bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimization of CD4+ T Lymphocyte Response to Human Cytomegalovirus Nuclear IE1 Protein through Modifications of Both Size and Cellular Localization
Authors: Delmas Sandra , Martin Laurence , Baron Michel , Nelson Jay A. , Streblow Daniel N. , Davignon Jean-Luc
Published: April 2014

We have previously reported that the CD4+ T lymphocyte response against nuclear human CMV IE1 protein depends in part on endogenous MHC class II presentation. To optimize presentation by HLA-DR of the nuclear IE1 protein and increase the response by CD4+ T cells, we have constructed two different adenovirus vectors containing mutant versions of IE1, containing a HLA-DR3 epitope, fused to GFP. The first construct consisted of a sequence of 46 aa encoded by exon 4, called GFP-IE1 (86–131). The second construct consisted of the whole IE1 mutated on exon 4 nuclear localization signals, identified in this study, and deleted of already known exon 2 nuclear localization signals (GFP-IE1M). Both of these IE1 vectors expressed proteins with cytoplasmic localization, as evidenced by GFP expression, as opposed to control GFP-IE1, which was nuclear. GFP-IE1 (86–131) induced IE1-specific CD4+ T cell clone response that was >30-fold more potent than that against GFP-IE1 and GFP-IE1M. The CD4+ T cell response was due to endogenous presentation followed by exogenous presentation at later time points. Presentation was dependent on both proteasome and acidic compartments. GFP-IE1 (86–131) was rapidly degraded by the APC, which may account for better presentation. Our data show potentiation of the CD4+ T cell response to a specific epitope through shortening and relocation of an otherwise nuclear protein and suggest applications in vaccination.
Chronic Ethanol Ingestion in Rats Decreases Granulocyte-Macrophage Colony-Stimulating Factor Receptor Expression and Downstream Signaling in the Alveolar Macrophage
Authors: Joshi Pratibha C. , Applewhite Lisa , Ritzenthaler Jeffrey D. , Roman Jesse , Fernandez Alberto L. , Eaton Douglas C. , Brown Lou Ann S. , Guidot David M.
Published: April 2014

Although it is well recognized that alcohol abuse impairs alveolar macrophage immune function and renders patients susceptible to pneumonia, the mechanisms are incompletely understood. Alveolar macrophage maturation and function requires priming by GM-CSF, which is produced and secreted into the alveolar space by the alveolar epithelium. In this study, we determined that although chronic ethanol ingestion (6 wk) in rats had no effect on GM-CSF expression within the alveolar space, it significantly decreased membrane expression of the GM-CSF receptor in alveolar macrophages. In parallel, ethanol ingestion decreased cellular expression and nuclear binding of PU.1, the master transcription factor that activates GM-CSF-dependent macrophage functions. Furthermore, treatment of ethanol-fed rats in vivo with rGM-CSF via the upper airway restored GM-CSF receptor membrane expression as well as PU.1 protein expression and nuclear binding in alveolar macrophages. Importantly, GM-CSF treatment also restored alveolar macrophage function in ethanol-fed rats, as reflected by endotoxin-stimulated release of TNF-α and bacterial phagocytosis. We conclude that ethanol ingestion dampens alveolar macrophage immune function by decreasing GM-CSF receptor expression and downstream PU.1 nuclear binding and that these chronic defects can be reversed relatively quickly with rGM-CSF treatment in vivo.
Decoupling of Carbohydrate Binding and MASP-2 Autoactivation in Variant Mannose-Binding Lectins Associated with Immunodeficiency
Authors: Wallis Russell , Lynch Nicholas J. , Roscher Silke , Reid Kenneth B. , Schwaeble Wilhelm J.
Published: April 2014

Mannan-binding lectin (MBL) initiates complement activation by binding to arrays of carbohydrates on the surfaces of pathogenic microorganisms and activating MBL-associated serine proteases (MASPs). Separate point mutations to the collagenous domain of human MBL are associated with immunodeficiency, caused by reduced complement activation by the variant MBLs as well as by lower serum MBL concentrations. In the work reported here, we have used the well characterized rat lectin pathway to analyze the molecular and functional defects associated with two of the variant proteins. Mutations Gly25 → Asp and Gly28 → Glu create comparable structural changes in rat MBL but the G28E variant activates complement >10-fold less efficiently than the G25D variant, which in turn has ∼7-fold lower activity than wild-type MBL. Analysis of mutant MBL · MASP-2 complexes assembled from recombinant components shows that reduced complement activation by both mutant MBLs is caused by failure to activate MASP-2 efficiently on binding to a mannan-coated surface. Disruption of MBL-MASP-2 interactions as well as to changes in oligomeric structure and reduced binding to carbohydrate ligands compared with wild-type MBL probably account for the intermediate phenotype of the G25D variant. However, carbohydrate binding and MASP-2 activation are ostensibly completely decoupled in complexes assembled from the G28E mutant, such that the rate of MASP-2 activation is no greater than the basal rate of zymogen MASP-2 autoactivation. Analogous molecular defects in human MBL probably combine to create the mutant phenotypes of immunodeficient individuals.
Both Hemopoietic and Resident Cells Are Required for MyD88-Dependent Pulmonary Inflammatory Response to Inhaled Endotoxin
Authors: Noulin Nicolas , Quesniaux Valérie F. J. , Schnyder-Candrian Silvia , Schnyder Bruno , Maillet Isabelle , Robert Thomas , Vargaftig B. Boris , Ryffel Bernhard , Couillin Isabelle
Published: April 2014

Inhaled endotoxin induces an inflammatory response that contributes to the development and severity of asthma and other forms of airway disease. Here, we show that inhaled endotoxin-induced acute bronchoconstriction, TNF, IL-12p40, and KC production, protein leak, and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecule MyD88. Bronchoconstriction, inflammation, and protein leak are normal in Toll/IL-1R domain-containing adaptor inducing IFN-β-deficient mice. MyD88 is involved in TLR, but also in IL-1R-associated kinase 1-mediated IL-1R and -18R signaling. We exclude a role for IL-1 and IL-18 pathways in this response, as IL-1R1 and caspase-1 (ICE)-deficient mice develop lung inflammation while TLR4-deficient mice are unresponsive to inhaled LPS. Significantly, using bone marrow chimera, we demonstrate that both hemopoietic and resident cells are necessary for a full MyD88-dependent response to inhaled endotoxin; bronchoconstriction depends on resident cells while cytokine secretion is mediated by hemopoietic cells.
Contrasting Effects of Cyclooxygenase-1 (COX-1) and COX-2 Deficiency on the Host Response to Influenza A Viral Infection
Authors: Carey Michelle A. , Bradbury J. Alyce , Seubert John M. , Langenbach Robert , Zeldin Darryl C. , Germolec Dori R.
Published: April 2014

Influenza is a significant cause of morbidity and mortality worldwide despite extensive research and vaccine availability. The cyclooxygenase (COX) pathway is important in modulating immune responses and is also a major target of nonsteroidal anti-inflammatory drugs (NSAIDs) and the newer COX-2 inhibitors. The purpose of the present study was to examine the effect of deficiency of COX-1 or COX-2 on the host response to influenza. We used an influenza A viral infection model in wild type (WT), COX-1−/−, and COX-2−/− mice. Infection induced less severe illness in COX-2−/− mice in comparison to WT and COX-1−/− mice as evidenced by body weight and body temperature changes. Mortality was significantly reduced in COX-2−/− mice. COX-1−/− mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the BAL fluid, whereas the inflammatory and cytokine responses were blunted in COX-2−/− mice. However, lung viral titers were markedly elevated in COX-2−/− mice relative to WT and COX-1−/− mice on day 4 of infection. Levels of PGE2 were reduced in COX-1−/− airways whereas cysteinyl leukotrienes were elevated in COX-2−/− airways following infection. Thus, deficiency of COX-1 and COX-2 leads to contrasting effects in the host response to influenza infection, and these differences are associated with altered production of prostaglandins and leukotrienes following infection. COX-1 deficiency is detrimental whereas COX-2 deficiency is beneficial to the host during influenza viral infection.
Involvement of TLR4/Type I IL-1 Receptor Signaling in the Induction of Inflammatory Mediators and Cell Death Induced by Ethanol in Cultured Astrocytes
Authors: Blanco Ana M. , Vallés Soraya L. , Pascual Maria , Guerri Consuelo
Published: April 2014

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1β, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1β, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-κB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-κB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.
Abnormally Differentiated Subsets of Intestinal Macrophage Play a Key Role in Th1-Dominant Chronic Colitis through Excess Production of IL-12 and IL-23 in Response to Bacteria
Authors: Kamada Nobuhiko , Hisamatsu Tadakazu , Okamoto Susumu , Sato Toshiro , Matsuoka Katsuyoshi , Arai Kumiko , Nakai Takaaki , Hasegawa Akira , Inoue Nagamu , Watanabe Noriaki , Akagawa Kiyoko S. , Hibi Toshifumi
Published: April 2014

Disorders in enteric bacteria recognition by intestinal macrophages (Mφ) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mφ in intestinal inflammation by using an IL-10-deficient (IL-10−/−) mouse colitis model. GM-CSF-induced bone marrow-derived Mφ (GM-Mφ) and M-CSF-induced bone marrow-derived Mφ (M-Mφ) were generated from bone marrow CD11b+ cells. M-Mφ from IL-10−/− mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mφ from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mφ was not significantly different between WT and IL-10−/− mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mφ (CLPMφ) but not splenic Mφ from WT mice was similar to that of M-Mφ, and CLPMφ but not splenic Mφ from IL-10−/− mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from M-Mφ from IL-10−/− mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMφ and M-Mφ act as anti-inflammatory Mφ and suppress excess inflammation induced by bacteria in WT mice. In IL-10−/− mice, however, such Mφ subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mφ may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mφ to gut flora relationship in the development of colitis in IL-10−/− mice.
Resolution of Inflammation: Prostaglandin E2 Dissociates Nuclear Trafficking of Individual NF-κB Subunits (p65, p50) in Stimulated Rheumatoid Synovial Fibroblasts
Authors: Gomez Paul F. , Pillinger Michael H. , Attur Mukundan , Marjanovic Nada , Dave Mander , Park Jean , Bingham Clifton O. , Al-Mussawir Hayf , Abramson Steven B.
Published: April 2014

NF-κB transcription factors regulate inflammatory responses to cytokines such as IL-1β and TNF-α. We tested whether PGE2 regulated nuclear localization of individual NF-κB subunits, p65 and p50, in synovial fibroblasts harvested from patients with rheumatoid arthritis (RA). IL-1β/TNF-α stimulated the translocation of p65 and p50 from the cytosol to the nucleus of human RA synovial fibroblasts, as well as NF-κB activation measured by luciferase reporter assay. PGE2 (10 nM, 6 h) enhanced p50, but inhibited p65 translocation and NF-κB activation. In contrast, depletion of endogenous PGE2 by ibuprofen (100 μM) and celecoxib (5 μM) enhanced p65, but inhibited p50 nuclear translocation as well as binding to NF-κB DNA binding sites. PGE2 also blocked IL-1β/TNF-α-stimulated ERK activation, and the ERK inhibitor, PD98059, mimicked PGE2 in blocking p65, but enhancing p50 nuclear translocation, suggesting that the effects of PGE2 on p65 and p50 are mediated via effects on ERK. PGE2 also enhanced the expression of IκBα in an ERK-independent manner, suggesting that PGE2 inhibits NF-κB activation by both ERK-dependent and -independent mechanisms. Our data indicate that PGE2 may act to attenuate cytokine-induced inflammatory responses in RA synovial fibroblasts via regulation of the localization of specific NF-κB family dimers.
Proliferation Response of Leukemic Cells to CD70 Ligation Oscillates with Recurrent Remission and Relapse in a Low-Grade Lymphoma
Authors: Kaufmann Yael , Amariglio Ninette , Rosenthal Esther , Hirsch Yasmine Jacob , Many Amira , Rechavi Gideon
Published: April 2014

Interactions of the TNF-related cell surface ligand CD70 with its receptor CD27 provide a costimulatory signal in B and T cell activation. Functional CD70-CD27 interactions could contribute to lymphoma and leukemia progression. This possibility was studied using DNA microarrays on a unique case of low-grade lymphoma/leukemia characterized by recurrent cycles of acute leukemic phase alternating with spontaneous remission. Upon induction of the acute phase expression of CD70 and CD27 in the leukemic cells increased 38- and 25-fold, respectively. Coexpression of membrane CD70 and CD27 on the leukemic (CD5+CD19+) cells was maximal 2–3 days following initiation of the attack. Soluble CD27 in the patient’s serum was elevated during remission and further increased in the attack. Functional tests showed that neither anti-CD70 nor anti-CD27 Abs affect the rate of apoptosis. However, the anti-CD70 Ab specifically enhanced proliferation of the remission phase leukemic cells, whereas proliferation of the acute-phase counterparts that express higher level of membrane CD70 was unaffected. Hence, in this lymphoma/leukemia, membrane CD70 is presented on the leukemic cells in a responsive state during the remission and a nonresponsive state during the attack. Presumably, CD70 in its responsive state provides a costimulatory receptor for initiating the next acute phase while its nonresponsive state enables the remission.
Expansion and Contraction of HIV-Specific CD4 T Cells with Short Bursts of Viremia, but Physical Loss of the Majority of These Cells with Sustained Viral Replication
Authors: Seth Nilufer , Kaufmann Daniel , Lahey Timothy , Rosenberg Eric S. , Wucherpfennig Kai W.
Published: April 2014

Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-γ-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7−CD45RA−) and functional properties (IFN-γ+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.
Transcription Factor Nrf2 Plays a Pivotal Role in Protection against Elastase-Induced Pulmonary Inflammation and Emphysema
Authors: Ishii Yukio , Itoh Ken , Morishima Yuko , Kimura Toru , Kiwamoto Takumi , IIzuka Takashi , Hegab Ahmed E. , Hosoya Tomonori , Nomura Akihiro , Sakamoto Tohru , Yamamoto Masayuki , Sekizawa Kiyohisa
Published: April 2014

Emphysema is one of the major pathological abnormalities associated with chronic obstructive pulmonary disease. The protease/antiprotease imbalance and inflammation resulting from oxidative stress have been attributed to the pathogenesis of emphysema. Nrf2 is believed to protect against oxidative tissue damage through the transcriptional activation of a battery of antioxidant enzymes. In this study, we investigated the protective role of Nrf2 in the development of emphysema using elastase-induced emphysema as our model system. We found that elastase-provoked emphysema was markedly exacerbated in Nrf2-knockout (KO) mice compared with wild-type mice. The severity of emphysema in Nrf2-KO mice correlated intimately with the degree of lung inflammation in the initial stage of elastase treatment. The highly inducible expression of antioxidant and antiprotease genes observed in wild-type alveolar macrophages was significantly attenuated in the lungs of Nrf2-KO mice. Interestingly, transplantation of wild-type bone marrow cells into Nrf2-KO mice retarded the development of initial lung inflammation and subsequent emphysema, and this improvement correlated well with the appearance of macrophages expressing Nrf2-regulated antiprotease and antioxidant genes. Thus, Nrf2 appears to exert its protective effects through the transcriptional activation of antiprotease and antioxidant genes in alveolar macrophages.
Inhibition of CX3CL1 (Fractalkine) Improves Experimental Autoimmune Myositis in SJL/J Mice
Authors: Suzuki Fumihito , Nanki Toshihiro , Imai Toshio , Kikuchi Hirotoshi , Hirohata Shunsei , Kohsaka Hitoshi , Miyasaka Nobuyuki
Published: April 2014

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-α, IFN-γ, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.
Stimulating Lymphotoxin β Receptor on the Dendritic Cells Is Critical for Their Homeostasis and Expansion
Authors: Wang Yu-Gang , Kim Kwang Dong , Wang Jing , Yu Ping , Fu Yang-Xin
Published: April 2014

The increased number of dendritic cells (DCs) inside lymphoid tissue may contribute to the enhanced priming of lymphocytes. The homeostasis of splenic DCs has mostly been attributed to their migration to the spleen via the chemokine microenvironment induced by lymphotoxin β receptor (LTβR) signaling on splenic stromal cells. In this study we show that the lack of direct LTβR signaling on DCs is associated with the reduction of the number of DCs in the spleen independently of chemokine gradients. LTβR−/− mice have reduced DCs and reduced BrdU incorporation on DCs, and fewer DCs from LTβR−/− mice are detected in the spleen. Furthermore, increased expression of LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpes virus entry mediator on T cells) on T cells, a member of the TNF family (TNFSF14) and a ligand for LTβR, could dramatically increase the number of T cells and DCs, which leads to severe autoimmune diseases in a LTβR-dependent fashion. In vitro, LIGHT could directly promote accumulation of bone marrow-derived DCs. Furthermore, intratumor expression of LIGHT can dramatically expand DCs in situ, and inoculation of DCs into tumor tissues enhanced tumor immunity. Therefore, LTβR signaling on DCs is required for their homeostasis during physiology and pathological conditions, and increased LIGHT-LTβR interaction could stimulate DC expansion for T cell-mediated immunity.
Sodium Stibogluconate Interacts with IL-2 in Anti-Renca Tumor Action via a T Cell-Dependent Mechanism in Connection with Induction of Tumor-Infiltrating Macrophages
Authors: Fan Keke , Zhou Ming , Pathak Manas K. , Lindner Daniel J. , Altuntas Cengiz Z. , Tuohy Vincent K. , Borden Ernest C. , Yi Taolin
Published: April 2014

IL-2 therapy results in 10–20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mφ). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mφ (4-fold), whereas IL-2 alone had little effect. Mφ increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mφ expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-γ T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-γ capable of activating tumoricidal Mφ. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-γ production and expansion/activation of Mφ. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.
Constitutive Activation of p38 and ERK1/2 MAPKs in Epithelial Cells of Myasthenic Thymus Leads to IL-6 and RANTES Overexpression: Effects on Survival and Migration of Peripheral T and B Cells
Authors: Colombara Michaela , Antonini Valeria , Riviera Anna Pia , Mainiero Fabrizio , Strippoli Raffaele , Merola Marcello , Fracasso Giulio , Poffe Ornella , Brutti Nadia , Tridente Giuseppe , Colombatti Marco , Ramarli Dunia
Published: April 2014

Myasthenia gravis (MG) is an autoimmune disease of neuromuscular junctions where thymus plays a pathogenetic role. Thymectomy benefits patients, and thymic hyperplasia, a lymphoid infiltration of perivascular spaces becoming site of autoantibody production, is recurrently observed. Cytokines and chemokines, produced by thymic epithelium and supporting survival and migration of T and B cells, are likely to be of great relevance in pathogenesis of thymic hyperplasia. In thymic epithelial cell (TEC) cultures derived “in vitro” from normal or hyperplastic age-matched MG thymuses, we demonstrate by gene profiling analysis that MG-TEC basally overexpress genes coding for p38 and ERK1/2 MAPKs and for components of their signaling pathways. Immunoblotting experiments confirmed that p38 and ERK1/2 proteins were overexpressed in MG-TEC and, in addition, constitutively activated. Pharmacological blockage with specific inhibitors confirmed their role in the control of IL-6 and RANTES gene expression. According to our results, IL-6 and RANTES levels were abnormally augmented in MG-TEC, either basally or upon induction by adhesion-related stimuli. The finding that IL-6 and RANTES modulate, respectively, survival and migration of peripheral lymphocytes of myasthenic patients point to MAPK transcriptional and posttranscriptional abnormalities of MG-TEC as a key step in the pathological remodelling of myasthenic thymus.
Telomere Length of Transferred Lymphocytes Correlates with In Vivo Persistence and Tumor Regression in Melanoma Patients Receiving Cell Transfer Therapy
Authors: Zhou Juhua , Shen Xinglei , Huang Jianping , Hodes Richard J. , Rosenberg Steven A. , Robbins Paul F.
Published: April 2014

Recent studies have indicated that adoptive immunotherapy with autologous antitumor tumor-infiltrating lymphocytes (TILs) following nonmyeloablative chemotherapy mediates tumor regression in ∼50% of treated patients with metastatic melanoma, and that tumor regression is correlated with the degree of persistence of adoptively transferred T cells in peripheral blood. These findings, which suggested that the proliferative potential of transferred T cells may play a role in clinical responses, led to the current studies in which telomere length as well as phenotypic markers expressed on the administered TILs were examined. TILs that were associated with objective clinical responses following adoptive transfer possessed a mean telomere length of 6.3 kb, whereas TILs that were not associated with significant clinical responses were significantly shorter, averaging 4.9 kb (p < 0.01). Furthermore, individual TIL-derived T cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that were longer than telomeres of T cell clonotypes that failed to persist (6.2 vs 4.5 kb, respectively; p < 0.001). Expression of the costimulatory molecule CD28 also appeared to be associated with long telomeres and T cell persistence. These results, indicating that the telomere length of transferred lymphocytes correlated with in vivo T cell persistence following adoptive transfer, and coupled with the previous observation that T cell persistence was associated with clinical responses in this adoptive immunotherapy trial, suggest that telomere length and the proliferative potential of the transferred T cells may play a significant role in mediating response to adoptive immunotherapy.
Granulocyte Colony-Stimulating Factor: A Novel Mediator of T Cell Tolerance
Authors: Rutella Sergio , Zavala Flora , Danese Silvio , Kared Hassen , Leone Giuseppe
Published: April 2014

In recent years, several investigators have unraveled a previously unrecognized role for G-CSF in the regulation of T cell and dendritic cell functions. The experimental evidence in favor of G-CSF-mediated immune regulation includes the ability to switch T cell cytokine secretion profile to Th2 responses and the promotion of regulatory T cell and tolerogenic dendritic cell differentiation. Interestingly, G-CSF is beneficial in animals for the prevention and/or treatment of immune-mediated diseases, e.g., graft-vs-host disease, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and diabetes, suggesting a potential role in human autoimmune diseases. This review summarizes the growing body of evidence that supports a critical role for G-CSF as a novel mediator of T cell tolerance.
B Cells Productively Engage Soluble Antigen-Pulsed Dendritic Cells: Visualization of Live-Cell Dynamics of B Cell-Dendritic Cell Interactions
Authors: Huang Ning-Na , Han Sang-Bae , Hwang Il-Young , Kehrl John H.
Published: April 2014

Interactions between B lymphocytes and Ag-bearing dendritic cells (DC) likely occur at inflammatory sites and within lymphoid organs. To better understand these interactions we imaged B cells (TgB) from hen egg lysozyme (HEL) transgenic mice and DC pulsed with HEL (DC-HEL) in collagen matrices. Analysis of live-cell dynamics revealed autonomous movements and repeated encounters between TgB cells and DC-HEL that are best described by a “kiss-run and engage” model, whereas control B cells had only short-lived interactions. Ag localized at contact sites between TgB cells and DC-HEL, and both cell types rearranged their actin cytoskeletons toward the contact zone. The interaction of a TgB cell with a HEL-bearing DC triggered strong Ca2+ transients in the B cells. Thus, B cells can productively interact with DC displaying their cognate Ag.
STAT4 Expression in Human T Cells Is Regulated by DNA Methylation but Not by Promoter Polymorphism
Authors: Shin Hyun-Jin , Park Hye-Young , Jeong Sook-Jung , Park Heung-Woo , Kim Yoon-Keun , Cho Sang-Heon , Kim You-Young , Cho Mi-La , Kim Ho-Youn , Min Kyung-Up , Lee Chang-Woo
Published: April 2014

STAT4, which plays a pivotal role in Th1 immune responses, enhances IFN-γ transcription in response to the interaction of IL-12 with the IL-12R. Mice deficient in STAT4 lack IL-12-induced IFN-γ production and Th1 differentiation and display a predominantly Th2 phenotype. Although these findings indicate that STAT4 expression levels are important for the development of cytokine-producing Th1 cells, the transcriptional and posttranscriptional mechanisms regulating STAT4 expression are unknown. We sought to identify and characterize the transcriptional regulatory elements in the promoter region of the human STAT4 gene. We found that disruption of multiple transcriptional regions covering the CREB, OCT1, and SP1 motifs significantly reduced STAT4 promoter activity. However, genomic DNA isolated from 91 patients with asthma or rheumatoid arthritis showed no evidence of mutations in the defined STAT4 essential promoter region. The 5′ flanking region of the promoter was found to contain a −149A/G change in ∼20–35% of patients, but this polymorphism had no effect on promoter activity. Interestingly, STAT4 expression was drastically increased in human T cells following treatment with a DNA methyltransferase inhibitor, and truncation of methylation sites in the proximal regulatory elements of the STAT4 promoter markedly enhanced transcriptional activity. Thus, our findings provide molecular insight into STAT4 expression and suggest that, in human T cells, STAT4 expressional regulation is associated with DNA hypermethylation, but not promoter polymorphisms.
Sphingosine-1-Phosphate Agonists Increase Macrophage Homing, Lymphocyte Contacts, and Endothelial Junctional Complex Formation in Murine Lymph Nodes
Authors: Singer Irwin I. , Tian Min , Wickham L. Alexandra , Lin Jeffrey , Matheravidathu Scaria S. , Forrest Michael J. , Mandala Suzanne , Quackenbush Elizabeth J.
Published: April 2014

The sphingosine-1-phosphate (S1P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S1P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S1P receptor 1 (S1P1)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S1P1-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S1P1, and treatment with FTY-P resulted in intense staining and colocalization of CD31, β-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.
Helicobacter pyloriActivates NF-κB via the Alternative Pathway in B Lymphocytes
Authors: Ohmae Tomoya , Hirata Yoshihiro , Maeda Shin , Shibata Wataru , Yanai Ayako , Ogura Keiji , Yoshida Haruhiko , Kawabe Takao , Omata Masao
Published: April 2014

Helicobacter pylori causes various gastroduodenal diseases including gastric MALT lymphoma, but the mechanism underlying H. pylori-induced carcinogenesis is not known. The alternative pathway for NF-κB activation, which involves the processing of NF-κB2/p100 to p52, has been implicated in lymphocyte survival, attenuated apoptosis, and secondary lymphoid tissue development. In this study, we investigated H. pylori-induced activation of NF-κB through the alternative pathway in B lymphocytes. In immunoblot and EMSA, H. pylori induced NF-κB2/p100 processing to p52 and subsequent nuclear accumulation in IM-9 (human B cell line) cells and human peripheral blood B cells, but not in AGS (human gastric cancer cell line) cells. The activation of the alternative pathway was LPS-dependent but not cag pathogenicity island-dependent. Alternative pathway activation by H. pylori was associated with attenuated apoptosis. The expression levels of B lymphocyte chemoattractant, EBI-1 ligand chemokine, and stromal cell-derived factor-1α mRNAs were up-regulated in cocultured human B cells and in infected human gastric mucosa. In the infected mucosa, NF-κB2/p100 and p52 were detected immunohistochemically in the cytoplasm and nuclear compartments of lymphocytes, but not in epithelial cells. In summary, H. pylori activates the alternative NF-κB pathway in B lymphocytes. The effects on chemokine production and antiapoptosis mediated by H. pylori-induced processing of NF-κB2/p100 to p52 may drive lymphocytes to acquire malignant potential.
Apoptosis of Antigen-Specific T Lymphocytes upon the Engagement of CD8 by Soluble HLA Class I Molecules Is Fas Ligand/Fas Mediated: Evidence for the Involvement of p56lck, Calcium Calmodulin Kinase II, and Calcium-Independent Protein Kinase C Signaling Pathways and for NF-κB and NF-AT Nuclear Translocation
Authors: Contini Paola , Ghio Massimo , Merlo Andrea , Poggi Alessandro , Indiveri Francesco , Puppo Francesco
Published: April 2014

The binding of soluble HLA class I (sHLA-I) molecules to CD8 on EBV-specific CTL induced up-regulation of Fas ligand (FasL) mRNA and consequent sFasL protein secretion. This, in turn, triggered CTL apoptosis by FasL/Fas interaction. Molecular analysis of the biochemical pathways responsible for FasL up-regulation showed that sHLA-I/CD8 interaction firstly induced the recruitment of src-like p56lck and syk-like Zap-70 protein tyrosine kinases (PTK). Interestingly, p59fyn was activated upon the engagement of CD3/TCR complex but not upon the interaction of sHLA-I with CD8. In addition, sHLA-I/CD8 interaction, which is different from signaling through the CD3/TCR complex, did not induce nuclear translocation of AP-1 protein complex. These findings suggest that CD8− and CD3/TCR-mediated activating stimuli can recruit different PTK and transcription factors. Indeed, the engagement of CD8 by sHLA-I led to the activation of Ca2+ calmodulin kinase II pathway, which eventually was responsible for the NF-AT nuclear translocation. In addition, we found that the ligation of sHLA-I to CD8 recruited protein kinase C, leading to NF-κB activation. Both NF-AT and NF-κB were responsible for the induction of FasL mRNA and consequent CTL apoptosis. Moreover, FasL up-regulation and CTL apoptotic death were down-regulated by pharmacological specific inhibitors of Ca2+/calmodulin/calcineurin and Ca2+-independent protein kinase C signaling pathways. These findings clarify the intracellular signaling pathways triggering FasL up-regulation and apoptosis in CTL upon sHLA-I/CD8 ligation and suggest that sHLA-I molecules can be proposed as therapeutic tools to modulate immune responses.
The Inhibition of Autoreactive T Cell Functions by a Peptide Based on the CDR1 of an Anti-DNA Autoantibody Is via TGF-β-Mediated Suppression of LFA-1 and CD44 Expression and Function
Authors: Sela Uri , Mauermann Nora , Hershkoviz Rami , Zinger Heidy , Dayan Molly , Cahalon Liora , Liu Jian Ping , Mozes Edna , Lider Ofer
Published: April 2014

Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-β secretion 10 days later. Addition of TGF-β suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFβ-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 (αLβ2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-β-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1α and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-β on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.
Anti-CD25 Antibody Enhancement of Vaccine-Induced Immunogenicity: Increased Durable Cellular Immunity with Reduced Immunodominance
Authors: Moore Anne C. , Gallimore Awen , Draper Simon J. , Watkins Katherine R. , Gilbert Sarah C. , Hill Adrian V. S.
Published: April 2014

An efficacious vaccine strategy must be capable of inducing strong responses of an appropriate phenotype that are long lasting and sufficiently broad to prevent pathogen escape mechanisms. In the present study, we use anti-CD25 mAb to augment vaccine-induced immunity in mice. We demonstrate that coformulation of Ab and poxviral- or adenoviral-vectored vaccines induces significantly increased T cell responses to a malaria Ag; prior anti-CD25 Ab administration was not required for this effect. Furthermore, this vaccination approach subverts immunodominant epitope hierarchies by enhancing responses to subdominant epitopes induced by recombinant modified vaccinia virus Ankara immunization. Administration of anti-CD25 with a vaccine also induces more durable immunity compared with vaccine alone; significantly higher T cell responses were observed 100 days after the primary immunization. Enhanced immunogenicity is observed for multiple vaccine types with enhanced CD4+ and CD8+ T cell responses induced by bacillus Calmette-Guérin and a recombinant subunit protein vaccine to hepatitis B virus and with multiple Ags of tumor, viral, bacterial, and parasitic origin. Vaccine strategies incorporating anti-CD25 lead to improved protection against pre-erythrocytic malaria challenge. These data underpin new strategies for the design and development of more efficacious vaccines in clinical settings.
Early Kinetic Window of Target T Cell Susceptibility to CD25+ Regulatory T Cell Activity
Authors: Sojka Dorothy K. , Hughson Angela , Sukiennicki Teresa L. , Fowell Deborah J.
Published: April 2014

Peripheral tolerance is maintained in part by thymically derived CD25+CD4+ T cells (regulatory T cells (Tregs)). Their mechanism of action has not been well characterized. Therefore, to get a better understanding of Treg action, we investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6–10 h of culture. Visualization of this time frame, using a sensitive single-cell assay for IL-2, revealed the early elaboration of target cell IL-2 producers in the first 6 h despite the presence of CD25+CD4+ Tregs. However, after 6 h, a rapid rise in the number of IL-2 producers in the absence of Tregs was dramatically abrogated by the presence of Tregs. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. Modulating target T cell activation signals with provision of CD28, IL-2, or high Ag dose all abrogated suppression of proliferation late in culture. However, only CD28 signals enabled target T cells to resist the early Treg-induced down-regulation of IL-2. Therefore the quality of early target T cell activation signals, in particular engagement of CD28, represents an important control point in the balance between vulnerability and resistance to Treg suppression.
A Novel Mucosal Vaccine Based on Live Lactococci Expressing E7 Antigen and IL-12 Induces Systemic and Mucosal Immune Responses and Protects Mice against Human Papillomavirus Type 16-Induced Tumors
Authors: Bermúdez-Humarán Luis G. , Cortes-Perez Naima G. , Lefèvre François , Guimarães Valeria , Rabot Sylvie , Alcocer-Gonzalez Juan M. , Gratadoux Jean-Jacques , Rodriguez-Padilla Cristina , Tamez-Guerra Reyes S. , Corthier Gérard , Gruss Alexandra , Langella Philippe
Published: April 2014

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When challenged with lethal levels of tumor cell line TC-1 expressing E7, immunized mice showed full prevention of TC-1-induced tumors, even after a second challenge, suggesting that this prophylactic immunization can provide long-lasting immunity. Therapeutic immunization with L. lactis recombinant strains, i.e., 7 days after TC-1 injection, induced regression of palpable tumors in treated mice. The antitumor effects of vaccination occurred through a CTL response, which is CD4+ and CD8+ dependent. Furthermore, immunized mice developed an E7-specific mucosal immune response. These preclinical results suggest the feasibility of the low-cost mucosal vaccination and/or immunotherapy strategies against HPV-related cervical cancer in humans.
Targeted Expression of Human CD1d in Transgenic Mice Reveals Independent Roles for Thymocytes and Thymic APCs in Positive and Negative Selection of Vα14i NKT Cells
Authors: Schümann Jens , Pittoni Paola , Tonti Elena , MacDonald H. Robson , Dellabona Paolo , Casorati Giulia
Published: April 2014

CD1d-dependent invariant Vα14 (Vα14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Vα14-Jα18 TCR α-chain paired mostly with Vβ8.2 and Vβ7. The cellular requirements for thymic positive and negative selection of Vα14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Vα14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Vα14i NKT cells that proved functional when activated ex vivo with the Ag α-galactosyl ceramide. Vα14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vβ8.2. The low number of Vβ8.2+ Vα14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vβ7+ Vα14i NKT cells. Vβ8.2+, but not Vβ7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Vα14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.