After infection with M. tuberculosis, we found that the genotype of the macrophages and/or DCs, but not that of the T cells, was critical for both T cell and myeloid cell migration to the lungs. Further investigation revealed a critical role for CCR2 in the recruitment of F4/80dim macrophages and CD11cdim/intermediate DCs to the infected lung.
NK Cells and Polymorphonuclear Neutrophils Are Both Critical for IL-2-Induced Pulmonary Vascular Leak Syndrome
Authors: Assier Eric , Jullien Valérie , Lefort Jean , Moreau Jean-Louis , Di Santo James P. , Vargaftig B. Boris , Lapa e Silva Jose R. , Thèze Jacques
Published: April 2014

The mechanism of IL-2-induced vascular leak syndrome (VLS) is still poorly understood. Cells of both innate and adaptive immune systems have been implicated, but no definitive conclusions have been reached concerning their respective roles. In this study we report a new mouse model of IL-2-induced pulmonary VLS used to obtain a detailed analysis of the early events (sequestration of polymorphonuclear neutrophils and bronchoconstriction) and late events (modifications in the cell and protein content of bronchoalveolar lavages, followed by edema) that characterize this lung injury. This model and knockout animals are used to reconsider the importance of the different leukocyte lineages in early and late events. Recombinase-activating gene 2−/− mice are used to demonstrate that adaptive lymphocytes, including NK T cells, are not required for pulmonary VLS induction. By contrast, results obtained with newly described recombinase-activating gene 2−/−/IL-15−/− mice indicate that NK cells play a key role in both early and late events. In parallel, polymorphonuclear neutrophil depletion is used to evaluate the contributions made by these cells to the late alterations occurring in the lung. Furthermore, when used in combination with inhibition of NO synthase, granulocyte depletion was completely effective in protecting mice from the late events of IL-2-induced pulmonary VLS. Together our results indicate that both NK and PMN cells play a central role in the late events of IL-2-induced VLS.
The Transcriptional Activation Program of Human Neutrophils in Skin Lesions Supports Their Important Role in Wound Healing
Authors: Theilgaard-Mönch Kim , Knudsen Steen , Follin Per , Borregaard Niels
Published: April 2014

To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds, we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin lesions. After migration to skin lesions, PMNs demonstrated a significant transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming of PMNs. Among the up-regulated genes were cytokines and chemokines critical for chemotaxis of macrophages, T cells, and PMNs, and for the modulation of their inflammatory responses. PMNs in skin lesions down-regulated receptors mediating chemotaxis and anti-microbial activity, but up-regulated other receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated cytokines/chemokines/enzymes were critical for wound healing. These included the breakdown of fibrin clots and degradation of extracellular matrix, the promotion of angiogenesis, the migration and proliferation of keratinocytes and fibroblasts, the adhesion of keratinocytes to the dermal layer, and finally, the induction of anti-microbial gene expression in keratinocytes. Notably, the up-regulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing.
IL-16 Is Constitutively Present in Peripheral Blood Monocytes and Spontaneously Released During Apoptosis
Authors: Elssner Andreas , Doseff Andrea I. , Duncan Michelle , Kotur Mark , Wewers Mark D.
Published: April 2014

Constitutive expression of the pro-molecule of IL-16 has been found in T cells, mast cells, eosinophils, epithelial cells, fibroblasts, and dendritic cells. Here we show that IL-16 is also constitutively present in >98% of freshly isolated human CD14-positive peripheral blood monocytes when analyzed by flow cytometry. Because pro-IL-16 is cleaved to its bioactive mature form by caspase-3, and caspase-3 is also the pivotal effector of apoptosis in monocytes, we asked whether IL-16 release occurs in monocytes that undergo spontaneous apoptosis. As expected, freshly isolated, unstimulated monocytes underwent spontaneous caspase-3 activation. This apoptosis was paralleled by the loss of intracellular IL-16, as detected by flow cytometry, and the concurrent release of IL-16, as detected by ELISA. In contrast, stimulation with bacterial LPS inhibited caspase-3 activation and significantly inhibited the release of IL-16. As a specificity control, IL-1β and IL-8 were not released during spontaneous monocyte apoptosis. In summary, our data demonstrate that monocytes contain IL-16 that is released during spontaneous apoptosis.