bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary Lipopolysaccharide (LPS)-Binding Protein Inhibits the LPS-Induced Lung Inflammation In Vivo
Authors: Knapp Sylvia , Florquin Sandrine , Golenbock Douglas T. , van der Poll Tom
Published: April 2014

LPS-binding protein (LBP) facilitates the interaction of the Gram-negative cell wall component LPS with CD14, thereby enhancing the immune response to LPS. Although lung epithelial cells have been reported to produce LBP in vitro, knowledge of the in vivo role of pulmonary LBP is limited. Therefore, in the present study we sought to determine the function of pulmonary LBP in lung inflammation induced by intranasal administration of LPS in vivo. Using LBP-deficient (LBP−/−) and normal wild-type mice, we show that the contribution of LBP to pulmonary LPS responsiveness depended entirely on the LPS dose. Although the inflammatory response to low dose (1 ng) LPS was attenuated in LBP−/− mice, neutrophil influx and cytokine/chemokine concentrations in the bronchoalveolar compartment were enhanced in LBP−/− mice treated with higher (>10 ng) LPS doses. This finding was specific for LBP, because the exogenous administration of LBP to LBP−/− mice reversed this phenotype and reduced the local inflammatory response to higher LPS doses. Our results indicate that pulmonary LBP acts as an important modulator of the LPS response in the respiratory tract in vivo. This newly identified function of pulmonary LBP might prove beneficial by enabling a protective immune response to low LPS doses while preventing an overwhelming, potentially harmful immune response to higher doses of LPS.
Fibulin-4 Is a Target of Autoimmunity Predominantly in Patients with Osteoarthritis
Authors: Xiang Yang , Sekine Taichi , Nakamura Hiroshi , Imajoh-Ohmi Shinobu , Fukuda Hiroyuki , Yudoh Kazuo , Masuko-Hongo Kayo , Nishioka Kusuki , Kato Tomohiro
Published: April 2014

Autoimmunity to chondrocyte-producing proteins has been reported in patients with osteoarthritis (OA) as well as in those with rheumatoid arthritis (RA). To answer whether or not OA-specific autoimmunity exist, we performed screening of chondrocyte-producing autoantigens by two-dimensional electrophoresis and Western blotting with each of 20 OA and 20 RA serum samples. We identified an apparently OA-specific autoantigen spot with a molecular mass of 52 kDa and a Isoelectric point of 4.1 as fibulin-4 by mass fingerprinting. By preparing recombinant proteins of fibulin-4, we determined prevalence of the autoantibodies to fibulin-4 in 92 patients with OA, 67 patients with RA, 40 patients with systemic lupus erythematosus, and 43 patients with systemic scleroderma. As a result, the IgG type anti-fibulin-4 autoantibodies were detected in 23.9% of sera from patients with OA, in 8.9% of sera from patients with RA, in 2.5% of sera from patients with systemic lupus erythematosus, and in 9.3% of sera from patients with systemic scleroderma. Furthermore, we immunized DBA/1J, ICR, BALB/c, and C57BL/6 mice with the recombinant fibulin-4 proteins to investigate arthritogenecity of fibulin-4. As a result, mild synovitis was detected in all of the four strains. In addition, we demonstrated expression of fibulin-4 in chondrocytes at both mRNA and protein levels in vivo and in vitro by RT-PCR, Western blotting, and immunohistochemistry. Taken together, fibulin-4, expressed in chondrocytes and recognized as an autoantigen mainly in OA rather than in RA, may play pathogenic roles in OA.
Inhibition of the Transcription Factor Foxp3 Converts Desmoglein 3-Specific Type 1 Regulatory T Cells into Th2-Like Cells
Authors: Veldman Christian , Pahl Andreas , Beissert Stefan , Hansen Wiebke , Buer Jan , Dieckmann Detlef , Schuler Gerold , Hertl Michael
Published: April 2014

Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disorder and is associated with autoantibodies against desmoglein (Dsg)3 that are regulated by Th2 cells. Recently, Dsg3-specific type 1 regulatory T cells (Tr1) were identified that are presumably critical for the maintenance of tolerance against Dsg3 because there is a much lower Dsg3-specific Tr1:Th2 ratio in the PV patients than in healthy individuals. The aim of this study was to down-regulate the transcription factor Foxp3 in Dsg3-specific Tr1 using antisense oligonucleotides because Foxp3 is constitutively expressed by the Dsg3-specific Tr1. Antisense-treated Dsg3-specific Tr1 clones lost expression of Foxp3, glucocorticoid-induced TNFR family-related receptor, and CTLA-4, and started to secrete IL-2, whereas the secretion of IL-5, TGF-β, and IL-10 remained unchanged. Moreover, antisense treatment induced a proliferative response to Dsg3 of the formerly anergic Tr1 and abrogated their suppressor activity on Dsg3-specific Th2 cell clones. Thus, inhibition of Foxp3 mRNA expression in the Tr1 induced a Th2-like phenotype. In conclusion, Foxp3 expression is inherent to Tr1 function, and modulation of Foxp3 expression in autoaggressive Th2 cells may provide a novel therapeutic approach aimed at restoring tolerance against Dsg3 in PV.
Visualization of p53264–272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor
Authors: Zhu Xiaoyun , Belmont Heather J. , Price-Schiavi Shari , Liu Bai , Lee Hyung-il , Fernandez Marilyn , Wong Richard L. , Builes Janette , Rhode Peter R. , Wong Hing C.
Published: April 2014

Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.
The Lupus-Susceptibility Locus, Sle3, Mediates Enhanced Resistance to Bacterial Infections
Authors: Mehrad Borna , Park Stacy J. , Akangire Gangaram , Standiford Theodore J. , Wu Tianfu , Zhu Jiankun , Mohan Chandra
Published: April 2014

The genetic predisposition to many autoimmune diseases is inherited as a polygenic trait. It is conceivable that some of the causative alleles in these diseases became prevalent in the population by conferring a survival benefit against environmental assaults, such as infections. We used mice cogenic for genetic loci predisposing to systemic lupus erythomatosus to test the hypothesis that some of these genetic loci protect the host from bacterial infections. Mice with the Sle3 lupus-susceptibility locus on a wild-type background were found to have enhanced antibacterial responses in the context of pneumonia and intra-abdominal sepsis than wild-type animals. This was associated with markedly augmented accumulation of neutrophils in infected tissues, and was bone marrow transferable and dependent on the presence of neutrophils, but not lymphocytes. There was no difference in in vitro leukocyte killing of bacteria nor influx of phagocytes between lupus-susceptible and wild-type animals, but neutrophils from lupus-susceptible mice displayed markedly reduced rate of apoptosis, associated with altered expression of Bcl-2 family proteins, contributing to their greater accumulation. Importantly, deliberate inhibition of apoptosis in wild-type animals significantly boosted the accumulation of neutrophils at the site of infection and resulted in an enhanced antimicrobial response. These observations support the concept that some of the genetic loci that mediate autoimmunity may also confer augmented antimicrobial innate immunity.
Regulatory Networks Induced by Live Parasites Impair Both Th1 and Th2 Pathways in Patent Lymphatic Filariasis: Implications for Parasite Persistence
Authors: Babu Subash , Blauvelt Carla P. , Kumaraswami V. , Nutman Thomas B.
Published: April 2014

Patent lymphatic filariasis is characterized by a profound down-regulation of immune responses with both parasite Ag-specific tolerance and bystander suppression. Although this down-regulation is confined to the Th1 arm of the immune system in response to parasite Ag, we hypothesized a more generalized suppression in response to live parasites. Indeed, when we examined the cytokine profile of a cohort of filaria-infected (n = 10) and uninfected (n = 10) individuals in response to live infective-stage larvae or microfilariae of Brugia malayi, we found significant impairment of both Th1 and Th2 cytokines characterized by diminished production of IFN-γ, TNF-α, IL-4, IL-5, and IL-10 in infected patients. The molecular basis of this impaired Th1/Th2 response was examined, and we identified three major networks of immunoregulation and tolerance. First, impaired induction of T-bet and GATA-3 mRNA underlies the Th1/Th2 deficiency in infected individuals. Second, regulatory networks, as evidenced by significantly increased expression of Foxp3 (natural regulatory T cell marker) and regulatory effectors such as TGF-β, CTLA-4, PD-1, ICOS, and indoleamine 2,3-dioxygenase play an important role in immunosuppression. Third, the compromise of effector T cell function is mediated by the enhanced induction of anergy-inducing factors cbl-b, c-cbl (cbl is abbreviation for Casitas B lymphoma), Itch, and Nedd4. Indeed, blocking CTLA-4 or neutralizing TGF-β restored the ability to mount Th1/Th2 responses to live parasites and reversed the induction of anergy-inducing factors. Hence, we conclude that a profound impairment of live parasite-specific Th1 and Th2 immune responses occurs in lymphatic filariasis that is governed at the transcriptional level by a complex interplay of inhibitory mediators.
HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes
Authors: Takaki Toshiyuki , Marron Michele P. , Mathews Clayton E. , Guttmann Stephen T. , Bottino Rita , Trucco Massimo , DiLorenzo Teresa P. , Serreze David V.
Published: April 2014

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic β cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.
IL-10: The Master Regulator of Immunity to Infection
Authors: Couper Kevin N. , Blount Daniel G. , Riley Eleanor M.
Published: April 2014

IL-10 is an anti-inflammatory cytokine. During infection it inhibits the activity of Th1 cells, NK cells, and macrophages, all of which are required for optimal pathogen clearance but also contribute to tissue damage. In consequence, IL-10 can both impede pathogen clearance and ameliorate immunopathology. Many different types of cells can produce IL-10, with the major source of IL-10 varying in different tissues or during acute or chronic stages of the same infection. The priming of these various IL-10-producing populations during infections is not well understood and it is not clear whether the cellular source of IL-10 during infection dictates its cellular target and thus its outcome. In this article we review the biology of IL-10, its cellular sources, and its role in viral, bacterial, and protozoal infections.
Cutting Edge: Dendritic Cells Prime a High Avidity CTL Response Independent of the Level of Presented Antigen
Authors: Kroger Charles J. , Amoah Samuel , Alexander-Miller Martha A.
Published: April 2014

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.
Cutting Edge: Enhanced IL-2 Signaling Can Convert Self-Specific T Cell Response from Tolerance to Autoimmunity
Authors: Waithman Jason , Gebhardt Thomas , Davey Gayle M. , Heath William R. , Carbone Francis R.
Published: April 2014

Naive and memory T cells show differences in their response to antigenic stimulation. We examined whether this difference extended to the peripheral deletion of T cells reactive to self-Ag or, alternatively, the induction of autoimmunity. Our results show that although both populations where susceptible to deletion, memory T cells, but not naive T cells, also gave rise to autoimmunity after in vivo presentation of skin-derived self-Ags. The same migratory dendritic cells presented self-Ag to both naive and memory T cell populations, but only the latter had significant levels of the effector molecule granzyme B. Memory T cells also expressed increased levels of the high affinity IL-2 receptor chain after self-Ag recognition. Provision of IL-2 signaling using a stimulatory complex of anti-IL-2 Ab and IL-2 drove the otherwise tolerant naive T cells toward an autoimmune response. Therefore, enhanced IL-2 signaling can act as a major selector between tolerance and autoimmunity.
Virus-Like Display of a Neo-Self Antigen Reverses B Cell Anergy in a B Cell Receptor Transgenic Mouse Model
Authors: Chackerian Bryce , Durfee Marisa R. , Schiller John T.
Published: April 2014

The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4+ Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.
Danger Signaling through the Inflammasome Acts as a Master Switch between Tolerance and Sensitization
Authors: Watanabe Hideki , Gehrke Samuel , Contassot Emmanuel , Roques Stéphanie , Tschopp Jürg , Friedmann Peter S. , French Lars E. , Gaide Olivier
Published: April 2014

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1β is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.
Conserved Mycobacterial Lipoglycoproteins Activate TLR2 but Also Require Glycosylation for MHC Class II-Restricted T Cell Activation
Authors: Sieling Peter A. , Hill Preston J. , Dobos Karen M. , Brookman Kerry , Kuhlman Andrew M. , Fabri Mario , Krutzik Stephan R. , Rea Thomas H. , Heaslip Darragh G. , Belisle John T. , Modlin Robert L.
Published: April 2014

CD4+ T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4+ T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by α-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.
Differential Control of T Regulatory Cell Proliferation and Suppressive Activity by Mature Plasmacytoid versus Conventional Spleen Dendritic Cells
Authors: Ouabed Asmahan , Hubert Francois-Xavier , Chabannes Dominique , Gautreau Laetitia , Heslan Michèle , Josien Régis
Published: April 2014

Anergy and suppression are cardinal features of CD4+CD25+Foxp3+ T cells (T regulatory cells (Treg)) which have been shown to be tightly controlled by the maturation state of dendritic cells (DC). However, whether lymphoid organ DC subsets exhibit different capacities to control Treg is unclear. In this study, we have analyzed, in the rat, the role of splenic CD4+ and CD4− conventional DC and plasmacytoid DC (pDC) in allogeneic Treg proliferation and suppression in vitro. As expected, in the absence of exogenous IL-2, Treg did not expand in response to immature DC. Upon TLR-induced maturation, all DC became potent stimulators of CD4+CD25− T cells, whereas only TLR7- or TLR9-matured pDC induced strong proliferation of CD4+CD25+Foxp3+ T cells in the absence of exogenous IL-2. This capacity of pDC to reverse Treg anergy required cell contact and was partially CD86 dependent and IL-2 independent. In suppression assays, Treg strongly suppressed proliferation and IL-2 and IFN-γ production by CD4+CD25− T cells induced by mature CD4+ and CD4− DC. In contrast, upon stimulation by mature pDC, proliferating Treg suppressed IL-2 production by CD25− cells but not their proliferation or IFN-γ production. Taken together, these results suggest that anergy and the suppressive function of Treg are differentially controlled by DC subsets.
Tumor-Induced Impairment of TCR Signaling Results in Compromised Functionality of Tumor-Infiltrating Regulatory T Cells
Authors: Lutsiak M. E. Christine , Tagaya Yutaka , Adams Anthony J. , Schlom Jeffrey , Sabzevari Helen
Published: April 2014

This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4+CD25+FoxP3+GITRhighCD27lowCD62L−. The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that 1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and 2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.
HIV-1-Specific T Cell Precursors with High Proliferative Capacity Correlate with Low Viremia and High CD4 Counts in Untreated Individuals
Authors: Calarota Sandra A. , Foli Andrea , Maserati Renato , Baldanti Fausto , Paolucci Stefania , Young Mary A. , Tsoukas Christos M. , Lisziewicz Julianna , Lori Franco
Published: April 2014

Evidences have recently suggested that the preservation of vaccine-induced memory rather than effector T cells is essential for better outcome and survival following pathogenic SIV challenge in macaques. However, an equivalent demonstration in humans is missing, and the immune correlates of HIV-1 control have been only partially characterized. We focused on the quantification of Ag-specific T cell precursors with high proliferative capacity (PHPC) using a peptide-based cultured IFN-γ ELISPOT assay (PHPC assay), which has been shown to identify expandable memory T cells. To determine which responses correlate with viral suppression and positive immunologic outcome, PBMC from 32 chronically untreated HIV-1-infected individuals were evaluated in response to peptide pools, representing the complete HIV-1 Gag, Nef, and Rev proteins, by PHPC and IFN-γ ELISPOT assay, which instead identifies effector T cells with low proliferative capacity. High magnitude of Gag-specific PHPC, but not ELISPOT, responses significantly correlated with low plasma viremia, due to responses directed toward p17 and p15 subunits. Only Gag p17-specific PHPC response significantly correlated with high CD4 counts. Analysis of 20 additional PBMC samples from an independent cohort of chronically untreated HIV-1-infected individuals confirmed the correlation between Gag p17-specific PHPC response and either plasma viremia (inverse correlation) or CD4 counts (direct correlation). Our results indicate that the PHPC assay is quantitatively and qualitatively different from the ELISPOT assay, supporting that different T cell populations are being evaluated. The PHPC assay might be an attractive option for individual patient management and for the design and testing of therapeutic and prophylactic vaccines.
Plasmacytoid Dendritic Cells Migrate in Afferent Skin Lymph
Authors: Pascale Florentia , Contreras Vanessa , Bonneau Michel , Courbet Alexandre , Chilmonczyk Stefan , Bevilacqua Claudia , Eparaud Mathieu , Niborski Violeta , Riffault Sabine , Balazuc Anne-Marie , Foulon Eliane , Guzylack-Piriou Laurence , Riteau Beatrice , Hope Jayne , Bertho Nicolas , Charley Bernard , Schwartz-Cornil Isabelle
Published: April 2014

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor BnegCD11cneg subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-γ production in allogeneic CD4pos T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4posSIRPpos subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.
SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes
Authors: Garaude Johan , Farrás Rosa , Bossis Guillaume , Charni Seyma , Piechaczyk Marc , Hipskind Robert A. , Villalba Martin
Published: April 2014

The AP-1 family member JunB is a critical regulator of T cell function. JunB is a transcriptional activator of various cytokine genes, such as IL-2, IL-4, and IL-10; however, the post-translational modifications that regulate JunB activity in T cells are poorly characterized. We show here that JunB is conjugated with small ubiquitin-like modifier (SUMO) on lysine 237 in resting and activated primary T cells and T cell lines. Sumoylated JunB associated with the chromatin-containing insoluble fraction of cells, whereas nonsumoylated JunB was also in the soluble fraction. Blocking JunB sumoylation by mutation or use of a dominant-negative form of the SUMO-E2 Ubc-9 diminished its ability to transactivate IL-2 and IL-4 reporter genes. In contrast, nonsumoylable JunB mutants showed unimpaired activity with reporter genes controlled by either synthetic 12-O-tetradecanoylphorbol-13-acetate response elements or NF-AT/AP-1 and CD28RE sites derived from the IL-2 promoter. Ectopic expression of JunB in activated human primary CD4+ T cells induced activation of the endogenous IL-2 promoter, whereas the nonsumoylable JunB mutant did not. Thus, our work demonstrates that sumoylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation.
Reovirus Activates Human Dendritic Cells to Promote Innate Antitumor Immunity
Authors: Errington Fiona , Steele Lynette , Prestwich Robin , Harrington Kevin J. , Pandha Hardev S. , Vidal Laura , de Bono Johann , Selby Peter , Coffey Matt , Vile Richard , Melcher Alan
Published: April 2014

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-α, TNF-α, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-κB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-γ production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-γ) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus’s direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.
Phospholipase D1 Plays a Key Role in TNF-α Signaling
Authors: Sethu Swaminathan , Mendez-Corao Grecia , Melendez Alirio J.
Published: April 2014

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-α is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-α. However, the signaling network initiated by TNF-α in human leukocytes is still poorly understood. In this study, we report that TNF-α activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFκB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-α signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-α to activation of the phosphorylation of ERK1/2 and the activation of NFκB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-α-induced production of several important cytokines, such as IL-1β, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-α and its functional role for coordinating the signals to inflammatory responses.
Protective Immunosurveillance and Therapeutic Antitumor Activity of γδ T Cells Demonstrated in a Mouse Model of Prostate Cancer
Authors: Liu Zhiyong , Eltoum Isam-Eldin A. , Guo Ben , Beck Benjamin H. , Cloud Gretchen A. , Lopez Richard D.
Published: April 2014

In contrast to Ag-specific αβ T cells, γδ T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that γδ T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of γδ T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that γδ T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with γδ T cell-deficient mice (TCRδ−/− mice) yielding TRAMP × TCRδ−/− mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic γδ T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with γδ T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous γδ T cells for the treatment of prostate cancer.
Long-Term Control of Alloreactive B Cell Responses by the Suppression of T Cell Help
Authors: Li Yijin , Ma Lianli , Yin Dengping , Shen JiKun , Chong Anita S.
Published: April 2014

Alloantibodies can play a key role in acute and chronic allograft rejection. However, relatively little is known of factors that control B cell responses following allograft tolerance induction. Using 3-83 Igi mice expressing an alloreactive BCR, we recently reported that allograft tolerance was associated with the sustained deletion of the alloreactive B cells at the mature, but not the immature, stage. We have now investigated the basis for the long-term control of alloreactive B cell responses in a non-BCR-transgenic model of C57BL/6 cardiac transplantation into BALB/c recipients treated with anti-CD154 and transfusion of donor-specific spleen cells. We demonstrate that the long-term production of alloreactive Abs by alloreactive B cells is actively regulated in tolerant BALB/c mice through the dominant suppression of T cell help. Deletion of CD25+ cells resulted in a loss of tolerance and an acquisition of the ability to acutely reject allografts. In contrast, the restoration of alloantibody responses required both the deletion of CD25+ cells and the reconstitution of alloreactive B cells. Collectively, these data suggest that alloreactive B cell responses in this model of tolerance are controlled by dominant suppression of T cell help as well as the deletion of alloreactive B cells in the periphery.
Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM
Authors: Kiefer Kerstin , Nakajima Pamela B. , Oshinsky Jennifer , Seeholzer Steven H. , Radic Marko , Bosma Gayle C. , Bosma Melvin J.
Published: April 2014

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM−/low). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM−/low anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vλx) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgMlow transitional splenic B cells of wild-type mice.
Immunological and Genetic Evidence for a Crucial Role of IL-10 in Cutaneous Lesions in Humans Infected with Leishmania braziliensis
Authors: Salhi Adnene , Rodrigues Virmondes , Santoro Ferrucio , Dessein Helia , Romano Audrey , Castellano Lucio Roberto , Sertorio Mathieu , Rafati Sima , Chevillard Christophe , Prata Aluisio , Alcaïs Alexandre , Argiro Laurent , Dessein Alain
Published: April 2014

In populations exposed to Leishmania braziliensis, certain subjects develop skin ulcers, whereas others are naturally protected against cutaneous leishmaniasis. We have evaluated which cytokines are most crucial in the development of skin lesions. We found that active lesions occur in subjects with polarized Th2 or mixed Th1/Th2 responses, both associated with elevated IL-10 production. IL-10 was strongly associated (p = 0.004, odd ratio (OR) = 6.8, confidence interval = 1.9–25) with lesions, excluding IFN-γ, IL-12, TNF, IL-13, and IL-4 from the regression model. IL-10 was produced by blood monocytes and CD4+CD25+ T lymphocytes (mostly Foxp3+). However, we did not observe any difference between the number of these cells present in the blood of subjects with active lesions and those present in resistant subjects. Genetic analysis of the IL10−819C/T polymorphism, located in the IL10 promoter, showed that the C allele increased the risk of lesions (OR = 2.5 (1.12–5.7), p = 0.003). Functional analysis of these variants showed allele-specific binding of nuclear factors. The IL10-819C/C genotype was associated with higher levels of IL-10 than C/T and T/T genotypes. These observations demonstrate an important role for IL-10 in skin lesions in humans infected with L. braziliensis, and identify circulating monocytes and Tregs as principal sources of IL-10 in these patients.
Alternatively Activated Myeloid Cells Limit Pathogenicity Associated with African Trypanosomiasis through the IL-10 Inducible Gene Selenoprotein P
Authors: Bosschaerts Tom , Guilliams Martin , Noel Wim , Hérin Michel , Burk Raymond F. , Hill Kristina E. , Brys Lea , Raes Geert , Ghassabeh Gholamreza Hassanzadeh , De Baetselier Patrick , Beschin Alain
Published: April 2014

Uncontrolled inflammation is a major cause of tissue injury/pathogenicity often resulting in death of a host infected with African trypanosomes. Thus, comparing the immune response in hosts that develop different degrees of disease severity represents a promising approach to discover processes contributing to trypanosomiasis control. It is known that limitation of pathogenicity requires a transition in the course of infection, from an IFN-γ-dependent response resulting in the development of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alternatively activated myeloid cells (M2). Herein, mechanisms and downstream effectors by which M2 contribute to lower the pathogenicity and the associated susceptibility to African trypanosomiasis have been explored. Gene expression analysis in IL-10 knockout and wild-type mice, that are susceptible and relatively resistant to Trypanosoma congolense infection, respectively, revealed a number of IL-10-inducible genes expressed by M2, including Sepp1 coding for selenoprotein P. Functional analyses confirm that selenoprotein P contributes to limit disease severity through anti-oxidant activity. Indeed, Sepp1 knockout mice, but not Sepp1Δ240-361 mice retaining the anti-oxidant motif but lacking the selenium transporter domain of selenoprotein P, exhibited increased tissue injury that associated with increased production of reactive oxygen species and increased apoptosis in the liver immune cells, reduced parasite clearance capacity of myeloid cells, and decreased survival. These data validate M2-associated molecules as functioning in reducing the impact of parasite infection on the host.
Neisseria gonorrhoeae-Induced Human Defensins 5 and 6 Increase HIV Infectivity: Role in Enhanced Transmission
Authors: Klotman Mary E. , Rapista Aprille , Teleshova Natalia , Micsenyi Amanda , Jarvis Gary A. , Lu Wuyuan , Porter Edith , Chang Theresa L.
Published: April 2014

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4+ T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.
T Cell Response Mediated by Myeloid Cell-Derived IL-12 Is Responsible for Porphyromonas gingivalis-Induced Periodontitis in IL-10-Deficient Mice
Authors: Sasaki Hajime , Suzuki Noriyuki , Kent Ralph , Kawashima Nobuyuki , Takeda Junji , Stashenko Philip
Published: April 2014

Periodontal disease is a chronic inflammatory disease in the oral cavity, which culminates in alveolar bone loss. Porphyromonas gingivalis is a consensus periodontal pathogen that has been implicated in adult forms of periodontitis. We previously demonstrated that IL-10-deficient mice exhibit a hyperinflammatory phenotype and are highly susceptible to P. gingivalis-induced periodontitis, indicating an important anti-inflammatory effect of IL-10 in suppressing bone loss. In this study, we analyzed the pathway(s) by which IL-10 deficiency leads to severe P. gingivalis-induced periodontitis. Because Stat3 is essential in IL-10 signaling, immune cell-specific Stat3-deficient mice were subjected to P. gingivalis infection to identify the key IL-10-responsive cells in preventing periodontitis. Myeloid cell-specific Stat3-deficient mice exhibited increased periodontal bone loss (p < 0.001), whereas T cell- and B cell-specific Stat3 mice were resistant, suggesting that macrophages (MP) and/or polymorphonuclear leukocytes are the key target cells normally suppressed by IL-10. Myeloid cell-specific Stat3-deficient mice exhibited elevated gingival CD40L gene expression in vivo compared with wild-type controls (p < 0.01), and Stat3-deficient MPs exhibited vigorous P. gingivalis-stimulated IL-12 production in vitro and induced elevated Ag-specific T cell proliferation compared with wild-type MPs (p < 0.01). Of importance, both IL-12p40/IL-10 and T cell/IL-10 double-deficient mice were resistant to P. gingivalis-induced periodontitis, demonstrating roles for both IL-12p40 and T cells in pathogenesis in a hyperinflammatory model of disease. These data demonstrate that P. gingivalis-induced periodontitis in IL-10-deficient mice is dependent upon IL-12p40-mediated proinflammatory T cell responses.
Inhibition of Yin Yang 1-Dependent Repressor Activity of DR5 Transcription and Expression by the Novel Proteasome Inhibitor NPI-0052 Contributes to its TRAIL-Enhanced Apoptosis in Cancer Cells
Authors: Baritaki Stavroula , Suzuki Eriko , Umezawa Kazuo , Spandidos Demetrios A. , Berenson James , Daniels Tracy R. , Penichet Manuel L. , Jazirehi Ali R. , Palladino Michael , Bonavida Benjamin
Published: April 2014

TRAIL promotes apoptotic tumor cell death; however, TRAIL-resistant tumors need to be sensitized to reverse resistance. Proteasome inhibitors potentiate TRAIL apoptosis in vitro and in vivo and correlate with up-regulation of death receptor 5 (DR5) via an unknown mechanism. We hypothesized that the proteasome inhibitor NPI-0052 inhibits the transcription repressor Yin Yang 1 (YY1) which regulates TRAIL resistance and negatively regulates DR5 transcription. Treatment of PC-3 and Ramos cells with NPI-0052 (≤2.5 nM) and TRAIL sensitizes the tumor cells to TRAIL-induced apoptosis. By comparison to bortezomib, a 400-fold less concentration of NPI-0052 was used. NPI-0052 up-regulated DR5 reporter activity and both surface and total DR5 protein expression. NPI-0052-induced inhibition of NF-κB activity was involved in TRAIL sensitization as corroborated by the use of the NF-κB inhibitor dehydroxymethylepoxyquinomicin. NPI-0052 inhibited YY1 promoter activity as well as both YY1 mRNA and protein expression. The direct role of NPI-0052-induced inhibition of YY1 and up-regulation of DR5 in the regulation of TRAIL sensitivity was demonstrated by the use of YY1 small interfering RNA. The NPI-0052-induced sensitization to TRAIL involved activation of the intrinsic apoptotic pathway and dysregulation of genes that regulate apoptosis. The NPI-0052 concentrations used for TRAIL sensitization were not toxic to human hematopoetic stem cells. The present findings demonstrate, for the first time, the potential mechanism by which a proteasome inhibitor, like NPI-0052, inhibits the transcription repressor YY1 involved in TRAIL resistance and DR5 regulation. The findings also suggest the therapeutic application of subtoxic NPI-0052 concentrations in combination with TRAIL/agonist DR4/DR5 mAbs in the treatment of TRAIL-resistant tumors.
Bactericidal Action of a Complement-Independent Antibody against Relapsing Fever Borrelia Resides in Its Variable Region
Authors: LaRocca Timothy J. , Katona Laura I. , Thanassi David G. , Benach Jorge L.
Published: April 2014

A single chain variable fragment (scFv) of CB515, a complement-independent bactericidal monoclonal IgM against a relapsing fever Borrelia, was constructed to investigate the region wherein the unique bactericidal function resides. Monomeric CB515 scFv (26 kDa) was capable of binding its Ag on whole organisms and by immunoblot. This binding was shown to be species and serotype-specific to the 19 kDa variable small protein, recognized by its parent monoclonal IgM. A dose-dependent bactericidal effect of the CB515 scFv was detected by direct enumeration of spirochetes. Spirochetes incubated with the CB515 scFv before inoculation into mice grew into escape mutants, whereas spirochetes incubated with an irrelevant scFv developed as the original infecting serotype. This bactericidal effect, as seen at the ultrastructural level, was due to disruption of the outer membrane and to severe membrane blebbing eventually progressing to lysis. These results indicate that the variable region of CB515 is responsible for this bactericidal activity and that the constant region of the Ab is dispensable.