bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial Colonization Drives Lymphocyte Accumulation and Differentiation in the Follicle-Associated Epithelium of Peyer’s Patches
Authors: Yamanaka Takeshi , Helgeland Lars , Farstad Inger Nina , Fukushima Hisanori , Midtvedt Tore , Brandtzaeg Per
Published: April 2014

Peyer’s patches (PPs) are lined by follicle-associated epithelium (FAE) with Ag-transporting M cells. To investigate the spatial relationships of B cells, T cells, and dendritic cells (DCs) in PPs during microbial colonization, their in situ redistribution was examined in germfree (GF) rats exposed to a conventional pathogen-free microflora (conventionalized, CV). Although occasional B and T cells occurred in the FAE of GF rats, it contained mainly immature DCs (CD4+CD86−), whereas mature DCs (CD86high) were seen in the interfollicular zones even under GF conditions. In CV rats, DCs had disappeared from the FAE, which instead contained clusters by B and T cells associated with induction of putative M cell pockets. CD86 was seen neither in the FAE nor in the follicles under GF conditions, but it became apparent on intraepithelial B cells 5 wk after colonization. The level of CD86 on these B cells was comparable to that on germinal center B cells, although the B cell follicles did not show direct contact with the M cell areas. B cells in the follicular mantles acquired Bcl-2 after 12 wk in CV rats, whereas B cells in the FAE did not express Bcl-2 at a substantial level throughout the experimental period. The cellular redistribution patterns and phenotypic characteristics observed after colonization suggested that immature DCs, but not B cells, are involved in Ag presentation during primary immune responses against intestinal bacteria. However, the spatial cellular relationships sequentially being established among DCs, B cells, and T cells in PPs, are most likely important for the induction of post-germinal center B cells subsequently residing within the M cell pockets.
Indoleamine 2,3-Dioxygenase Is Regulated by IFN-γ in the Mouse Placenta DuringListeria monocytogenesInfection
Authors: Mackler Ari M. , Barber Ellen M. , Takikawa Osamu , Pollard Jeffrey W.
Published: April 2014

The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is expressed in macrophages that have been differentiated in the presence of CSF-1 and is important in the containment of intracellular pathogens. IDO also appears to play a role in suppression of T cell responses in a variety of contexts. In the placenta, its enzymatic activity is believed to establish a chemical barrier that protects the fetal allograft from T cell-mediated immune aggression. We have studied the regulation of IDO in the utero-placental unit of mice following infection with the Gram-positive, intracellular bacterium Listeria monocytogenes that has a predilection for replication in the decidua basalis. IDO mRNA and protein expression is enhanced in the utero-placental unit following infection with L. monocytogenes. However, in contrast to the human where IDO is expressed by the CSF-1R-positive syncytial trophoblast, IDO is not expressed in murine trophoblastic tissue but instead is found in stromal cells of the decidua basalis and metrial gland and following infection, in endothelial cells. Using mice carrying null mutations in cytokine/growth factor genes, we explored the regulation of IDO in the placenta. Consistent with the absence of CSF-1R expression in the IDO-expressing cells of mice, neither the basal levels of IDO nor its induction following infection is affected by the absence of CSF-1. However, although the basal level of IDO is normal, the enhanced expression during Listeriosis is completely abrogated in the absence of IFN-γ, a cytokine required for the resolution of this infection. These data suggest that IDO plays a role in resolving bacterial infection in the placenta while at the same time maintaining a barrier to T cells whose presence might result in fetal rejection.
A Critical Role for Syk Protein Tyrosine Kinase in Fc Receptor-Mediated Antigen Presentation and Induction of Dendritic Cell Maturation
Authors: Sedlik Christine , Orbach Daniel , Veron Philippe , Schweighoffer Edina , Colucci Francesco , Gamberale Romina , Ioan-Facsinay Andrea , Verbeek Sjef , Ricciardi-Castagnoli Paola , Bonnerot Christian , Tybulewicz Victor L. J. , Di Santo James , Amigorena Sebastian
Published: April 2014

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called “maturation”, which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.
Suppressor of Cytokine Signaling-1 Has IFN-γ-Independent Actions in T Cell Homeostasis
Authors: Cornish Ann L. , Davey Gayle M. , Metcalf Donald , Purton Jared F. , Corbin Jason E. , Greenhalgh Christopher J. , Darwiche Rima , Wu Li , Nicola Nicos A. , Godfrey Dale I. , Heath William R. , Hilton Douglas J. , Alexander Warren S. , Starr Robyn
Published: April 2014

Suppressor of cytokine signaling (SOCS)-1 is a member of a family of proteins that negatively regulate cytokine signaling pathways. We have previously established that SOCS-1 is a key regulator of IFN-γ signaling and that IFN-γ is responsible for the complex inflammatory disease that leads to the death of SOCS-1-deficient mice. In this study, we provide evidence that SOCS-1 is also a critical regulator of IFN-γ-independent immunoregulatory factors. Mice lacking both SOCS-1 and IFN-γ, although outwardly healthy, have clear abnormalities in their immune system, including a reduced ratio of CD4:CD8 T cells in lymphoid tissues and increased expression of T cell activation markers. To examine the contribution of TCR Ag specificity to these immune defects, we have generated two lines of SOCS-1-deficient mice expressing a transgenic TCR specific for an exogenous Ag, OVA (OT-I and OT-II). Although TCR transgenic SOCS-1−/− mice have a longer lifespan than nontransgenic SOCS-1−/− mice, they still die as young adults with inflammatory disease and the TCR transgenic SOCS-1−/− T cells appear activated despite the absence of OVA. This suggests that both Ag-dependent and -independent mechanisms contribute to the disease in SOCS-1-deficient mice. Thus, SOCS-1 is a critical regulator of T cell activation and homeostasis, and its influence extends beyond regulating IFN-γ signaling.
Up-Regulation of Early Growth Response Gene-1 Via the CXCR3 Receptor Induces Reactive Oxygen Species and Inhibits Na+/K+-ATPase Activity in an Immortalized Human Proximal Tubule Cell Line
Authors: Bek Martin J. , Reinhardt Hans C. , Fischer Karl-Georg , Hirsch Jochen R. , Hupfer Charlotte , Dayal Eileen , Pavenstädt Hermann
Published: April 2014

The CXCR3 chemokine receptor, a member of the CXCR family, has been linked to a pathological role in autoimmune disease, inflammatory disease, allograft rejection, and ischemia. In the kidney, expression of the CXCR3 receptor and its ligands is up-regulated in states of glomerulonephritis and in allograft rejection, but little is known about the expression and functional role the CXCR3 receptor might play. Here, we study the function of the CXCR3 chemokine receptor in an immortalized human proximal tubular cell line (IHKE-1). Stimulation of the CXCR3 receptor by its selective agonist monokine induced by IFN-γ leads via a Ca2+-dependent mechanism to an up-regulation of early growth response gene (EGR)-1. Overexpression of EGR-1 induces down-regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase and stimulates the generation of reactive oxygen species (ROS) via the NADH/NADPH-oxidase system. EGR-1 overexpression or treatment with monokine induced by IFN-γ resulted in a ROS-dependent inhibition of basolateral Na+/K+-ATPase activity, compromising sodium transport in these cells. Thus, activation of the CXCR3 receptor in proximal tubular cells might disturb natriuresis during inflammatory and ischemic kidney disease via EGR-1-mediated imbalance of ROS.
Systematic Analysis of the Combinatorial Nature of Epitopes Recognized by TCR Leads to Identification of Mimicry Epitopes for Glutamic Acid Decarboxylase 65-Specific TCRs
Authors: Uemura Yasushi , Senju Satoru , Maenaka Katsumi , Iwai Leo Kei , Fujii Shinji , Tabata Hiroki , Tsukamoto Hirotake , Hirata Shinya , Chen Yu-Zhen , Nishimura Yasuharu
Published: April 2014

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4+ T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.
Targeting with Bovine CD154 Enhances Humoral Immune Responses Induced by a DNA Vaccine in Sheep
Authors: Manoj Sharmila , Griebel Philip J. , Babiuk Lorne A. , van Drunen Littel-van den Hurk Sylvia
Published: April 2014

CD40-CD154 interactions play an important role in regulating humoral and cell-mediated immune responses. Recently, these interactions have been exploited for the development of therapeutic and preventive treatments. The objective of this study was to test the ability of bovine CD154 to target a plasmid-encoded Ag to CD40-expressing APCs. To achieve this, a plasmid coding for bovine CD154 fused to a truncated secreted form of bovine herpesvirus 1 glycoprotein D (tgD), pSLIAtgD-CD154, was constructed. The chimeric tgD-CD154 was expressed in vitro in COS-7 cells and reacted with both glycoprotein D- and CD154-specific Abs. Both tgD and tgD-CD154 were capable of binding to epithelial cells, whereas only tgD-CD154 bound to B cells. Furthermore, dual-labeling of ovine PBMCs revealed that tgD-CD154 was bound by primarily B cells. The functional integrity of the tgD-CD154 chimera was confirmed by the induction of both IL-4-dependent B cell proliferation and tgD-specific lymphoproliferative responses in vitro. Finally, sheep immunized with pSLIAtgD-CD154 developed a more rapid primary tgD-specific Ab response and a significantly stronger tgD-specific secondary response when compared with animals immunized with pSLIAtgD and control animals. Similarly, virus-neutralizing Ab titers were significantly higher after secondary immunization with pSLIAtgD-CD154. These results demonstrate that using CD154 to target plasmid-expressed Ag can significantly enhance immune responses induced by a DNA vaccine.
Cutting Edge: Perforin Down-Regulates CD4 and CD8 T Cell-Mediated Immune Responses to a Transplanted Organ
Authors: Bose Anirban , Inoue Yoshihiko , Kokko Kenneth E. , Lakkis Fadi G.
Published: April 2014

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.
Cutting Edge: Immediate RANTES Secretion by Resting Memory CD8 T Cells Following Antigenic Stimulation
Authors: Walzer Thierry , Marçais Antoine , Saltel Frédéric , Bella Chantal , Jurdic Pierre , Marvel Jacqueline
Published: April 2014

The efficiency of CD8 memory response relies partially on the modification of cellular functional capacities. To identify effector functions that can be modified following priming, we have compared the chemokines produced by naive and memory CD8 T cells. Our results show that in contrast to naive cells, resting memory CD8 T cells contain high levels of RANTES mRNA. As a result, they have the capacity to rapidly secrete RANTES upon ex vivo antigenic stimulation. In contrast to that of IFN-γ, RANTES secretion is mainly due to the translation of the pre-existing mRNA.
Cutting Edge: Distinct Toll-Like Receptor 2 Activators Selectively Induce Different Classes of Mediator Production from Human Mast Cells
Authors: McCurdy Jeffrey D. , Olynych Timothy J. , Maher Lauren H. , Marshall Jean S.
Published: April 2014

Mast cells play a critical role in host defense against bacterial infection. Murine mast cells produce cytokines in response to bacterial peptidoglycan and LPS via Toll-like receptor (TLR) TLR2- and TLR4-dependent mechanisms. The expression of TLRs by human mast cells and responses to known TLR activators was examined. Human mast cells expressed mRNA for TLR1, TLR2, and TLR6 but not TLR4. Bacterial peptidoglycan and yeast zymosan were potent inducers of GM-CSF and IL-1β and also induced substantial short-term cysteinyl leukotriene generation. In contrast, a synthetic triacylated lipopeptide induced short-term degranulation but failed to induce cysteinyl leukotriene production. The TLR4 activator Escherichia coli LPS did not induce a GM-CSF, IL-1β leukotriene, or degranulation response. These data demonstrate highly selective production of different classes of mast cell mediators in response to distinct TLR activators of potential importance to the host response to bacterial or fungal pathogens.
Cutting Edge: Both Activating and Inhibitory Fc Receptors Expressed on Mast Cells Regulate Experimental Allergic Encephalomyelitis Disease Severity
Authors: Robbie-Ryan Michaela , Tanzola Melinda B. , Secor Virginia H. , Brown Melissa A.
Published: April 2014

Mast cell-deficient mice (W/Wv) exhibit significantly reduced severity of experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. In this study, the contribution of FcR-mediated mast cell activation to disease was examined. W/Wv mice were reconstituted i.v. with bone marrow-derived mast cells (BMMCs) from wild-type mice or those lacking functional FcRs. Eight weeks later, EAE was induced by immunization with the myelin oligodendrocyte glycoprotein 35–55 peptide. Disease scores were analyzed in reconstituted mice and compared with age-matched W/Wv mice and wild-type littermates. Mice reconstituted with FcRγ−/− BMMCs or FcγRIII−/− BMMCs exhibited less severe clinical symptoms similar to W/Wv controls, while reconstitution with FcRIIB−/− BMMCs resulted in disease significantly more severe than wild-type controls. Notably, mice reconstituted with FcγRIII−/− BMMC exhibit a relapsing-remitting course of disease. These data demonstrate that both activating and inhibitory FcRs expressed on mast cells influence the course of EAE.
Cutting Edge: Carbohydrate Profiling Identifies New Pathogens That Interact with Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin on Dendritic Cells
Authors: Appelmelk Ben J. , van Die Irma , van Vliet Sandra J. , Vandenbroucke-Grauls Christina M. J. E. , Geijtenbeek Teunis B. H. , van Kooyk Yvette
Published: April 2014

Dendritic cells (DC) are instrumental in handling pathogens for processing and presentation to T cells, thus eliciting an appropriate immune response. C-type lectins expressed by DC function as pathogen-recognition receptors; yet their specificity for carbohydrate structures on pathogens is not fully understood. In this study, we analyzed the carbohydrate specificity of DC-specific ICAM-3-grabbing nonintegrin (SIGN)/CD209, the recently documented HIV-1 receptor on DC. Our studies show that DC-SIGN binds with high affinity to both synthetic mannose- and fucose-containing glycoconjugates. These carbohydrate structures are abundantly expressed by pathogens as demonstrated by the affinity of DC-SIGN for natural surface glycans of the human pathogens Mycobacterium tuberculosis, Helicobacter pylori, Leishmania mexicana, and Schistosoma mansoni. This analysis expands our knowledge on the carbohydrate and pathogen-specificity of DC-SIGN and identifies this lectin to be central in pathogen-DC interactions.
CD40 Ligand in Pathogenesis of Autoimmune Ovarian Disease of Day 3-Thymectomized Mice: Implication for CD40 Ligand Antibody Therapy
Authors: Sharp Colin , Thompson Claire , Samy Eileen T. , Noelle Randolph , Tung Kenneth S. K.
Published: April 2014

The blockade of CD40 ligand (CD40L) is effective in autoimmune disease prevention. Recently, a brief period of CD40L mAb treatment was reported to induce tolerance and enhancement of CD4+CD25+ regulatory T cell activity. We therefore determined the efficacy of CD40L mAb treatment in autoimmunity that resulted from CD4+CD25+ regulatory T cell deficiency. Autoimmune ovarian disease (AOD) and oocyte autoantibody response of day 3-thymectomized (d3tx) mice were inhibited by continuous CD40L mAb treatment from day 3, or from days 10–14, whereas CD40L mAb treatment confined to the neonatal week was ineffective. The enhanced expression of memory markers (CD44 and CD62Llow) on CD4+ T cells of the d3tx mice was unaffected by CD40L mAb treatment. In contrast, their increased T cell activation markers (CD69 and CD25) were eliminated by CD40L mAb treatment. Moreover, ex vivo activated T cells of d3tx mice expressed elevated intracellular IFN-γ, and this was also blocked by CD40L mAb. The memory T cells, although nonpathogenic in CD40L mAb-positive environment, transferred severe AOD to CD40L mAb− neonatal recipients. Most importantly, CD40L mAb treatment inhibited AOD in recipients of T cells from d3tx donors with severe AOD and led to regression of AOD in d3tx mice documented at 4 wk. Therefore, 1) the continuous presence of CD40L mAb both prevents and causes regression of AOD in the d3tx mice; and 2) the multiple steps of the d3tx autoimmune disease, including T cell activation, cytokine production, T cell-mediated inflammation, and tissue injury, are CD40L dependent.
Allergen-Independent Maternal Transmission of Asthma Susceptibility
Authors: Hamada Kaoru , Suzaki Yasue , Goldman Alejandra , Ning Yao Yu , Goldsmith Carroll , Palecanda Aiyappa , Coull Brent , Hubeau Cedric , Kobzik Lester
Published: April 2014

Maternal asthma is a risk factor for development of asthma in children, but mechanisms remain unclear. Offspring of asthmatic mother mice (sensitized and repeatedly exposed to OVA Ag) showed airway hyperresponsiveness and allergic pulmonary inflammation after an intentionally suboptimal OVA sensitization and exposure protocol that had little effect on normal offspring. Similar results were obtained when offspring of OVA-allergic mothers were exposed to an unrelated allergen, casein, indicating that the maternal effect is allergen independent and not transferred by OVA-specific Abs. Premating treatment with neutralizing anti-IL-4 Ab or reduction of maternal allergen exposure abrogated the maternal effect, showing a critical mechanistic role for IL-4 and suggesting an additional benefit of allergen avoidance.
Epitope Spreading Is Not Required for Relapses in Experimental Autoimmune Encephalomyelitis
Authors: Jones Richard E. , Bourdette Dennis , Moes Nicole , Vandenbark Arthur , Zamora Alex , Offner Halina
Published: April 2014

The sequential emergence of specific T lymphocyte-mediated immune reactivity directed against multiple distinct myelin epitopes (epitope spreading) has been associated with clinical relapses in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Based on this association, an appealing and plausible model for immune-mediated progression of the advancing clinical course in MS and EAE has been proposed in which epitope spreading is the cause of clinical relapses in T cell-mediated CNS inflammatory diseases. However, the observed association between epitope spreading and disease progression is not universal, and absolute requirements for epitope spreading in progressive EAE have not been tested in the absence of multiple T cell specificities, because most prior studies have been conducted in immunocompetent mouse strains that possessed broad TCR repertoires. Consequently, the precise nature of a causal relationship between epitope spreading and disease progression remains uncertain. To determine whether relapsing or progressive EAE can occur in the absence of epitope spreading, we evaluated the course of disease in mice which possessed only a single myelin-specific TCR. These mice (transgenic/SCID +/+) exhibited a progressive and sometimes remitting/relapsing disease course in the absence of immune reactivity to multiple, spreading myelin epitopes. The results provide direct experimental evidence relevant to discussions on the mechanisms of disease progression in MS and EAE.
HIV-1 Nef Induces the Release of Inflammatory Factors from Human Monocyte/Macrophages: Involvement of Nef Endocytotic Signals and NF-κB Activation
Authors: Olivetta Eleonora , Percario Zulema , Fiorucci Gianna , Mattia Gianfranco , Schiavoni Ilaria , Dennis Caitriona , Jäger Joachim , Harris Mark , Romeo Gianna , Affabris Elisabetta , Federico Maurizio
Published: April 2014

It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1α and 1β, IL-1β, IL-6, and TNF-α. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-κB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.
The Leishmania mexicana Cysteine Protease, CPB2.8, Induces Potent Th2 Responses
Authors: Pollock Kevin G. J. , McNeil Katherine S. , Mottram Jeremy C. , Lyons Russell E. , Brewer James M. , Scott Phillip , Coombs Graham H. , Alexander James
Published: April 2014

We have previously identified that Leishmania mexicana cysteine proteases (CPs) are virulence factors. We have now produced a recombinant L. mexicana CP, CPB2.8, which has similar enzymatic activity to native enzyme. Inoculation of CPB2.8 (≤5 μg) into the footpads of BALB/c mice not only up-regulated mRNA transcripts for IL-4 and IL-4 production in the draining popliteal lymph nodes, but also polarized splenocyte anti-CD3 stimulated responses toward a Th2 bias as measured by increased IL-5 production compared with controls. In agreement with promoting a Th2 response, CPB2.8 also induced strong specific IgE responses in treated mice as well as increasing whole IgE levels. Inhibition of the enzyme activity of CPB2.8 by treatment with E-64 ablated the enzyme’s ability to induce IgE. Significantly, infection of mice with CPB-deficient parasites failed to stimulate production of IgE, unlike infection with wild-type parasites. Furthermore, enzymatically active (<0.1 U/ml) but not E-64-inactivated CPB2.8 was able to proteolytically cleave CD23 and CD25, although not B220 or CD4 from murine lymphocytes. These properties are similar to those demonstrated by the house dust mite allergen Der p I and provide an explanation for the immunomodulatory activity of the CPB2.8 virulence factor. Vaccination with CPB2.8 enhanced L. mexicana lesion growth compared with control animals. Nevertheless, vaccination with IL-12 and CPB2.8 resulted in a degree of protection associated with inhibition of lesion growth and a Th1 response. Thus, CPB2.8 is a potent Th2-inducing molecule capable of significant vaccine potential if administered with a suitable adjuvant.
NF-κB Hyperactivation Has Differential Effects on the APC Function of Nonobese Diabetic Mouse Macrophages
Authors: Sen Pradip , Bhattacharyya Sandip , Wallet Mark , Wong Carmen P. , Poligone Brian , Sen Maitreyee , Baldwin Albert S. , Tisch Roland
Published: April 2014

Type 1 diabetes is characterized by a chronic inflammatory response resulting in the selective destruction of the insulin-producing β cells. We have previously demonstrated that dendritic cells (DCs) prepared from nonobese diabetic (NOD) mice, a model for spontaneous type 1 diabetes, exhibit hyperactivation of NF-κB resulting in an increased capacity to secrete proinflammatory cytokines and stimulate T cells compared with DCs of nondiabetic strains of mice. In the current study, the activational status of NF-κB and its role in regulating the APC function of macrophages (Mφ) prepared from NOD, nonobese resistant (NOR), and BALB/c mice was investigated. Independent of the stimulus, splenic and bone marrow-derived Mφ prepared from NOD mice exhibited increased NF-κB activation relative to NOR and BALB/c Mφ. This hyperactivation was detected for different NF-κB complexes and correlated with increased IκBα degradation. Furthermore, increased NF-κB activation resulted in an enhanced capacity of NOD vs NOR or BALB/c Mφ to secrete IL-12(p70), TNF-α, and IL-1α, which was inhibited upon infection with an adenoviral recombinant encoding a modified form of IκBα. In contrast, elevated NF-κB activation had no significant effect on the capacity of NOD Mφ to stimulate CD4+ or CD8+ T cells in an Ag-specific manner. These results demonstrate that in addition to NOD DCs, NOD Mφ exhibit hyperactivation of NF-κB, which correlates with an increased ability to mediate a proinflammatory response. Furthermore, NF-κB influences Mφ APC function by regulating cytokine secretion but not T cell stimulation.
Antibody Repertoire Development in Fetal and Neonatal Piglets. VI. B Cell Lymphogenesis Occurs at Multiple Sites with Differences in the Frequency of In-frame Rearrangements
Authors: Šinkora Marek , Sun Jishan , Šinkorová Jana , Christenson Ronald K. , Ford Steven P. , Butler John E.
Published: April 2014

B cell lymphogenesis in mammals occurs in various tissues during development but it is generally accepted that it operates by the same mechanism in all tissues. We show that in swine, the frequency of in-frame (IF) VDJ rearrangements differs among yolk sac, fetal liver, spleen, early thymus, bone marrow, and late thymus. All VDJ rearrangements recovered and analyzed on the 20th day of gestation (DG20) from the yolk sac were 100% IF. Those recovered at DG30 in the fetal liver were >90% IF, and this predominance of cells with apparently a single IF rearrangement continued in all organs until approximately DG45, which corresponds to the time when lymphopoiesis begins in the bone marrow. Thereafter, the proportion of IF rearrangements drops to ∼71%, i.e., the value predicted whether VDJ rearrangement is random and both chromosomes were involved. Unlike other tissues, VDJs recovered from thymus after DG50 display a pattern suggesting no selection for IF rearrangements. Regardless of differences in the proportion of IF rearrangements, we observed no significant age- or tissue-dependent changes in CDR3 diversity, N region additions, or other characteristics of fetal VDJs during ontogeny. These findings indicate there are multiple sites of B cell lymphogenesis in fetal piglets and differences in the frequency of productive VDJ rearrangements at various sites. We propose the latter to result from differential selection or a developmentally dependent change in the intrinsic mechanism of VDJ rearrangement.
The Th2 Cell Cytokines IL-4 and IL-13 Regulate Found in Inflammatory Zone 1/Resistin-Like Molecule α Gene Expression by a STAT6 and CCAAT/Enhancer-Binding Protein-Dependent Mechanism
Authors: Stütz Adrian M. , Pickart Louise A. , Trifilieff Alexandre , Baumruker Thomas , Prieschl-Strassmayr Eva , Woisetschläger Maximilian
Published: April 2014

The onset of allergic inflammation in the lung is driven by a complex genetic program. This study shows that found in inflammatory zone (FIZZ)1 and FIZZ2, but not FIZZ3, gene expression was up-regulated 6 h after Ag challenge in a mouse model of acute pulmonary inflammation. Induction of both genes was abolished in allergen-challenged STAT6-deficient mice. FIZZ1, but not FIZZ2, mRNA was up-regulated upon incubation of the myeloid cell line BMnot with IL-4. The promoter region of FIZZ1 contains functional binding sites for STAT6 and C/EBP. FIZZ1 promoter reporter gene constructs responded to IL-4 and IL-13 stimulation in transiently transfected cells. Point mutations in the STAT6 or the C/EBP site led to loss of cytokine responsiveness indicating that IL-4-mediated induction of murine FIZZ1 is orchestrated by the coordinate action of STAT6 and C/EBP. It is concluded that the expression of the genes encoding FIZZ1 and FIZZ2, but not FIZZ3, is induced in allergen-challenged lungs in a STAT6-dependent fashion. STAT6 directly regulates IL-4- and IL-13-triggered induction of FIZZ1 expression at the transcriptional level by cooperation with C/EBP. Induction of FIZZ2 gene expression most likely occurs independent of a direct effect by these cytokines and may be due to indirect STAT6-driven mechanisms.
Cooperation of TNF Family Members CD40 Ligand, Receptor Activator of NF-κB Ligand, and TNF-α in the Activation of Dendritic Cells and the Expansion of Viral Specific CD8+ T Cell Memory Responses in HIV-1-Infected and HIV-1-Uninfected Individuals
Authors: Yu Qigui , Gu Jenny X. , Kovacs Colin , Freedman John , Thomas Elaine K. , Ostrowski Mario A.
Published: April 2014

Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-κB ligand (RANKL), and TNF-α, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of individuals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF-α alone to mature DCs and to expand CTL. In healthy volunteers, TNF-α or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative effects between TNF-α or RANKL in combination with CD40L were variable. TNF-α and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF-α enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4+ T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4+ T cells can provide help to CD8+ T cells independently of CD40L, RANKL, or TNF-α.
Naturally Occurring Human IgM Antibody That Binds B7-DC and Potentiates T Cell Stimulation by Dendritic Cells
Authors: Radhakrishnan Suresh , Nguyen Loc T. , Ciric Bogoljub , Ure Daren R. , Zhou Bin , Tamada Koji , Dong Haidong , Tseng Su-Yi , Shin Tahiro , Pardoll Drew M. , Chen Lieping , Kyle Robert A. , Rodriguez Moses , Pease Larry R.
Published: April 2014

A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.
Role of Double-Negative Regulatory T Cells in Long-Term Cardiac Xenograft Survival
Authors: Chen Wenhao , Ford Megan S. , Young Kevin J. , Cybulsky Myron I. , Zhang Li
Published: April 2014

A novel subset of CD3+CD4−CD8− (double negative; DN) regulatory T cells has recently been shown to induce donor-specific skin allograft acceptance following donor lymphocyte infusion (DLI). In this study, we investigated the effect of DLI on rat to mouse cardiac xenotransplant survival and the ability of DN T cells to regulate xenoreactive T cells. B6 mice were given either DLI from Lewis rats, a short course of depleting anti-CD4 mAb, both DLI and anti-CD4 treatment together, or left untreated. DLI alone did not prolong graft survival when compared with untreated controls. Although anti-CD4-depleting mAb alone significantly prolonged graft survival, grafts were eventually rejected by all recipients. However, the combination of DLI and anti-CD4 treatment induced permanent cardiac xenograft survival. We demonstrate that recipients given both DLI and anti-CD4 treatment had a significant increase in the total number of DN T cells in their spleens when compared with all other treatment groups. Furthermore, DN T cells harvested from the spleens of DLI plus anti-CD4-treated mice could dose-dependently inhibit the proliferation of syngeneic antidonor T cells. Suppression mediated by these DN T cells was specific for antidonor T cells as T cells stimulated by third-party Ags were not suppressed. These results demonstrate for the first time that a combination of pretransplant DLI and anti-CD4-depleting mAb can induce permanent survival of rat to mouse cardiac xenografts and that DN T regulatory cells play an important role in preventing long-term concordant xenograft rejection through the specific suppression of antidonor T cells.
Human Activation-Induced Cytidine Deaminase Is Induced by IL-4 and Negatively Regulated by CD45: Implication of CD45 as a Janus Kinase Phosphatase in Antibody Diversification
Authors: Zhou Cheng , Saxon Andrew , Zhang Ke
Published: April 2014

Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and VH gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.
Locus-Specific Constitutive and Cytokine-Induced HLA Class I Gene Expression
Authors: Johnson David R.
Published: April 2014

Cytokine induction of the MHC class I genes increases the nascent molecules available for binding potentially antigenic peptides. The human H chain loci, HLA-A, -B, and -C, encode highly homologous and polymorphic mRNAs. Here, these transcripts were resolved and measured by competitive PCR of cDNA using locus-specific primers. Endothelial cells expressed many HLA-A and -B, but fewer HLA-C, transcripts. In contrast, HeLa cells expressed many HLA-A and -C, but fewer HLA-B, transcripts. The inflammatory cytokines TNF-α, IFN-β, and IFN-γ induced HLA-B strongly, but HLA-A and -C weakly in both cell types. Combined treatment with IFNs and TNF further increased HLA-A and -B, but not HLA-C transcripts. The constitutive and inducible activities of transfected promoters correlated well with mRNA levels. The weak IFN response of the HLA-A2 promoter was not due to variations in the IFN consensus sequence, the site α, or a 3-bp insertion between them. The HLA-Cw6 promoter was less TNF responsive due to a variant κB enhancer, which also reduced the IFN responses. The NF-κB subunit RelA strongly activated the HLA-A2 and -B7 promoters but only weakly activated the HLA-Cw6 promoter due to the variant κB. Cotransfecting NF-κB1 with RelA further increased activity of the HLA-A2 and -B7, but not HLA-Cw6, promoters. All three promoters were activated by MHC class II trans-activator, but not CREB-binding protein, whereas IFN regulatory factor-1 and -2 weakly activated the HLA-B7 and -Cw6, but not HLA-A2, promoters. These studies illustrate common and locus-specific mechanisms that may be targeted to modulate immune reactions.
CTLA-4 in Filarial Infections: Implications for a Role in Diminished T Cell Reactivity
Authors: Steel Cathy , Nutman Thomas B.
Published: April 2014

To determine the role that CTLA-4 might play in mediating the diminished parasite Ag-specific T cell responsiveness that is characteristically seen in filaria-infected patients, several study populations and methods were used. First, quantitative assessment of mRNA expression determined that PBMC from uninfected adolescents exposed in utero to microfilarial (Mf) Ag demonstrated a strong up-regulation of CTLA-4 to the Mf stage of the parasite in contrast to that observed in cells from children born of uninfected mothers (p = 0.005). Next, the frequency of CTLA-4 expression was examined using flow cytometry in cells from filaria-infected and -uninfected individuals ex vivo. Individuals born in filarial endemic regions of the world (with long-standing infections) had greater percentages of CD4+CTLA-4+ cells than did expatriate infected or uninfected individuals (p = 0.005 and 0.05, respectively); in addition, Mf+ patients demonstrated higher frequencies of CD4+CTLA-4+ and CD8+CTLA-4+ cells (p = 0.027 and 0.037, respectively) than did Mf− infected individuals. Of interest, the greatest intensity of CTLA-4 expression occurred in CD4+CD25+ cells, a population purported to include suppressor cells. Finally, in vitro blocking of CTLA-4 expression in PBMC from filaria-infected individuals induced a mean increase of 44% in IL-5 production to Mf Ag, whereas there was a concurrent mean decrease of 42% in IFN-γ production, suggesting that CTLA-4 also acts to alter the Th1/Th2 balance in filaria-infected individuals. Together, these data indicate a significant role for CTLA-4 in regulating the host response to filarial infections and that factors such as length of exposure and patency are important codeterminants.
Mouse SWAM1 and SWAM2 Are Antibacterial Proteins Composed of a Single Whey Acidic Protein Motif
Authors: Hagiwara Koichi , Kikuchi Tohru , Endo Yoshiyuki , Huqun  , Usui Kazuhiro , Takahashi Mitsu , Shibata Naoko , Kusakabe Takashi , Xin Hong , Hoshi Sachiko , Miki Makoto , Inooka Nozomu , Tokue Yutaka , Nukiwa Toshihiro
Published: April 2014

Antibacterial proteins are important participants in the innate immunity system. Elafin and SLPI are the whey acidic protein (WAP) motif proteins with both antibacterial activity and antiprotease activity, and their role in innate immunity is under intense investigation. We cloned two novel antibacterial WAP motif proteins from mice, SWAM1 and SWAM2. SWAM1 and SWAM2 are composed of a signal sequence and a single WAP motif that has high homologies with the WAP motifs of elafin and SLPI. SWAM1 is constitutively expressed in kidney and epididymis, and is induced in the pneumonic lung. SWAM2 is constitutively expressed in tongue. SWAM1 and SWAM2 inhibit the growth of both Escherichia coli and Staphylococcus aureus at a IC90 (concentration that achieves 90% inhibition) of 10 μM. Human genes LOC149709 and huWAP2 are considered to be human SWAM1 and SWAM2, respectively. These and several WAP motif proteins (WAP1, elafin, SLPI, HE4, eppin, C20orf170, LOC164237, and WFDC3) form a gene cluster on human chromosome 20, suggesting that they may be derived from the same ancestral gene by gene duplication. Our results underscore the role of the WAP motif as a skeletal motif to form antibacterial proteins, and warrant the study of antibacterial activity in other WAP motif proteins.
CD8+ T Cells Accumulate in the Lungs of Mycobacterium tuberculosis-Infected Kb−/−Db−/− Mice, But Provide Minimal Protection
Authors: Urdahl Kevin B. , Liggitt Denny , Bevan Michael J.
Published: April 2014

Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (Kb−/−Db−/− mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (β2m−/− mice). The susceptibility of Kb−/−Db−/− mice cannot be explained by the failure of CD8+ T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8+ T cells were a relatively small population in uninfected Kb−/−Db−/− mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN-γ following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of Kb−/−Db−/− mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8α−/− mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8+ effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.
NF-κB1 Is Required for Optimal CD4+ Th1 Cell Development and Resistance to Leishmania major
Authors: Artis David , Speirs Kendra , Joyce Karen , Goldschmidt Michael , Caamaño Jorge , Hunter Christopher A. , Scott Phillip
Published: April 2014

The NF-κB family of transcription factors regulates the expression of a wide range of immune response genes involved in immunity to pathogens. However, the need for individual family members in regulating innate and adaptive immune responses in vivo has yet to be clearly defined. We investigated the role of NF-κB1 in the induction of protective IL-12-dependent Th1 cell responses following infection with the intracellular protozoan parasite Leishmania major. Whereas wild-type C57BL/6 mice controlled parasite replication, NF-κB1 knockout (KO) mice were susceptible to infection, developing chronic unresolving lesions associated with persistent parasites. There was a profound defect in Ag-specific CD4+ T cell proliferation and IFN-γ production in infected KO mice, although innate responses—including IL-12 production and control of intracellular parasite replication by macrophages—were intact. In vitro polyclonal stimulation of purified naive KO T cells revealed an intrinsic defect in CD4+ T cell proliferation associated with reduced IL-2 receptor expression, but operating independently of APC function and IL-2 production. Critically, the frequency of proliferating KO CD4+ T cells secreting IFN-γ matched that of wild-type cells, suggesting that NF-κB1 was not required for efficient transcription of the IFN-γ gene. Taken together, these results identify a novel role for NF-κB1 in CD4+ T cell proliferation and the development of Th1 cell responses required for protective immunity against intracellular pathogens.