bluuebunny/crossref_metadata_2025 huggingface dataset:
TNF-α Controls Intrahepatic T Cell Apoptosis and Peripheral T Cell Numbers
Authors: Murray Debbie A. , Crispe I. Nicholas
Published: April 2014

At the end of an immune response, activated lymphocyte populations contract, leaving only a small memory population. The deletion of CD8+ T cells from the periphery is associated with an accumulation of CD8+ T cells in the liver, resulting in both CD8+ T cell apoptosis and liver damage. After adoptive transfer and in vivo activation of TCR transgenic CD8+ T cells, an increased number of activated CD8+ T cells was observed in the lymph nodes, spleen, and liver of mice treated with anti-TNF-α. However, caspase activity was decreased only in CD8+ T cells in the liver, not in those in the lymphoid organs. These results indicate that TNF-α is responsible for inducing apoptosis in the liver and suggest that CD8+ T cells escaping this mechanism of deletion can recirculate into the periphery.
CD40 Engagement Enhances Antigen-Presenting Langerhans Cell Priming of IFN-γ-Producing CD4+ and CD8+ T Cells Independently of IL-12
Authors: Gorbachev Anton V. , Fairchild Robert L.
Published: April 2014

The delivery of CD40 signaling to APCs during T cell priming enhances many T cell-mediated immune responses. Although CD40 signaling up-regulates APC production of IL-12, the impact of this increased production on T cell priming is unclear. In this study an IL-12-independent T cell-mediated immune response, contact hypersensitivity (CHS), was used to further investigate the effect of CD40 ligation on the phenotypic development of Ag-specific CD4+ and CD8+ T cells. Normally, sensitization for CHS responses induces hapten-specific CD4+ T cells producing type 2 cytokines and CD8+ T cells producing IFN-γ. Treatment of mice with agonist anti-CD40 mAb during sensitization with the hapten 2,4-dinitrofluorobenzene resulted in CHS responses of increased magnitude and duration. These augmented responses in anti-CD40 Ab-treated mice correlated with increased numbers of hapten-specific CD4+ and CD8+ T cells producing IFN-γ in the skin draining lymph nodes. Identical results were observed using IL-12−/− mice, indicating that CD40 ligation promotes CHS responses and development of IFN-γ-producing CD4+ and CD8+ T cells in the absence of IL-12. Engagement of CD40 on hapten-presenting Langerhans cells (hpLC) up-regulated the expression of both class I and class II MHC and promoted hpLC migration into the T cell priming site. These results indicate that hpLC stimulated by CD40 ligation use a mechanism distinct from increased IL-12 production to promote Ag-specific T cell development to IFN-γ-producing cells.
Induction of IgG2a Class Switching in B Cells by IL-27
Authors: Yoshimoto Takayuki , Okada Keiko , Morishima Noriko , Kamiya Sadahiro , Owaki Toshiyuki , Asakawa Masayuki , Iwakura Yoichiro , Fukai Fumio , Mizuguchi Junichiro
Published: April 2014

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-γ-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-γ-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.
Functional Characterization of MHC Class II-Restricted CD8+CD4− and CD8−CD4− T Cell Responses to Infection in CD4−/− Mice
Authors: Pearce Erika L. , Shedlock Devon J. , Shen Hao
Published: April 2014

Classical CD4+ and CD8+ T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4−/− mice mount a strong, readily detectable CD8+ T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8+CD4− T cells are more similar to classical CD8+ T cells than to CD4+ T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8+ T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4−/− mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4−/− mice consists mainly of CD8−CD4− T cells. In contrast to CD8+ T cells, MHCII-restricted CD8−CD4− T cells are capable of producing IL-2 in addition to IFN-γ and thus appear to have attributes characteristic of CD4+ T cells rather than CD8+ T cells. Therefore, MHCII-restricted T cells in CD4−/− mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8+ T cells or with MHCII-restricted CD4+ T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.
Complement C5a Receptor Is Essential for the Optimal Generation of Antiviral CD8+ T Cell Responses
Authors: Kim Alfred H. J. , Dimitriou Ioannis D. , Holland M. Claire H. , Mastellos Dimitrios , Mueller Yvonne M. , Altman John D. , Lambris John D. , Katsikis Peter D.
Published: April 2014

The complement system has been long regarded as an important effector of the innate immune response. Furthermore, complement contributes to various aspects of B and T cell immunity. Nevertheless, the role of complement in CD8+ T cell antiviral responses has yet to be fully delineated. We examined the CD8+ T cell response in influenza type A virus-infected mice treated with a peptide antagonist to C5aR to test the potential role of complement components in CD8+ T cell responses. We show that both the frequency and absolute numbers of flu-specific CD8+ T cells are greatly reduced in C5aR antagonist-treated mice compared with untreated mice. This reduction in flu-specific CD8+ T cells is accompanied by attenuated antiviral cytolytic activity in the lungs. These results demonstrate that the binding of the C5a component of complement to the C5a receptor plays an important role in CD8+ T cell responses.
Requirement for Both H-2Db and H-2Kd for the Induction of Diabetes by the Promiscuous CD8+ T Cell Clonotype AI4
Authors: Takaki Toshiyuki , Lieberman Scott M. , Holl Thomas M. , Han Bingye , Santamaria Pere , Serreze David V. , DiLorenzo Teresa P.
Published: April 2014

The NOD mouse is a model for autoimmune type 1 diabetes in humans. CD8+ T cells are essential for the destruction of the insulin-producing pancreatic β cells characterizing this disease. AI4 is a pathogenic CD8+ T cell clone, isolated from the islets of a 5-wk-old female NOD mouse, which is capable of mediating overt diabetes in the absence of CD4+ T cell help. Recent studies using MHC-congenic NOD mice revealed marked promiscuity of the AI4 TCR, as the selection of this clonotype can be influenced by multiple MHC molecules, including some class II variants. The present work was designed, in part, to determine whether similar promiscuity also characterizes the effector function of mature AI4 CTL. Using splenocyte and bone marrow disease transfer models and in vitro islet-killing assays, we report that efficient recognition and destruction of β cells by AI4 requires the β cells to simultaneously express both H-2Db and H-2Kd class I MHC molecules. The ability of the AI4 TCR to interact with both H-2Db and H-2Kd was confirmed using recombinant peptide libraries. This approach also allowed us to define a mimotope peptide recognized by AI4 in an H-2Db-restricted manner. Using ELISPOT and mimotope/H-2Db tetramer analyses, we demonstrate for the first time that AI4 represents a readily detectable T cell population in the islet infiltrates of prediabetic NOD mice. Our identification of a ligand for AI4-like T cells will facilitate further characterization and manipulation of this pathogenic and promiscuous T cell population.
Human Lymphocytes Interact Directly with CD47 through a Novel Member of the Signal Regulatory Protein (SIRP) Family
Authors: Brooke Gary , Holbrook Joanna D. , Brown Marion H. , Barclay A. Neil
Published: April 2014

Two closely related proteins, signal regulatory protein α (SIRPα; SHPS-1/CD172) and SIRPβ, have been described in humans. The existence of a third SIRP protein has been suggested by cDNA sequence only. We show that this third SIRP is a separate gene that is expressed as a protein with unique characteristics from both α and β genes and suggest that this gene should be termed SIRPγ. We have expressed the extracellular region of SIRPγ as a soluble protein and have shown that, like SIRPα, it binds CD47, but with a lower affinity (Kd, ∼23 μM) compared with SIRPα (Kd, ∼2 μM). mAbs specific to SIRPγ show that it was not expressed on myeloid cells, in contrast to SIRPα and -β, being expressed instead on the majority of T cells and a proportion of B cells. The short cytoplasmic tail of SIRPγ does not contain any known signaling motifs, nor does it contain a characteristic lysine, as with SIRPβ, that is required for DAP12 interaction. DAP12 coexpression is a requirement for SIRPβ surface expression, whereas SIRPγ is expressed in its absence. The SIRPγ-CD47 interaction may therefore not be capable of bidirectional signaling as with the SIRPα-CD47, but, instead, use unidirectional signaling via CD47 only.
c-Fos as a Regulator of Degranulation and Cytokine Production in FcεRI-Activated Mast Cells
Authors: Lee Yu-Nee , Tuckerman Jan , Nechushtan Hovav , Schutz Gunter , Razin Ehud , Angel Peter
Published: April 2014

The AP-1 complex is composed of c-Jun and c-Fos and is a key component in the regulation of proinflammatory genes. Mast cells play a significant role in the initiation of many inflammatory responses, such as allergy and allergy-associated diseases. In the present work, we characterized the role of c-Fos in mast cell function by investigating IL-3-dependent cell proliferation, degranulation capability, and cytokine expression in c-Fos-deficient mice. In c-Fos-deficient mast cells, we found that FcεRI-mediated degranulation was significantly inhibited, which correlates with the reduced expression of SWAP-70, VAMP-7, and Synaptotagmin I genes, which are involved directly in the degranulation process. These findings show that c-Fos plays an important role in FcεRI-mediated regulation of mast cell function.
Endoplasmic Reticulum-Associated Degradation-Induced Dissociation of Class II Invariant Chain Complexes Containing a Glycosylation-Deficient Form of p41
Authors: Sevilla Lisa M. , Comstock Sarah S. , Swier Kevin , Miller Jim
Published: April 2014

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.
Human Papillomavirus Type-16 Virus-Like Particles Activate Complementary Defense Responses in Key Dendritic Cell Subpopulations
Authors: Yang Rongcun , Murillo Francisco Martinez , Lin Ken-Yu , Yutzy William H. , Uematsu Satoshi , Takeda Kiyoshi , Akira Shizuo , Viscidi Raphael P. , Roden Richard B. S.
Published: April 2014

Human papillomavirus type-16 (HPV16) L1 virus-like particles (VLPs) activate dendritic cells (DCs) and induce protective immunity. In this study, we demonstrate, using global gene expression analysis, that HPV16 VLPs produce quite distinct innate responses in murine splenic DC subpopulations. While HPV16 VLPs increase transcription of IFN-γ and numerous Th1-related cytokines and chemokines in CD8α+CD11c+ DCs, CD4+CD11c+ DCs up-regulate only type I IFN and a different set of Th2-associated cytokines and chemokines. Type I IFN, but not IFN-γ, potentiates humoral immunity, notably production of VLP-specific IgG2a. However, HPV16 VLP-stimulated IL-12 production by CD8α+CD11c+ DCs is augmented by autocrine IFN-γ signaling. Thus, before adaptive immunity, HPV16 VLPs signal complementary defense responses in key DC subpopulations, indicating specialized DC lineages with predetermined polarization.
Functional Inactivation of Immature Dendritic Cells by the Intracellular Parasite Toxoplasma gondii
Authors: McKee Amy S. , Dzierszinski Florence , Boes Marianne , Roos David S. , Pearce Edward J.
Published: April 2014

Despite its noted ability to induce strong cellular immunity, and its known susceptibility to IFN-γ-dependent immune effector mechanisms, the protozoan Toxoplasma gondii is a highly successful parasite, able to replicate, disseminate, and either kill the host or, more commonly, establish resistant encysted life forms before the emergence of protective immune responses. We sought to understand how the parasite gains the advantage. Using transgenic clonal parasite lines engineered to express fluorescent markers in combination with dendritic cells (DC) grown from the bone marrow of wild-type mice or transgenic mice expressing fluorescent protein-tagged MHC class II molecules, we used flow cytometry and fluorescence microscopy to analyze the responses of infected DC to both invasion by the parasite and subsequent DC maturation signals. We found that T. gondii preferentially invades immature dendritic cells but fails to activate them in the process, and renders them resistant to subsequent activation by TLR ligands or the immune-system-intrinsic maturation signal CD40L. The functional consequences of T. gondii-mediated suppression of DC activation are manifested in a relative inability of infected immature DC to activate naive CD4+ Th lymphocytes, or to secrete cytokines, such IL-12 and TNF-α, that play important roles in innate and/or adaptive immunity. The findings reveal that T. gondii suppresses the ability of immature DC to participate in innate immunity and to induce adaptive immune responses. The ability of T. gondii to temporarily evade recognition could provide a selective advantage that permits dissemination and establishment before adaptive immune response initiation.
Mycobacterium tuberculosisLprG (Rv1411c): A Novel TLR-2 Ligand That Inhibits Human Macrophage Class II MHC Antigen Processing
Authors: Gehring Adam J. , Dobos Karen M. , Belisle John T. , Harding Clifford V. , Boom W. Henry
Published: April 2014

MHC class II (MHC-II)-restricted CD4+ T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-α production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4+ T cells.
A Secreted Protein from the Human Hookworm Necator americanus Binds Selectively to NK Cells and Induces IFN-γ Production
Authors: Hsieh George C.-F. , Loukas Alex , Wahl Allison M. , Bhatia Monica , Wang Yan , Williamson Angela L. , Kehn Kylene W. , Maruyama Haruhiko , Hotez Peter J. , Leitenberg David , Bethony Jeff , Constant Stephanie L.
Published: April 2014

Parasitic helminths induce chronic infections in their hosts although, with most human helminthiases, protective immunity gradually develops with age or exposure of the host. One exception is infection with the human hookworm, Necator americanus, where virtually no protection ensues over time. Such observations suggest these parasites have developed unique mechanisms to evade host immunity, leading us to investigate the role of the excretory/secretory (ES) products of adult N. americanus in manipulating host immune responses. Specifically, we found that a protein(s) from ES products of adult N. americanus bound selectively to mouse and human NK cells. Moreover, incubation of purified NK cells with N. americanus ES products stimulated the production of augmented (4- to 30-fold) levels of IFN-γ. This augmentation was dependent on the presence of both IL-2 and IL-12 and was endotoxin-independent. This is the first report of a pathogen protein that binds exclusively to NK cells and the first report of a nematode-derived product that induces abundant levels of cytokines from NK cells. Such an interaction could provide a means of cross-regulating deleterious Th2 immune responses in the host, thereby contributing to the long-term survival of N. americanus.
Suppression of Innate Immunity by Acute Ethanol Administration: A Global Perspective and a New Mechanism Beginning with Inhibition of Signaling through TLR3
Authors: Pruett Stephen B. , Schwab Carlton , Zheng Qiang , Fan Ruping
Published: April 2014

Excessive consumption of ethanol (EtOH) suppresses innate immunity, but the mechanisms have not been fully delineated. The present study was conducted to determine whether EtOH suppresses TLR signaling in vivo in mice and to characterize the downstream effects of such suppression. Degradation of IL-1R-associated kinase 1 induced by a TLR3 ligand in peritoneal cells (∼90% macrophages) was suppressed by EtOH. Phosphorylation of p38 kinase in peritoneal macrophages (F4/80+) was suppressed, as was nuclear translocation of p-c-Jun and p65 in peritoneal cells. EtOH decreased IL-6 and IL-12 (p40), but did not significantly affect IL-10 in peritoneal lavage fluid or in lysates of peritoneal cells. Changes in cytokine mRNAs (by RNase protection assay) in macrophages isolated by cell sorting or using Ficoll were generally consistent with changes in protein levels in cell lysates and peritoneal lavage fluid. Thus, suppression of TLR signaling and cytokine mRNA occurred in the same cells, and this suppression generally corresponded to changes in i.p. and intracellular cytokine concentrations. DNA microarray analysis revealed the suppression of an IFN-related amplification loop in peritoneal macrophages, associated with decreased expression of numerous innate immune effector genes (including cytokines and a chemokine also suppressed at the protein level). These results indicate that EtOH suppresses innate immunity at least in part by suppressing TLR3 signaling, suppressing an IFN-related amplification loop, and suppressing the induction of a wide range of innate effector molecules in addition to proinflammatory cytokines and chemokines.
The Induction of Acute Ileitis by a Single Microbial Antigen of Toxoplasma gondii
Authors: Rachinel Nicolas , Buzoni-Gatel Dominique , Dutta Chaitali , Mennechet Franck J. D. , Luangsay Souphalone , Minns Laurie A. , Grigg Michael E. , Tomavo Stanislas , Boothroyd John C. , Kasper Lloyd H.
Published: April 2014

The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7–9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Δsag1) engineered. We investigated the ability of Δsag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Δsag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1−/−) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4+ effector T lymphocytes isolated from C57BL/6-infected mice into RAG−/− mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4+ effector T lymphocytes from mice infected with the mutant Δsag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.
Impact of Fibronectin Fragments on the Transendothelial Migration of HIV-Infected Leukocytes and the Development of Subendothelial Foci of Infectious Leukocytes
Authors: Birdsall Holly H. , Porter Wendy J. , Green David M. , Rubio Jose , Trial JoAnn , Rossen Roger D.
Published: April 2014

Leukocyte infiltrates that can serve as viral reservoirs, and sites for viral replication are found in many organs of HIV-1-infected patients. Patients whose blood leukocytes migrate across confluent endothelial monolayers ex vivo and transmit infectious virus to mononuclear leukocytes (MNLs) lodged beneath this endothelial barrier have a worse prognosis. We evaluated the ability of 110- to 120-kDa fibronectin fragments (FNf), which are found in the blood of >60% of HIV-1-infected patients, to stimulate transendothelial migration and drive productively infected MNLs into a potential perivascular space. FNf induced MNLs to release TNF-α in a dose-dependent fashion; the resulting increase in lymphocyte and monocyte transendothelial migration could be blocked with soluble TNF receptor I. Rather than penetrate deeply into the subendothelial matrix, as is seen with untreated controls, FNf-treated MNLs clustered just below the endothelial monolayer. Treatment with FNf during migration increased subsequent recovery of HIV-infected cells from the subendothelial compartment. FNf treatment also significantly increased the numbers of HLA-DRbright, dendritic-type cells that reverse-migrated from the subendothelial depot to the apical endothelial surface 48 h after migration. Fibronectin fragments can be produced by viral and host proteases in the course of inflammatory conditions. The ability of FNf to stimulate transendothelial migration of HIV-1-infected MNLs may help to explain the dissemination of this infection into cardiac, renal, and CNS tissues.
IL-9-Mediated Induction of Eotaxin1/CCL11 in Human Airway Smooth Muscle Cells
Authors: Gounni Abdelilah Soussi , Hamid Qutayba , Rahman Sahidur M. , Hoeck Jutta , Yang Jie , Shan Lianyu
Published: April 2014

Recent work has shown the potential importance of IL-9 in allergic diseases. The development of transgenic mice overexpressing IL-9 has suggested a key role for this cytokine in the development of the asthmatic phenotype including airway eosinophilia. In this study, we evaluated the expression of the IL-9R and the effects of IL-9 on human ASM cells by examining the release of Th2-associated chemokines (eotaxin1/CCL11 and thymus- and activation-regulated chemokine (TARC)/CCL17). IL-9R α-chain mRNA and surface expression were detected in cultured human airway smooth muscle (ASM) cells. In addition, primary cultured ASM cells, as well as bronchial smooth muscle cells within biopsies of asthmatics and not control subjects, revealed IL-9R protein expression. IL-9 stimulation of human ASM cells resulted in release of eotaxin1/CCL11, but had no effect on the release of TARC/CCL17, in time- and dose-dependent manner. Moreover, in vitro chemotaxis assay demonstrated that conditioned medium from IL-9-stimulated ASM cells attracted human eosinophils. Neutralizing Abs to IL-9, but not to IL-4 or IL-13, reduced significantly IL-9-induced production of eotaxin1/CCL11 from ASM cells. Interestingly, real-time RT-PCR showed that IL-9 up-regulated eotaxin1/CCL11 mRNA expression, but had no effect on TARC/CCL17. Treatment with Act D abrogates IL-9-induced eotaxin1/CCL11 mRNA and protein release by ASM cells. Finally, transfection study using eotaxin1/CCL11 promoter luciferase construct confirmed that IL-9 induced eotaxin1/CCL11 at the transcriptional level. Taken together, these data provide new evidence demonstrating that IL-9-dependent activation of ASM cells contributes to eosinophilic inflammation observed in asthma.
Ex Vivo Homeostatic Proliferation of CD4+ T Cells in Rheumatoid Arthritis Is Dysregulated and Driven by Membrane-Anchored TNFα
Authors: Wagner Ulf , Pierer Matthias , Wahle Matthias , Moritz Falk , Kaltenhäuser Sylke , Häntzschel Holm
Published: April 2014

The systemic CD4+ T cell compartment in patients with rheumatoid arthritis (RA) is characterized by TCR repertoire contraction, shortened telomere lengths, and decreased numbers of recent thymic emigrants, suggesting a disturbed CD4+ T cell homeostasis. In mice, homeostatic proliferation of peripheral CD4+ T cells is regulated by TCR interaction with self peptide-MHC complexes (pMHC) and can be reproduced in vitro. We have established an ex vivo model of homeostatic proliferation, in which self-replication of human CD4+ T cells is induced by cell-cell contact with autologous monocytes. In healthy individuals, blockade of TCR-pMHC class II contact resulted in decreased CD4+ T cell division. In contrast, homeostatic proliferation in RA patients was not inhibited by pMHC blockade, but increased during the initial culture period. The anti-TNF-α Ab cA2 inhibited homeostasis-driven ex vivo proliferation in healthy controls and in RA patients. In addition, treatment of RA patients with infliximab decreased the ex vivo rate of homeostatic proliferation of CD4+ T cells. Our results suggest a disturbed regulation of CD4+ T cell homeostasis leading to the repertoire aberrations reported in RA. Membrane-anchored TNF-α appears to be a cell-cell contact-dependent stimulus of homeostatic proliferation of CD4+ T cells, possibly favoring self-replication of autoreactive CD4+ T cells in patients with RA.
Molecular Recognition Patterns of Serum Anti-DNA Topoisomerase I Antibody in Systemic Sclerosis
Authors: Hu Paul Q. , Fertig Noreen , Medsger Thomas A. , Wright Timothy M.
Published: April 2014

Autoreactive anti-DNA topoisomerase I (anti-Topo I) Abs are commonly detected in sera of systemic sclerosis (SSc) patients. Our studies have established a positive correlation between the levels of serum anti-Topo I Abs and both disease severity and activity of SSc. The molecular targets of anti-Topo I Ab on Topo I domains remain to be further defined. In this report, we studied the molecular recognition pattern of serum anti-Topo I Ab in 52 SSc patients. The highest reactivity of serum anti-Topo I Abs was against the core subdomains I and II (aa 207–441) and, to a lesser extent, against the core subdomain III (aa 433–636) of Topo I. The linker domain (aa 636–712) and the C-terminal domain (aa 713–765) had much less reactivity than the core domain (aa 207–636). Strikingly, very little reactivity was directed against the N-terminal domain (aa 1–213) by serum anti-Topo I Ab. This molecular recognition pattern was consistent among all SSc serum samples studied. Results from patients with serial serum samples indicated that this pattern remained unchanged over time. Interestingly, some naive B cells from healthy controls, upon transformation by EBV, produced IgM Abs against Topo I. These Abs had low affinity for Topo I and reacted equally to all domains of Topo I. The molecular recognition pattern of serum anti-Topo I Ab in SSc suggests the presence of a unique antigenic stimulation in vivo in this disease.
Leukemia-Derived Immature Dendritic Cells Differentiate into Functionally Competent Mature Dendritic Cells That Efficiently Stimulate T Cell Responses
Authors: Cignetti Alessandro , Vallario Antonella , Roato Ilaria , Circosta Paola , Allione Bernardino , Casorzo Laura , Ghia Paolo , Caligaris-Cappio Federico
Published: April 2014

Primary acute myeloid leukemia cells can be induced to differentiate into dendritic cells (DC). In the presence of GM-CSF, TNF-α, and/or IL-4, leukemia-derived DC are obtained that display features of immature DC (i-DC). The aim of this study was to determine whether i-DC of leukemic origin could be further differentiated into mature DC (m-DC) and to evaluate the possibility that leukemic m-DC could be effective in vivo as a tumor vaccine. Using CD40L as maturating agent, we show that leukemic i-DC can differentiate into cells that fulfill the phenotypic criteria of m-DC and, compared with normal counterparts, are functionally competent in vitro in terms of: 1) production of cytokines that support T cell activation and proliferation and drive Th1 polarization; 2) generation of autologous CD8+ CTLs and CD4+ T cells that are MHC-restricted and leukemia-specific; 3) migration from tissues to lymph nodes; 4) amplification of Ag presentation by monocyte attraction; 5) attraction of naive/resting and activated T cells. Irradiation of leukemic i-DC after CD40L stimulation did not affect their differentiating and functional capacity. Our data indicate that acute myeloid leukemia cells can fully differentiate into functionally competent m-DC and lay the ground for testing their efficacy as a tumor vaccine.
Targeted CTLA-4 Engagement Induces CD4+CD25+CTLA-4high T Regulatory Cells with Target (Allo)antigen Specificity
Authors: Vasu Chenthamarakshan , Prabhakar Bellur S. , Holterman Mark J.
Published: April 2014

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4+CD25+CTLA-4high regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-γ and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-β1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-β1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4+CD25+ Treg cells. The CD4+CD25+CTLA-4high Treg cells from tolerant mice suppressed the effector function of CD25− T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L−) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.
Effects of Cytokines on Acetylcholine Receptor Expression: Implications for Myasthenia Gravis
Authors: Poëa-Guyon Sandrine , Christadoss Premkumar , Le Panse Rozen , Guyon Thierry , De Baets Marc , Wakkach Abdelilah , Bidault Jocelyne , Tzartos Socrates , Berrih-Aknin Sonia
Published: April 2014

Myasthenia gravis is an autoimmune disease associated with thymic pathologies, including hyperplasia. In this study, we investigated the processes that may lead to thymic overexpression of the triggering Ag, the acetylcholine receptor (AChR). Using microarray technology, we found that IFN-regulated genes are more highly expressed in these pathological thymic tissues compared with age- and sex-matched normal thymus controls. Therefore, we investigated whether proinflammatory cytokines could locally modify AChR expression in myoid and thymic epithelial cells. We found that AChR transcripts are up-regulated by IFN-γ, and even more so by IFN-γ and TNF-α, as assessed by real-time RT-PCR, with the α-AChR subunit being the most sensitive to this regulation. The expression of AChR protein was increased at the cytoplasmic level in thymic epithelial cells and at the membrane in myoid cells. To examine whether IFN-γ could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-γ gene knock-out mice, and found a significant decrease in AChR transcript levels in the thymus but not in the muscle, compared with wild-type mice. However, up-regulation of AChR protein expression was found in the muscles of animals with myasthenic symptoms treated with TNF-α. Altogether, these results indicate that proinflammatory cytokines influence the expression of AChR in vitro and in vivo. Because proinflammatory cytokine activity is evidenced in the thymus of myasthenia gravis patients, it could influence AChR expression and thereby contribute to the initiation of the autoimmune anti-AChR response.
TGF-β1 Uses Distinct Mechanisms to Inhibit IFN-γ Expression in CD4+ T Cells at Priming and at Recall: Differential Involvement of Stat4 and T-bet
Authors: Lin Jack T. , Martin Stacey L. , Xia Luxi , Gorham James D.
Published: April 2014

TGF-β1 plays a critical role in restraining pathogenic Th1 autoimmune responses in vivo, but the mechanisms that mediate TGF-β1’s suppressive effects on CD4+ T cell expression of IFN-γ expression remain incompletely understood. To evaluate mechanisms by which TGF-β1 inhibits IFN-γ expression in CD4+ T cells, we primed naive wild-type murine BALB/c CD4+ T cells in vitro under Th1 development conditions in the presence or the absence of added TGF-β1. We found that the presence of TGF-β1 during priming of CD4+ T cells suppressed both IFN-γ expression during priming as well as the development of Th1 effector cells expressing IFN-γ at a recall stimulation. TGF-β1 inhibited the development of IFN-γ-expressing cells in a dose-dependent fashion and in the absence of APC, indicating that TGF-β1 can inhibit Th1 development by acting directly on the CD4+ T cell. During priming, TGF-β1 strongly inhibited the expression of both T-bet (T box expressed in T cells) and Stat4. We evaluated the importance of these two molecules in the suppression of IFN-γ expression at the two phases of Th1 responses. Enforced expression of T-bet by retrovirus prevented TGF-β1’s inhibition of Th1 development, but did not prevent TGF-β1’s inhibition of IFN-γ expression at priming. Conversely, enforced expression of Stat4 partly prevented TGF-β1’s inhibition of IFN-γ expression during priming, but did not prevent TGF-β1’s inhibition of Th1 development. These data show that TGF-β1 uses distinct mechanisms to inhibit IFN-γ expression in CD4+ T cells at priming and at recall.
Intravenous Infusion of Syngeneic Apoptotic Cells by Photopheresis Induces Antigen-Specific Regulatory T Cells
Authors: Maeda Akira , Schwarz Agatha , Kernebeck Kerstin , Gross Nicole , Aragane Yoshinori , Peritt David , Schwarz Thomas
Published: April 2014

The basis of extracorporeal photopheresis is the reinfusion of leukocytes previously exposed to 8-methoxypsoralen (8-MOP) and UVA radiation. It has been approved for the palliative treatment of cutaneous T cell lymphoma and has reported benefits in autoimmune diseases, transplant rejection, and graft-vs-host disease. However, the underlying mechanism of photopheresis remains unresolved. Because UVB radiation can cause immune tolerance via induction of regulatory T cells, we studied whether photopheresis exerts a similar effect extracorporeally. Therefore, we established a model of photopheresis using a murine model of contact hypersensitivity. Splenocytes and lymph node cells of mice that were sensitized with dinitrofluorobenzene were exposed to 8-MOP plus UVA in vitro. Intravenous injection of these cells into naive mice caused inhibition of a hapten immune response, which was lost upon depletion of CD11c+ cells but not T cells. Mice that received untreated cells or cells exposed to UVA or 8-MOP alone were not affected. Inhibition was cell-mediated and Ag-specific as demonstrated by transfer of tolerance from the primary recipients into naive animals, which could, however, properly respond to the unrelated hapten oxazolone. Transfer activity was lost when cells were depleted of CD4+ or CD25+ subpopulations. These data suggest that photopheresis exerts its immunomodulatory effects via the induction of Ag-specific regulatory T cells.
Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229
Authors: Martín Margarita , Del Valle Juana M. , Saborit Ifigènia , Engel Pablo
Published: April 2014

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.
γδ T Cells Respond Directly to Pathogen-Associated Molecular Patterns
Authors: Hedges Jodi F. , Lubick Kirk J. , Jutila Mark A.
Published: April 2014

γδ T cells recognize unprocessed or non-peptide Ags, respond rapidly to infection, and localize to mucosal surfaces. We have hypothesized that the innate functions of γδ T cells may be more similar to those of cells of the myeloid lineage than to other T cells. To begin to test this assumption, we have analyzed the direct response of cultured human and peripheral blood bovine γδ T cells to pathogen associated molecular patterns (PAMPs) in the absence of APCs using microarray, real-time RT-PCR, proteome array, and chemotaxis assays. Our results indicate that purified γδ T cells respond directly to PAMPs by increasing expression of chemokine and activation-related genes. The response was distinct from that to known γδ T cell Ags and different from the response of myeloid cells to PAMPs. In addition, we have analyzed the expression of a variety of PAMP receptors in γδ T cells. Freshly purified bovine γδ T cells responded more robustly to PAMPs than did cultured human cells and expressed measurable mRNA encoding a variety of PAMP receptors. Our results suggest that rapid response to PAMPs through the expression of PAMP receptors may be another innate role of γδ T cells.
Pertussis Toxin (PTX) B Subunit and the Nontoxic PTX Mutant PT9K/129G Inhibit Tat-Induced TGF-β Production by NK Cells and TGF-β-Mediated NK Cell Apoptosis
Authors: Zocchi M. Raffaella , Contini Paola , Alfano Massimo , Poggi Alessandro
Published: April 2014

We show that the pertussis toxin B oligomer (PTX-B), and the PTX mutant PT9K/129G, which is safely administered in vivo, inhibit both transcription and secretion of TGF-β elicited by HIV-1 Tat in NK cells. Tat-induced TGF-β mRNA synthesis is also blocked by the ERK1 inhibitor PD98059, suggesting that ERK1 is needed for TGF-β production. Moreover, Tat strongly activates the c-Jun component of the multimolecular complex AP-1, whereas TGF-β triggers c-Fos and c-Jun. Of note, treatment of NK cells with PTX-B or PT9K/129G inhibits Tat- and TGF-β-induced activation of AP-1. TGF-β enhances starvation-induced NK cell apoptosis, significantly reduces transcription of the antiapoptotic protein Bcl-2, and inhibits Akt phosphorylation induced by oligomerization of the triggering NK cell receptor NKG2D. All these TGF-β-mediated effects are prevented by PTX-B or PT9K/129G through a PI3K-dependent mechanism, as demonstrated by use of the specific PI3K inhibitor, LY294002. Finally, PTX-B and PT9K/129G up-regulate Bcl-xL, the isoform of Bcl-x that protects cells from starvation-induced apoptosis. It is of note that in NK cells from patients with early HIV-1 infection, mRNA expression of Bcl-2 and Bcl-xL was consistently lower than that in healthy donors; interestingly, TGF-β and Tat were detected in the sera of these patients. Together, these data suggest that Tat-induced TGF-β production and the consequent NK cell failure, possibly occurring during early HIV-1 infection, may be regulated by PTX-B and PT9K/129G.
B Cell Receptor (BCR) Cross-Talk: CD40 Engagement Creates an Alternate Pathway for BCR Signaling That Activates IκB Kinase/IκBα/NF-κB without the Need for PI3K and Phospholipase Cγ
Authors: Mizuno Takuya , Rothstein Thomas L.
Published: April 2014

BCR signaling is propagated by a series of intermediaries and eventuates in NF-κB activation, among other outcomes. Interruption of several mediators that constitute the signalosome, such as PI3K and phospholipase Cγ2, completely blocks BCR signaling for NF-κB. We show here that this accepted, conventional paradigm is, in fact, limited to naive B cells. CD40L treatment reprograms normal B cells such that a novel, alternate pathway for BCR signaling is created. Through this alternate pathway BCR triggering induces nuclear NF-κB without the need for PI3K or for phospholipase Cγ2. Induction of NF-κB via the alternate pathway is accompanied by IκB kinase β (IKKβ) phosphorylation, IκBα phosphorylation, and IκBα degradation, and inhibition of IKKβ blocked IκBα degradation. Several key events in the conventional pathway, including early protein tyrosine phosphorylation, were unimpeded by generation of the alternate pathway which appears to operate in parallel, rather than in competition, with classical BCR signaling. These results demonstrate cross-talk between CD40 and BCR, such that the requirements for BCR signaling are altered by prior B cell exposure to CD40L. The alternate BCR signaling pathway bypasses multiple signalosome elements and terminates in IKKβ activation.
Expression of Killer Cell Lectin-Like Receptor G1 on Antigen-Specific Human CD8+ T Lymphocytes during Active, Latent, and Resolved Infection and its Relation with CD57
Authors: Ibegbu Chris C. , Xu Yong-Xian , Harris Wayne , Maggio David , Miller Joseph D. , Kourtis Athena P.
Published: April 2014

Killer cell lectin-like receptor G1 (KLRG1) is one of several inhibitory killer cell lectin-like receptors expressed by NK cells and T lymphocytes, mainly CD8+ effector/memory cells that can secrete cytokines but have poor proliferative capacity. Using multiparameter flow cytometry, we studied KLRG1 expression on CD8+ T cells specific for epitopes of CMV, EBV, influenza, and HIV. Over 92% of CD8+ cells specific for CMV or EBV expressed KLRG1 during the latent stage of these chronic infections. CD8+ T cell cells specific for HIV epitopes were mostly (72–89%) KLRG1+, even though not quite at the level of predominance noted with CMV or EBV. Lower frequency of KLRG1 expression was observed among CD8+ cells specific for influenza (40–73%), a resolved infection without a latent stage. We further observed that CD8+ cells expressing CD57, a marker of replicative senescence, also expressed KLRG1; however, a population of CD57−KLRG1+ cells was also identified. This population may represent a “memory” phenotype, because they also expressed CD27, CD28, CCR7, and CD127. In contrast, CD57+KLRG1+ cells did not express CD27, CD28, and CCR7, and expressed CD127 at a much lower frequency, indicating that they represent effector cells that are truly terminally differentiated. The combination of KLRG1 and CD57 expression might thus aid in refining functional characterization of CD8+ T cell subsets.