bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Selective Tyrosine Dephosphorylation of Interferon-Activated Nuclear STAT5 by the VHR Phosphatase
Authors: Hoyt Richard , Zhu Wei , Cerignoli Fabio , Alonso Andres , Mustelin Tomas , David Michael
Published: April 2014

Cytokine-induced tyrosine phosphorylation of the transcription factor STAT5 is required for its transcriptional activity. In this article we show that the small dual-specificity phosphatase VHR selectively dephosphorylates IFN-α- and β-activated, tyrosine-phosphorylated STAT5, leading to the subsequent inhibition of STAT5 function. Phosphorylation of VHR at Tyr138 was required for its phosphatase activity toward STAT5. In addition, the Src homology 2 domain of STAT5 was required for the effective dephosphorylation of STAT5 by VHR. The tyrosine kinase Tyk2, which mediates the phosphorylation of STAT5, was also responsible for the phosphorylation of VHR at Tyr138.
Histamine Improves Antigen Uptake and Cross-Presentation by Dendritic Cells
Authors: Amaral Maria Marta , Davio Carlos , Ceballos Ana , Salamone Gabriela , Cañones Cristian , Geffner Jorge , Vermeulen Mónica
Published: April 2014

Previous studies have shown that histamine is able to modulate the function of dendritic cells (DCs). Histamine seems to be required for the normal differentiation of DCs. Moreover, it is capable of stimulating the chemotaxis of immature DCs and of promoting the differentiation of T CD4+ cells into a Th2 profile. In this study, we analyzed whether histamine was able to modulate endocytosis and cross-presentation mediated by immature DCs. Our results show that both functions are stimulated by histamine. Endocytosis of soluble HRP and FITC-OVA and cross-presentation of soluble OVA were markedly increased by histamine. Interestingly, stimulation of endocytosis and cross-presentation appeared to be mediated through different histamine receptors. In fact, the enhancement of endocytosis was prevented by the histamine2 receptor (H2R) antagonist cimetidine, whereas the stimulation of cross-presentation was prevented by the H3R/H4R antagonist thioperamide. Of note, contrasting with the observations made with soluble Ags, we found that histamine did not increase either the uptake of OVA-attached to latex beads, or the cross-presentation of OVA immobilized on latex beads. This suggests that the ability of histamine to increase endocytosis and cross-presentation is dependent on the Ag form and/or the mechanisms through which the Ag is internalized by DCs. Our results support that histamine may favor cross-presentation of soluble allergens by DCs enabling the activation of allergen-specific T CD8+ cells, which appears to play an important role in the development of allergic responses in the airway.
Dectin-1 Interaction with Mycobacterium tuberculosis Leads to Enhanced IL-12p40 Production by Splenic Dendritic Cells
Authors: Rothfuchs Antonio Gigliotti , Bafica Andre , Feng Carl G. , Egen Jackson G. , Williams David L. , Brown Gordon D. , Sher Alan
Published: April 2014

Dectin-1 is a fungal pattern recognition receptor that binds to β-glucans and triggers cytokine production by facilitating interaction with TLR2 or by directly activating spleen tyrosine kinase (Syk). To assess the possible role of Dectin-1 in the innate response to mycobacteria, we used an in vitro system in which IL-12p40 production is measured in splenic dendritic cells (SpDC) following exposure to live Mycobacterium tuberculosis bacilli. Treatment of SpDC with laminarin or glucan phosphate, two molecules known to block Dectin-1-dependent activity, led to a reduction in M. tuberculosis-induced IL-12p40 as well as IL-12p70 production. Moreover, SpDC from Dectin-1−/− chimeric mice displayed reduced IL-12p40 production in response to mycobacteria when compared with Dectin-sufficient DC. Laminarin treatment also inhibited mycobacterial-induced IL-12p40 production in DC from TLR2−/− mice, arguing that Dectin-1 functions independently of TLR2 signaling in this system. Importantly, a Dectin-1 fusion protein was found to directly bind to live mycobacteria in a laminarin-inhibitable manner indicating the presence of ligands for the receptor in the bacterium and laminarin pretreatment resulted in reduced association of mycobacteria to SpDC. In additional experiments, mycobacterial stimulation was shown to be associated with increased phosphorylation of Syk and this response was inhibited by laminarin. Furthermore, pharmacologic inhibition of Syk reduced the M. tuberculosis-induced IL-12p40 response. Together, these findings support a role for Dectin-1 in promoting M. tuberculosis-induced IL-12p40 production by DC in which the receptor augments bacterial-host cell interaction and enhances the subsequent cytokine response through an unknown mechanism involving Syk signaling.
Impaired Induction of CD27 and CD28 Predicts Naive CD4 T Cell Proliferation Defects in HIV Disease
Authors: Luciano Angel A. , Lederman Michael M. , Valentin-Torres Alice , Bazdar Douglas A. , Sieg Scott F.
Published: April 2014

Many immunological defects have been described in HIV disease, including a diminished capacity of naive CD4+ T cells to expand after TCR stimulation. The mechanisms underlying impaired naive CD4+ T cell expansion in HIV disease are not well described. Using a rigorous phenotypic definition of naive T cells, we found that cell cycle entry after TCR engagement was restricted to cells that increased surface expression of costimulatory molecules CD27 and CD28. Induction of these receptors, however, was not sufficient to result in cell cycle entry among the CD4+CD31− naive T cell subset. Analyses of cells from HIV-infected persons indicated that naive CD4+CD31+ T cells from these subjects were impaired in their ability to enter the cell cycle after stimulation and this impairment was predicted by the relatively poor induction of costimulatory molecules on these cells. Thus, failure to increase surface expression of costimulatory molecules may contribute to the naive T cell expansion failure that characterizes HIV infection.
IL-21 Induces Apoptosis of Antigen-Specific CD8+ T Lymphocytes
Authors: Barker Brianne R. , Parvani Jenny G. , Meyer Debra , Hey Adam S. , Skak Kresten , Letvin Norman L.
Published: April 2014

IL-21, a member of the common γ-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common γ-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.
A Key Role for TGF-β Signaling to T Cells in the Long-Term Acceptance of Allografts
Authors: Daley Stephen R. , Ma Jianbo , Adams Elizabeth , Cobbold Stephen P. , Waldmann Herman
Published: April 2014

TGF-β is a key immunoregulatory cytokine which supports self-tolerance by signaling to T cells. In this report, we show a crucial role for TGF-β signaling to T cells in enabling the long-term acceptance of allografts, whether natural or induced therapeutically by coreceptor and costimulation blockade. The requirement for TGF-β appears most pronounced during the initial exposure to alloantigens. We demonstrate the ability of TGF-β to direct the development in vitro of regulatory cells that suppress graft rejection in vivo. Such suppression was not affected by anti-TGF-β treatment of the recipient mice. Despite this, TGF-β may still have a role in CD4+ cell-mediated suppression of antiallograft responses in vivo, since its neutralization can, in some cases, abrogate suppression. These results show that TGF-β signaling to T cells is dispensable for mounting destructive responses against skin allografts while appearing to be an essential intermediary in establishing long-term tolerance.
Intracellular Cysteine Residues in the Tail of MHC Class I Proteins Are Crucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1
Authors: Gruda Raizy , Achdout Hagit , Stern-Ginossar Noam , Gazit Roi , Betser-Cohen Gili , Manaster Irit , Katz Gil , Gonen-Gross Tsufit , Tirosh Boaz , Mandelboim Ofer
Published: April 2014

The activity of NK cells is regulated by activating receptors that recognize mainly stress-induced ligands and by inhibitory receptors that recognize mostly MHC class I proteins on target cells. Comparing the cytoplasmic tail sequences of various MHC class I proteins revealed the presence of unique cysteine residues in some of the MHC class I molecules which are absent in others. To study the role of these unique cysteines, we performed site specific mutagenesis, generating MHC class I molecules lacking these cysteines, and demonstrated that their expression on the cell surface was impaired. Surprisingly, we demonstrated that these cysteines are crucial for the surface binding of the leukocyte Ig-like receptor 1 inhibitory receptor to the MHC class I proteins, but not for the binding of the KIR2DL1 inhibitory receptor. In addition, we demonstrated that the cysteine residues in the cytoplasmic tail of MHC class I proteins are crucial for their egress from the endoplasmic reticulum and for their palmitoylation, thus probably affecting their expression on the cell surface. Finally, we show that the cysteine residues are important for proper extracellular conformation. Thus, although the interaction between leukocyte Ig-like receptor 1 and MHC class I proteins is formed between two extracellular surfaces, the intracellular components of MHC class I proteins play a crucial role in this recognition.
A Biochemical Signature for Rapid Recall of Memory CD4 T Cells
Authors: Chandok Meena R. , Okoye Francesca I. , Ndejembi Modesta P. , Farber Donna L.
Published: April 2014

Mechanisms for the rapid recall response mediated by memory T cells remain unknown. In this study, we present a novel, multiparameter analysis of TCR-coupled signaling and function in resting and activated naive and memory CD4 T cells, revealing a biochemical basis for immunological recall. We identify a striking elevation in expression of the proximal tyrosine kinase Zap70 in resting Ag-specific and polyclonal mouse memory vs naive CD4 T cells that is stably maintained independent of protein synthesis. Elevated Zap70 protein levels control effector function as IFN-γ production occurs exclusively from the Zap70high fraction of activated T cells in vitro and in vivo, and specific down-modulation of Zap70 expression in memory CD4 T cells by small interfering RNA or protein inhibition significantly reduces rapid IFN-γ production. Downstream of Zap70, we show quantitative differences in distal phosphorylation associated with effector function in naive and memory subsets, with low accumulation of phosphorylation in memory T cells producing IFN-γ at early time points, contrasting extensive phosphorylation associated with IFN-γ production following sustained activation of naive T cells. Our results reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficacious responses through increased Zap70 expression and reduced accumulation of downstream signaling events.
T Cell Lineage Determination Precedes the Initiation of TCRβ Gene Rearrangement
Authors: Masuda Kyoko , Kakugawa Kiyokazu , Nakayama Toshinori , Minato Nagahiro , Katsura Yoshimoto , Kawamoto Hiroshi
Published: April 2014

Loss of dendritic cell potential is one of the major events in intrathymic T cell development, during which the progenitors become determined to the T cell lineage. However, it remains unclear whether this event occurs in synchrony with another important event, TCRβ chain gene rearrangement, which has been considered the definitive sign of irreversible T cell lineage commitment. To address this issue, we used transgenic mice in which GFP expression is controlled by the lck proximal promoter. We found that the double-negative (DN) 2 stage can be subdivided into GFP− and GFP+ populations, representing functionally different developmental stages in that the GFP−DN2, but not GFP+DN2, cells retain dendritic cell potential. The GFP+DN2 cells were found to undergo several rounds of proliferation before the initiation of TCRβ rearrangement as evidenced by the diversity of D-Jβ rearrangements seen in T cells derived from a single GFP+DN2 progenitor. These results indicated that the determination step of progenitors to the T cell lineage is a separable event from TCRβ rearrangement.
IFN-α Is Not Sufficient to Drive Th1 Development Due to Lack of Stable T-bet Expression
Authors: Ramos Hilario J. , Davis Ann M. , George Thaddeus C. , Farrar J. David
Published: April 2014

During inflammatory immune responses, the innate cytokine IL-12 promotes CD4+ Th-1 development through the activation of the second messenger STAT4 and the subsequent expression of T-bet. In addition, type I IFN (IFN-αβ), secreted primarily during viral and intracellular bacterial infections, can promote STAT4 activation in human CD4+ T cells. However, the role of IFN-αβ in regulating Th1 development is controversial, and previous studies have suggested a species-specific pathway leading to Th1 development in human but not mouse CD4+ T cells. In this study, we found that although both IFN-α and IL-12 can promote STAT4 activation, IFN-α failed to promote Th1 commitment in human CD4+ T cells. The difference between these innate signaling pathways lies with the ability of IL-12 to promote sustained STAT4 tyrosine phosphorylation, which correlated with stable T-bet expression in committed Th1 cells. IFN-α did not promote Th1 development in human CD4+ T cells because of attenuated STAT4 phosphorylation, which was insufficient to induce stable expression of T-bet. Further, the defect in IFN-α-driven Th1 development was corrected by ectopic expression of T-bet within primary naive human CD4+ T cells. These results indicate that IL-12 remains unique in its ability to drive Th1 development in human CD4+ T cells and that IFN-α lacks this activity due to its inability to promote sustained T-bet expression.
Bruton’s Tyrosine Kinase Mediates NF-κB Activation and B Cell Survival by B Cell-Activating Factor Receptor of the TNF-R Family
Authors: Shinners Nicholas P. , Carlesso Gianluca , Castro Iris , Hoek Kristen L. , Corn Radiah A. , Woodland Robert L. , Scott Martin L. , Wang Demin , Khan Wasif N.
Published: April 2014

Loss of Bruton’s tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-κB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-κB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-κB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IκB kinase γ-containing complexes and defective IκBα degradation. In addition, Btk-deficient B cells produce reduced levels of NF-κB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-κB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-κB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-κB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-κB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.
CD8+ T Cell Protective Immunity against Chlamydia pneumoniae Includes an H2-M3-Restricted Response That Is Largely CD4+ T Cell-Independent
Authors: Tvinnereim Amy , Wizel Benjamin
Published: April 2014

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-γ production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb−/−/Db−/− mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb−/−/Db−/− mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.
Critical Negative Regulation of Type 1 T Cell Immunity and Immunopathology by Signaling Adaptor DAP12 during Intracellular Infection
Authors: Divangahi Maziar , Yang Tony , Kugathasan Kapilan , McCormick Sarah , Takenaka Shunsuke , Gaschler Gordon , Ashkar Ali , Stampfli Martin , Gauldie Jack , Bramson Jonathan , Takai Toshiyuki , Brown Earl , Yokoyama Wayne M. , Aoki Naoko , Xing Zhou
Published: April 2014

Transmembrane signaling adaptor DAP12 has increasingly been recognized for its important role in innate responses. However, its role in the regulation of antimicrobial T cell responses has remained unknown. In our current study, we have examined host defense, T cell responses, and tissue immunopathology in models of intracellular infection established in wild-type and DAP12-deficient mice. During mycobacterial infection, lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses, overactivation of Ag-specific CD4 and CD8 T cells of type 1 phenotype, and heightened immunopathology both in the lung and lymphoid organs. DAP12-deficient airway APC display enhanced NF-κB activation and cytokine responses upon TLR stimulation or mycobacterial infection in vitro. Of importance, adoptive transfer of Ag-loaded DAP12-deficient APC alone could lead to overactivation of transferred transgenic or endogenous wild-type T cells in vivo. We have further found that the immune regulatory role by DAP12 is not restricted only to intracellular bacterial infection, since lack of this molecule also leads to uncontrolled type 1 T cell activation and severe immunopathology and tissue injury during intracellular viral infection. Our study thus identifies DAP12 as an important novel immune regulatory molecule that acts, via APC, to control the level of antimicrobial type 1 T cell activation and immunopathology.
Plasmid-Deficient Chlamydia muridarum Fail to Induce Immune Pathology and Protect against Oviduct Disease
Authors: O’Connell Catherine M. , Ingalls Robin R. , Andrews Charles W. , Scurlock Amy M. , Darville Toni
Published: April 2014

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.
Chloride Movements in Human Neutrophils during Phagocytosis: Characterization and Relationship to Granule Release
Authors: Busetto Sara , Trevisan Elisa , Decleva Eva , Dri Pietro , Menegazzi Renzo
Published: April 2014

Chloride ion efflux is an early event occurring after exposure of human neutrophils to several soluble agonists. Under these circumstances, a rapid and reversible fall in the high basal intracellular chloride (Cl−i) levels is observed. This event is thought to play a crucial role in the modulation of several critical neutrophil responses including activation and up-regulation of adhesion molecules, cell attachment and spreading, cytoplasmic alkalinization, and activation of the respiratory burst. At present, however, no data are available on chloride ion movements during neutrophil phagocytosis. In this study, we provide evidence that phagocytosis of Candida albicans opsonized with either whole serum, complement-derived opsonins, or purified human IgG elicits an early and long-lasting Cl− efflux accompanied by a marked, irreversible loss of Cl−i. Simultaneous assessment of Cl− efflux and phagocytosis in cytochalasin D-treated neutrophils indicated that Cl− efflux occurs without particle ingestion. These results suggest that engagement of immune receptors is sufficient to promote chloride ion movements. Several structurally unrelated chloride channel blockers inhibited phagocytosis-induced Cl− efflux as well as the release of azurophilic—but not specific—granules. It implicates that different neutrophil secretory compartments display distinct sensitivity to Cl−i modifications. Intriguingly, inhibitors of Cl− exchange inhibited cytosolic Ca2+ elevation, whereas Cl− efflux was not impaired in Ca2+-depleted neutrophils. We also show that FcγR(s)- and CR3/CR1-mediated Cl− efflux appears to be dependent on protein tyrosine phosphorylation but independent of PI3K and phospholipase C activation.
IL-17 Enhances Chemokine Gene Expression through mRNA Stabilization
Authors: Hartupee Justin , Liu Caini , Novotny Michael , Li Xiaoxia , Hamilton Thomas
Published: April 2014

IL-17 plays an important role in host defense and autoimmunity via the induction of proinflammatory gene expression, particularly in combination with TNF-α. The molecular mechanisms by which IL-17 regulates such expression are not well understood. Using the mouse chemokine CXCL1 (KC) gene as a model, we have examined the effects of IL-17 alone or in combination with TNF-α on transcriptional and posttranscriptional events. Although treatment of mouse embryonic fibroblasts with IL-17 alone only modestly increased KC expression, the combination of IL-17 with TNF-α induced a synergistic response. IL-17 treatment exerted a strong posttranscriptional effect by extending the t1/2 of the highly unstable, TNF-α-induced KC mRNA. Using a tetracycline-regulated transgene in HeLa cells, we determined that IL-17 treatment alone promoted stabilization of KC mRNA in the absence of TNF-α. IL-17 treatment exerted little effect on KC transcription or NF-κB activation, suggesting that it primarily acts posttranscriptionally. We identified a number of other mRNAs whose t1/2 are prolonged in response to IL-17, suggesting that this is a common mechanism by which IL-17 promotes enhanced gene expression. Finally, activator of NF-κB1 protein (Act1), an adaptor protein recently implicated in IL-17 signaling, was necessary for IL-17-induced stabilization, and overexpression of Act1 resulted in stabilization of KC mRNA, indicating that events downstream of Act1 are sufficient to initiate this process. Thus, the synergy between TNF-α and IL-17 reflects their independent actions on KC gene expression; TNF-α serves as a stimulus to initiate transcription through activation of NF-κB, whereas IL-17 drives mRNA stabilization through an Act1-dependent pathway.
Hydrogen Sulfide Up-Regulates Substance P in Polymicrobial Sepsis-Associated Lung Injury
Authors: Zhang Huili , Hegde Akhil , Ng Siaw Wei , Adhikari Sharmila , Moochhala Shabbir M. , Bhatia Madhav
Published: April 2014

Hydrogen sulfide (H2S) has been shown to induce the activation of neurogenic inflammation especially in normal airways and urinary bladder. However, whether endogenous H2S would regulate sepsis-associated lung inflammation via substance P (SP) and its receptors remains unknown. Therefore, the aim of the study was to investigate the effect of H2S on the pulmonary level of SP in cecal ligation and puncture (CLP)-induced sepsis and its relevance to lung injury. Male Swiss mice or male preprotachykinin-A gene knockout (PPT-A−/−) mice and their wild-type (PPT-A+/+) mice were subjected to CLP-induced sepsis. DL-propargylglycine (50 mg/kg i.p.), an inhibitor of H2S formation was administered either 1 h before or 1 h after the induction of sepsis, while NaHS, an H2S donor, was given at the same time as CLP. L703606, an inhibitor of the neurokinin-1 receptor was given 30 min before CLP. DL-propargylglycine pretreatment or posttreatment significantly decreased the PPT-A gene expression and the production of SP in lung whereas administration of NaHS resulted in a further rise in the pulmonary level of SP in sepsis. PPT-A gene deletion and pretreatment with L703606 prevented H2S from aggravating lung inflammation. In addition, septic mice genetically deficient in PPT-A gene or pretreated with L703606 did not exhibit further increase in lung permeability after injection of NaHS. The present findings show for the first time that in sepsis, H2S up-regulates the generation of SP, which contributes to lung inflammation and lung injury mainly via activation of the neurokinin-1 receptor.
Zinc-Dependent Suppression of TNF-α Production Is Mediated by Protein Kinase A-Induced Inhibition of Raf-1, IκB Kinase β, and NF-κB
Authors: von Bülow Verena , Dubben Svenja , Engelhardt Gabriela , Hebel Silke , Plümäkers Birgit , Heine Holger , Rink Lothar , Haase Hajo
Published: April 2014

Excessive and permanent cytokine production in response to bacterial LPS causes cell and tissue damage, and hence organ failure during sepsis. We have previously demonstrated that zinc treatment prevents LPS-induced TNF-α expression and production in human monocytes by inhibiting cyclic nucleotide phosphodiesterase (PDE) activity and expression, and subsequent elevation of the cyclic nucleotide cGMP. In the present study, we investigated the molecular mechanism by which cGMP signaling affects the LPS-induced signaling cascade to suppress TNF-α transcription and release from monocytes. Zinc-mediated cGMP elevation led to cross activation of protein kinase A. This zinc-induced protein kinase A activation inhibited Raf-1 activity by phosphorylation at serine 259, preventing activation of Raf-1 by phosphorylation of serine 338. By this mechanism, zinc suppressed LPS-induced activation of IκB kinase β (IKKβ) and NF-κB, and subsequent TNF-α production. Our study shows that PDE inhibition by zinc modulates the monocytic immune response by selectively intervening in the Raf-1/IKKβ/NF-κB pathway, which may constitute a common mechanism for the anti-inflammatory action of PDE inhibitors.
Impaired Plasmacytoid Dendritic Cell Innate Immune Responses in Patients with Herpes Virus-Associated Acute Retinal Necrosis
Authors: Kittan Nicolai A. , Bergua Antonio , Haupt Sabrina , Donhauser Norbert , Schuster Philipp , Korn Klaus , Harrer Thomas , Schmidt Barbara
Published: April 2014

Plasmacytoid dendritic cells (PDC), the main producers of type I IFNs in the blood, are important for the recognition and control of viral and bacterial infections. Because several viruses induce IFN-α production, severe courses of herpes virus infections in nonimmunocompromised patients may be related to numerical or functional PDC deficits. To evaluate this hypothesis, PBMC and PDC were repeatedly isolated from nine patients with acute retinal necrosis (ARN), caused by herpes simplex or varicella zoster virus. The patients experienced meningitis/encephalitis and frequent infections in childhood (n = 2), recurrent herpes virus infections at unusual localizations (n = 2), ocular surgery (n = 1), infections (n = 4), and stress around ARN (n = 6). The median percentage of isolated PDC was significantly lower in patients compared with 18 age-matched healthy controls (p < 0.001), confirmed by FACS analysis using peripheral blood, and was extremely low during acute disease. PDC counts dropped in five controls suffering from respiratory infections or diarrhea. IFN-α production in PDC and PBMC exposed to different stimuli was significantly lower in patients than in controls (p < 0.05). Anergy to these stimuli was observed on four occasions, in particular during acute disease. PDC of patients showed up-regulated IFN regulatory factor-7 mRNA levels and evidence of in vivo activation (CD80) and maturation (CD83) (p < 0.05). CD8+ cell responses were significantly lower in patients vs controls (p = 0.04). These data support a risk factor model in which numerical and functional deficits in PDC-mediated innate immune responses contribute to an impaired control of latent herpes virus infections and subsequent development of ARN.
Cutting Edge: An In Vivo Requirement for STAT3 Signaling in TH17 Development and TH17-Dependent Autoimmunity
Authors: Harris Timothy J. , Grosso Joseph F. , Yen Hung-Rong , Xin Hong , Kortylewski Marcin , Albesiano Emilia , Hipkiss Edward L. , Getnet Derese , Goldberg Monica V. , Maris Charles H. , Housseau Franck , Yu Hua , Pardoll Drew M. , Drake Charles G.
Published: April 2014

STAT3 activation has been observed in several autoimmune diseases, suggesting that STAT3-mediated pathways promote pathologic immune responses. We provide in vivo evidence that the fundamental role of STAT3 signaling in autoimmunity relates to its absolute requirement for generating TH17 T cell responses. We show that STAT3 is a master regulator of this pathogenic T cell subtype, acting at multiple levels in vivo, including TH17 T cell differentiation and cytokine production, as well as induction of RORγt and the IL-23R. Neither naturally occurring TH17 cells nor TH17-dependent autoimmunity occurs when STAT3 is ablated in CD4 cells. Furthermore, ablation of STAT3 signaling in CD4 cells results in increased TH1 responses, indicating that STAT3 signaling skews TH responses away from the TH1 pathway and toward the TH17 pathway. Thus, STAT3 is a candidate target for TH17-dependent autoimmune disease immunotherapy that could selectively inhibit pathogenic immune pathways.
CD8+CD28− T Regulatory Lymphocytes Inhibiting T Cell Proliferative and Cytotoxic Functions Infiltrate Human Cancers
Authors: Filaci Gilberto , Fenoglio Daniela , Fravega Marco , Ansaldo Gianluca , Borgonovo Giacomo , Traverso Paolo , Villaggio Barbara , Ferrera Alessandra , Kunkl Annalisa , Rizzi Marta , Ferrera Francesca , Balestra Piercesare , Ghio Massimo , Contini Paola , Setti Maurizio , Olive Daniel , Azzarone Bruno , Carmignani Giorgio , Ravetti Jean Louis , Torre Giancarlo , Indiveri Francesco
Published: April 2014

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+CD28− T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+CD28− T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+CD25+ T regulatory lymphocytes associate with CD8+CD28− T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.
TLR2 Signaling Renders Quiescent Naive and Memory CD4+ T Cells More Susceptible to Productive Infection with X4 and R5 HIV-Type 1
Authors: Thibault Sandra , Tardif Mélanie R. , Barat Corinne , Tremblay Michel J.
Published: April 2014

It has been recently demonstrated that circulating microbial products are responsible for a systemic immune activation in individuals infected with HIV-type 1. Bacterial products carry structural conserved motifs recognized by TLRs. Some TLR members are expressed in primary human CD4+ T cells but the precise functional role played by these pattern recognition receptors is still imprecise. In this study, we report that engagement of TLR2 in quiescent naive and memory CD4+ T cells leads to the acquisition of an effector-like phenotype. Interestingly, engagement of TLR2 renders both cell subsets more susceptible to productive infection with X4 virions and a higher virus production was seen with R5 viruses. It can be proposed that exposure of resting CD4+ T cells to pathogen-derived products that can engage TLR2 induces the acquisition of an effector-like phenotype in naive and memory CD4+ T lymphocytes, a phenomenon that might result in an acceleration of virus replication, immune dysregulation, and HIV-type 1-mediated disease progression.
Neonatal FcR Expression in Bone Marrow-Derived Cells Functions to Protect Serum IgG from Catabolism
Authors: Akilesh Shreeram , Christianson Gregory J. , Roopenian Derry C. , Shaw Andrey S.
Published: April 2014

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.
Genetic Variation in PTPN22 Corresponds to Altered Function of T and B Lymphocytes
Authors: Rieck Mary , Arechiga Adrian , Onengut-Gumuscu Suna , Greenbaum Carla , Concannon Patrick , Buckner Jane H.
Published: April 2014

A variant of the PTPN22 gene, 1858C/T, is associated with an increased risk for the development of a wide array of autoimmune disorders. It is known that the protein tyrosine phosphatase Lyp encoded by this gene has an inhibitory effect on the proximal TCR signaling pathways. However, the consequences of carrying this variant and the mechanism by which it contributes to the development of autoimmunity are poorly understood. In this study, we demonstrate that homozygosity for this variant results in a profound deficit in T cell responsiveness to Ag stimulation. Heterozygosity for the variant allele is associated with reduced responsiveness of CD4+ memory T cells, characterized by diminished calcium mobilization, expression of CD25, and IL-10 production upon TCR stimulation. Additionally, the presence of the variant allele is associated with an increase in circulating memory T cells. We further demonstrate that these effects are not limited to the T cell compartment. Individuals with the variant allele have fewer memory B cells and these cells display a reduced response to stimulation via the BCR indicative of a B cell intrinsic defect. By identifying an immunologic phenotype in healthy subjects which correlates with the PTPN22 1858C/T genotype, we can now explore specific hypotheses regarding pathogenesis of diseases associated with the PTPN22 1858T variant.
Live Attenuated Lentivirus Infection Elicits Polyfunctional Simian Immunodeficiency Virus Gag-Specific CD8+ T Cells with Reduced Apoptotic Susceptibility in Rhesus Macaques that Control Virus Replication after Challenge with Pathogenic SIVmac239
Authors: Genescà Meritxell , Rourke Tracy , Li Jun , Bost Kristen , Chohan Barinderpaul , McChesney Michael B. , Miller Christopher J.
Published: April 2014

HIV-specific CD8+ T cells that secrete multiple cytokines in response to Ag stimulation are associated with the control of virus replication during chronic HIV infection. To determine whether the presence of polyfunctional CD8+ T cell responses distinguishes protected and unprotected monkeys in a live attenuated lentivirus model, SIV Gag peptide-specific CD8+ T cell responses of simian HIV (SHIV) 89.6-vaccinated, SIVmac239-challenged rhesus macaques were compared in two monkeys that controlled challenge virus replication and two that did not. The ratio of Bcl-2+ Gag-specific CD8+ T cells to caspase-3+ Gag-specific CD8+ T cells was higher in the vaccinated-protected animals compared with unprotected monkeys. In addition, polyfunctional SIV-specific CD8+ T cells were consistently detected through 12 wk postchallenge in the protected animals but not in the unprotected animals. In the unprotected monkeys, there was an increased frequency of CD8+ T cells expressing markers associated with effector memory T cells. Further, there was increased annexin V expression in central memory T cells of the unprotected animals before challenge. Thus, monkeys that control viral replication after live attenuated SHIV infection have polyfunctional SIV-specific CD8+ T cells with an increased survival potential. Importantly, the differences in the nature of the SIV-specific CD8+ T cell response in the protected and unprotected animals are present during acute stages postchallenge, before different antigenic levels are established. Thus, the polyfunctional capacity and increased survival potential of CD8+ SIV-specific T cells may account for live attenuated, SHIV89.6-mediated protection from uncontrolled SIV replication.
TLR3-Dependent Induction of Nitric Oxide Synthase in RAW 264.7 Macrophage-Like Cells via a Cytosolic Phospholipase A2/Cyclooxygenase-2 Pathway
Authors: Pindado José , Balsinde Jesús , Balboa María A.
Published: April 2014

dsRNA is a by-product of viral replication capable of inducing an inflammatory response when recognized by phagocyte cells. In this study, we identify group IVA cytosolic phospholipase A2 (cPLA2α) as an effector of the antiviral response. Treatment of RAW 264.7 murine macrophage-like cells with the dsRNA analog polyinosinic:polycytidylic acid (poly-IC) promotes the release of free arachidonic acid that is subsequently converted into PGE2 by the de novo-synthesized cyclooxygenase-2 (COX-2) enzyme. These processes are blocked by the selective cPLA2α inhibitor pyrrophenone, pointing out to cPLA2α as the effector involved. In keeping with this observation, the cPLA2α phosphorylation state increases after cellular treatment with poly-IC. Inhibition of cPLA2α expression and activity by either small interfering RNA (siRNA) or pyrrophenone leads to inhibition of the expression of the inducible NO synthase (iNOS) gene. Moreover, COX-2-derived PGE2 production appears to participate in iNOS expression, because siRNA inhibition of COX-2 also leads to inhibition of iNOS, the latter of which is restored by exogenous addition of PGE2. Finally, cellular depletion of TLR3 by siRNA inhibits COX-2 expression, PGE2 generation, and iNOS induction by poly-IC. Collectively, these findings suggest a model for macrophage activation in response to dsRNA, whereby engagement of TLR3 leads to cPLA2α-mediated arachidonic acid mobilization and COX-2-mediated PGE2 production, which cooperate to induce the expression of iNOS.
Regulatory T Cells Prevent CD8 T Cell Maturation by Inhibiting CD4 Th Cells at Tumor Sites
Authors: Chaput Nathalie , Darrasse-Jèze Guillaume , Bergot Anne-Sophie , Cordier Corinne , Ngo-Abdalla Stacie , Klatzmann David , Azogui Orly
Published: April 2014

Natural regulatory T cells (Tregs) are present in high frequencies among tumor-infiltrating lymphocytes and in draining lymph nodes, supposedly facilitating tumor development. To investigate their role in controlling local immune responses, we analyzed intratumoral T cell accumulation and function in the presence or absence of Tregs. Tumors that grew in normal BALB/c mice injected with the 4T1 tumor cell line were highly infiltrated by Tregs, CD4 and CD8 cells, all having unique characteristics. Most infiltrating Tregs expressed low levels of CD25Rs and Foxp3. They did not proliferate even in the presence of IL-2 but maintained a strong suppressor activity. CD4 T cells were profoundly anergic and CD8 T cell proliferation and cytotoxicity were severely impaired. Depletion of Tregs modified the characteristics of tumor infiltrates. Tumors were initially invaded by activated CD4+CD25− T cells, which produced IL-2 and IFN-γ. This was followed by the recruitment of highly cytotoxic CD8+ T cells at tumor sites leading to tumor rejection. The beneficial effect of Treg depletion in tumor regression was abrogated when CD4 helper cells were also depleted. These findings indicate that the massive infiltration of tumors by Tregs prevents the development of a successful helper response. The Tregs in our model prevent Th cell activation and subsequent development of efficient CD8 T cell activity required for the control of tumor growth.
Maintenance of Surrogate Light Chain Expression Induces Developmental Delay in Early B Cell Compartment
Authors: Martin Denise A. , Lu Liwei , Cascalho Marilia , Wu Gillian E.
Published: April 2014

The production of a mature B cell requires passage through a number of developmental checkpoints. The pre-BCR plays a critical role in passage through the pro-B cell/pre-B cell checkpoint, and thus plays a central role in regulating the differentiation of a B cell. Due to the significance of this receptor, it is imperative that pre-BCR expression and function are precisely regulated. In this study, we have investigated a system in which the regulation of the pre-BCR is altered. We have found that continued expression of components of the pre-BCR (λ5) resulted in a delay in the kinetics of B cell maturation. Pro-B cells from normal mouse bone marrow retrovirally infected with λ5 exhibited a delay in differentiation. As compared with wild-type cells at the same time point, there is a reduction in the presence of cell surface markers that indicate developmental progression, and there is a 6- to 16-fold decrease in the production of Ig-positive cells in B cell maturation assays. The capacity to alter B cell progression by modifying and extending pre-BCR expression argues that the receptor and its associated signals play a unique role in directing developmental outcomes.
Discriminating between Different Pathways of Memory CD8+ T Cell Differentiation
Authors: Ganusov Vitaly V.
Published: April 2014

Despite the rapid accumulation of quantitative data on the dynamics of CD8+ T cell responses following acute viral or bacterial infections of mice, the pathways of differentiation of naive CD8+ T cells into memory during an immune response remain controversial. Currently, three models have been proposed. In the “stem cell-associated differentiation” model, following activation, naive T cells differentiate into stem cell-like memory cells, which then convert into terminally differentiated short-lived effector cells. In the “linear differentiation” model, following activation, naive T cells first differentiate into effectors, and after Ag clearance, effectors convert into memory cells. Finally, in the “progressive differentiation” model, naive T cells differentiate into memory or effector cells depending on the amount of specific stimulation received, with weaker stimulation resulting in formation of memory cells. This study investigates whether the mathematical models formulated from these hypotheses are consistent with the data on the dynamics of the CD8+ T cell response to lymphocytic choriomeningitis virus during acute infection of mice. Findings indicate that two models, the stem cell-associated differentiation model and the progressive differentiation model, in which differentiation of cells is strongly linked to the number of cell divisions, fail to describe the data at biologically reasonable parameter values. This work suggests additional experimental tests that may allow for further discrimination between different models of CD8+ T cell differentiation in acute infections.
Antigen Presentation by Exosomes Released from Peptide-Pulsed Dendritic Cells Is not Suppressed by the Presence of Active CTL
Authors: Luketic Lea , Delanghe Jordan , Sobol Paul T. , Yang Pingchang , Frotten Erin , Mossman Karen L. , Gauldie Jack , Bramson Jonathan , Wan Yonghong
Published: April 2014

Despite the potency of dendritic cells (DCs) as a vaccine carrier, they are short-lived and sensitive to CTL-mediated elimination. Thus, it is believed that the longevity of Ag presentation by peptide-pulsed DC is limited in vivo. Surprisingly, however, we found that although the majority of injected DCs disappeared from the draining lymph nodes within 7 days, Ag presentation persisted for at least 14 days following DC immunization. This prolonged Ag presentation was not mediated by the remaining injected DCs or through Ag transfer to endogenous APCs. We provide evidence that exosomes released by DCs might be responsible for the persistence of Ag presentation. Functional exosomes could be recovered from the draining lymph nodes of C57BL/6 mice following DC vaccination and, in contrast to DCs, T cell stimulation by exosomes in vivo was not affected by the presence of CTL. Our findings demonstrate that Ag presentation following delivery of DC vaccines persists for longer than expected and indicate that the exosome may play a previously unrecognized role in Ag presentation following DC vaccination. Furthermore, our study reinforces the application of exosomes as a vaccination platform and suggests that exosome-based vaccines may be advantageous for booster immunizations due to their resistance to CTL.