bluuebunny/crossref_metadata_2025 huggingface dataset:
IFN-α-Induced Signal Transduction, Gene Expression, and Antitumor Activity of Immune Effector Cells Are Negatively Regulated by Suppressor of Cytokine Signaling Proteins
Authors: Zimmerer Jason M. , Lesinski Gregory B. , Kondadasula Sri Vidya , Karpa Volodymyr I. , Lehman Amy , RayChaudhury Abhik , Becknell Brian , Carson William E.
Published: April 2014

Proteins belonging to the suppressors of cytokine signaling (SOCS) family have been shown to regulate cytokine signal transduction in various cell types but their role in modulating the response of immune cells to IFN-α has not been fully explored. We hypothesized that SOCS proteins would inhibit the antitumor activity of IFN-α-stimulated immune cells. Transcripts for SOCS1, SOCS2, SOCS3, and cytokine-inducible Src homology 2-containing protein were identified in total human PBMC (PBMCs, NK cells, and T cells) within 1–2 h of stimulation with IFN-α (103–105 U/ml). Immunoblot analysis confirmed the expression of these factors at the protein level. Transcripts for SOCS proteins were rapidly but variably induced in PBMCs from patients with metastatic melanoma following the i.v. administration of IFN-α-2b (20 million units/m2). Overexpression of SOCS1 and SOCS3, but not SOCS2, in the Jurkat T cell line inhibited IFN-α-induced phosphorylated STAT1 and the transcription of IFN-stimulated genes. Conversely, small inhibitory RNA-mediated down-regulation of SOCS1 and SOCS3 in Jurkat cells and normal T cells enhanced the transcriptional response to IFN-α. Loss of SOCS1 or SOCS3 in murine immune effectors was associated with enhanced IFN-induced phosphorylated STAT1, transcription of IFN-stimulated genes, and antitumor activity. Of note, IFN-α treatment eliminated melanoma tumors in 70% of SOCS1-deficient mice, whereas IFN-treated SOCS-competent mice all died. The antitumor effects of IFN-α in tumor-bearing SOCS1-deficient mice were markedly inhibited following depletion of CD8+ T cells. These results indicate that the antitumor response of immune effector cells to exogenous IFN-α is regulated by SOCS proteins.
Type 1 Sphingosine 1-Phosphate G Protein-Coupled Receptor (S1P1) Mediation of Enhanced IL-4 Generation by CD4 T Cells from S1P1 Transgenic Mice
Authors: Wang Wengang , Huang Mei-Chuan , Goetzl Edward J.
Published: April 2014

Sphingosine 1-phosphate (S1P) is a natural lipid mediator that regulates immune cell traffic, Ab production, and T cell cytokine generation by mechanisms that enhance Th2 activities. Responses to S1P are controlled principally by the diverse expression patterns of its receptors in different cells. In T cells, the type 1 (S1P1) and type 4 (S1P4) G protein-coupled receptors are predominant. S1P1 mainly transduces effects on T cell migration and trafficking, whereas S1P4 transduces immunosuppression via its effects on T cell proliferation and cytokine production. Using T cell-specific S1P1 transgenic (TG) mice, we investigated the regulatory effects of the S1P-S1P1 axis on T cell cytokine production. The production of IL-4, but not IL-2 or IFN-γ, was significantly up-regulated >10-fold in activated CD4 T cells from S1P1 TG mice compared with those from wild-type mice. Quantitative real-time PCR analysis revealed that IL-4 up-regulation was initiated at the mRNA level as early as 4 h after T cell activation. The up-regulation of IL-4 mRNA was mediated by c-Maf, Jun B, and Gata3 as demonstrated by increases in their protein expression and DNA-binding activities. In contrast, the expression and DNA-binding activities of T-bet, FosB, C-Fos, Jun D, Fra-1, Fra-2, and c-Jun all were identical in wild-type and TG CD4 T cells. Immunological assays showed that increased IL-4 levels induced greater production of IgE. Thus, the S1P-S1P1 axis specifically up-regulates c-Maf, Jun B, and Gata3, which consequently enhance IL-4 production that may lead to a Th2 phenotype.
Stat3 and Stat4 Direct Development of IL-17-Secreting Th Cells
Authors: Mathur Anubhav N. , Chang Hua-Chen , Zisoulis Dimitrios G. , Stritesky Gretta L. , Yu Qing , O’Malley John T. , Kapur Reuben , Levy David E. , Kansas Geoffrey S. , Kaplan Mark H.
Published: April 2014

IL-17-secreting CD4+ T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFβ1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFβ1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORγt expression in TGFβ1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFβ1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types.
Quantitative Network Signal Combinations Downstream of TCR Activation Can Predict IL-2 Production Response
Authors: Kemp Melissa L. , Wille Lucia , Lewis Christina L. , Nicholson Lindsay B. , Lauffenburger Douglas A.
Published: April 2014

Proximal signaling events activated by TCR-peptide/MHC (TCR-pMHC) binding have been the focus of intense ongoing study, but understanding how the consequent downstream signaling networks integrate to govern ultimate avidity-appropriate TCR-pMHC T cell responses remains a crucial next challenge. We hypothesized that a quantitative combination of key downstream network signals across multiple pathways must encode the information generated by TCR activation, providing the basis for a quantitative model capable of interpreting and predicting T cell functional responses. To this end, we measured 11 protein nodes across six downstream pathways, along five time points from 10 min to 4 h, in a 1B6 T cell hybridoma stimulated by a set of three myelin proteolipid protein 139–151 altered peptide ligands. A multivariate regression model generated from this data compendium successfully comprehends the various IL-2 production responses and moreover successfully predicts a priori the response to an additional peptide treatment, demonstrating that TCR binding information is quantitatively encoded in the downstream network. Individual node and/or time point measurements less effectively accounted for the IL-2 responses, indicating that signals must be integrated dynamically across multiple pathways to adequately represent the encoded TCR signaling information. Of further importance, the model also successfully predicted a priori direct experimental tests of the effects of individual and combined inhibitors of the MEK/ERK and PI3K/Akt pathways on this T cell response. Together, our findings show how multipathway network signals downstream of TCR activation quantitatively integrate to translate pMHC stimuli into functional cell responses.
Severe Defect in Thymic Development in an Insertional Mutant Mouse Model
Authors: Assarsson Erika , Chambers Benedict J. , Högstrand Kari , Berntman Emma , Lundmark Carin , Fedorova Ludmila , Imreh Stefan , Grandien Alf , Cardell Susanna , Rozell Björn , Ljunggren Hans-Gustaf
Published: April 2014

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4+ and CD8+ T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.
Autoantigen-B Cell Antigen Receptor Interactions That Regulate Expression of B Cell Antigen Receptor Loci
Authors: Liu Xiaohe , Wysocki Lawrence J. , Manser Tim
Published: April 2014

Levels of AgR (BCR) expression are regulated during B cell development, activation, and induction of tolerance. The mechanisms responsible for and consequences of this regulation are poorly understood. We have described a class of DNA-based autoantigen-reactive B cell that down-regulates BCR expression during development to mature follicular phenotype. In this study, we show that at immature stages of primary differentiation, individual B cells of this type can dynamically modulate levels of expression of BCR in inverse proportion to degree of autoantigen engagement and induced BCR signaling. These adjustments in BCR expression are not associated with cell death, BCR revision, or altered development, and do not require TLR 9. Strikingly, modulation of BCR subunit gene RNA levels and transcription parallels these changes in BCR expression, indicating a direct link between autoantigen-BCR interactions of this type and regulation of transcription of BCR-encoding loci. We propose that this adaptive process allows this class of autoreactive B cell to avoid conventional tolerance pathways and promotes development to mature phenotype.
Both the Suppressor of Cytokine Signaling 1 (SOCS-1) Kinase Inhibitory Region and SOCS-1 Mimetic Bind to JAK2 Autophosphorylation Site: Implications for the Development of a SOCS-1 Antagonist
Authors: Waiboci Lilian W. , Ahmed Chulbul M. , Mujtaba Mustafa G. , Flowers Lawrence O. , Martin James P. , Haider Mohammed I. , Johnson Howard M.
Published: April 2014

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-γ by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-γ-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, (53)DTHFRTFRSHSDYRRI(68) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001–1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr1007, pJAK2(1001–1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-γ-induced STAT1-α phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-α activation. Both Tkip and SOCS1-KIR inhibited IFN-γ activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001–1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001–1013) enhanced suboptimal IFN-γ activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-γ activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001–1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.
CD8+ T Cell Dysfunction and Increase in Murine Gammaherpesvirus Latent Viral Burden in the Absence of 4-1BB Ligand
Authors: Fuse Shinichiro , Bellfy Sarah , Yagita Hideo , Usherwood Edward J.
Published: April 2014

Studies of costimulatory receptors belonging to the TNFR family have revealed their diverse roles in affecting different stages of the T cell response. The 4-1BB ligand (4-1BBL)/4-1BB pathway has emerged as a receptor-ligand pair that impacts not the initial priming, but later phases of the T cell response, such as sustaining clonal expansion and survival, maintaining memory CD8+ T cells, and supporting secondary expansion upon Ag challenge. Although the role of this costimulatory pathway in CD8+ T cell responses to acute viral infections has been well-studied, its role in controlling chronic viral infections in vivo is not known to date. Using the murine gammaherpesvirus-68 (MHV-68) model, we show that 4-1BBL-deficient mice lack control of MHV-68 during latency and show significantly increased latent viral loads. In contrast to acute influenza infection, the numbers of MHV-68-specific memory CD8+ T cells were maintained during latency. However, the virus-specific CD8+ T cells showed defects in function, including decreased cytolytic function and impaired secondary expansion. Thus, 4-1BBL deficiency significantly affects the function, but not the number, of virus-specific CD8+ T cells during gammaherpesvirus latency, and its absence results in an increased viral burden. Our study suggests that the 4-1BB costimulatory pathway plays an important role in controlling chronic viral infections.
IFN-γ Induces Cysteinyl Leukotriene Receptor 2 Expression and Enhances the Responsiveness of Human Endothelial Cells to Cysteinyl Leukotrienes
Authors: Woszczek Grzegorz , Chen Li-Yuan , Nagineni Sahrudaya , Alsaaty Sara , Harry Anya , Logun Carolea , Pawliczak Rafal , Shelhamer James H.
Published: April 2014

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN-γ in human primary endothelial cells. IFN-γ increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN-γ signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-γ. To determine mechanisms of IFN-γ-induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t1/2 assays in response to IFN-γ, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN-γ response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to Gq/11, activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN-γ stimulation. Thus, IFN-γ induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.
Knockout of Mkp-1 Enhances the Host Inflammatory Responses to Gram-Positive Bacteria
Authors: Wang Xianxi , Meng Xiaomei , Kuhlman Joshua R. , Nelin Leif D. , Nicol Kathleen K. , English B. Keith , Liu Yusen
Published: April 2014

MAPK phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAPK. We have previously demonstrated that MKP-1 acts as a negative regulator of p38 and JNK in immortalized macrophages after stimulation with peptidoglycan isolated from Gram-positive bacteria. To define the physiological function of MKP-1 during Gram-positive bacterial infection, we studied the innate immune responses to Gram-positive bacteria using Mkp-1 knockout (KO) mice. We found that Mkp-1−/− macrophages exhibited prolonged activation of p38 and JNK, but not of ERK, following exposure to either peptidoglycan or lipoteichoic acid. Compared with wild-type (WT) macrophages, Mkp-1−/− macrophages produced more proinflammatory cytokines such as TNF-α and IL-6. Moreover, after challenge with peptidoglycan, lipoteichoic acid, live or heat-killed Staphylococcus aureus bacteria, Mkp-1 KO mice also mounted a more robust production of cytokines and chemokines, including TNF-α, IL-6, IL-10, and MIP-1α, than did WT mice. Accordingly, Mkp-1 KO mice also exhibited greater NO production, more robust neutrophil infiltration, and more severe organ damage than did WT mice. Surprisingly, WT and Mkp-1 KO mice exhibited no significant difference in either bacterial load or survival rates when infected with live S. aureus. However, in response to challenge with heat-killed S. aureus, Mkp-1 KO mice exhibited a substantially higher mortality rate compared with WT mice. Our studies indicate that MKP-1 plays a critical role in the inflammatory response to Gram-positive bacterial infection. MKP-1 serves to limit the inflammatory reaction by inactivating JNK and p38, thus preventing multiorgan failure caused by exaggerated inflammatory responses.
Amelioration of Experimental Autoimmune Myasthenia Gravis in Rats by Neonatal FcR Blockade
Authors: Liu Liming , Garcia Ana Maria , Santoro Helen , Zhang Yixia , McDonnell Kevin , Dumont Jennifer , Bitonti Alan
Published: April 2014

The neonatal FcR (FcRn) plays a critical role in IgG homeostasis by protecting it from a lysosomal degradation pathway. It has been shown that IgG has an abnormally short half-life in FcRn-deficient mice and that FcRn blockade significantly increases the catabolism of serum IgG in mice. Therefore, reduction of serum IgG half-life may have therapeutic benefits in Ab-mediated autoimmune diseases. We have studied the therapeutic effects of an anti-rat FcRn mAb, 1G3, in two rat models of myasthenia gravis, a prototypical Ab-mediated autoimmune disease. Passive experimental autoimmune myasthenia gravis was induced by administration of an anti-acetylcholine receptor (AChR) mAb, and it was shown that treatment with 1G3 resulted in dose-dependent amelioration of the disease symptoms. In addition, the concentration of pathogenic Ab in the serum was reduced significantly. The effect of 1G3 was also studied in an active model of experimental autoimmune myasthenia gravis in which rats were immunized with AChR. Treatment with 1G3 significantly reduced the severity of the disease symptoms as well as the levels of total IgG and anti-AChR IgG relative to untreated animals. These data suggest that FcRn blockade may be an effective way to treat Ab-mediated autoimmune diseases.
Cutting Edge: Alternative Signaling of Th17 Cell Development by Sphingosine 1-Phosphate
Authors: Liao Jia-Jun , Huang Mei-Chuan , Goetzl Edward J.
Published: April 2014

Sphingosine 1-phosphate (S1P) in blood and lymph controls T cell traffic and proliferation through type 1 S1P receptor (S1P1) signals, but suppression of IFN-γ generation has been the only consistently observed effect on T cell cytokines. The fact that S1P enhances the development of Th17 cells from Ag-challenged transgenic S1P1-overexpressing CD4 T cells suggested that the S1P-S1P1 axis may promote the expansion of Th17 cells in wild-type mice. In a model of Th17 cell development from CD4 T cells stimulated by anti-CD3 plus anti-CD28 Abs and a mixture of TGF-β1, IL-1, and IL-6, S1P enhanced their number and IL-17-generating activity the same as IL-23. As for IL-23 enhancement of Th17 cell development, that by S1P was prevented by IL-4 plus IFN-γ and by IL-27. The prevention of S1P augmentation of Th17 cell development by the S1P receptor agonist and down-regulator FTY720 implies that FTY720 immunosuppression is attributable partially to inhibition of Th17-mediated inflammation.
The Pro-Th1 Cytokine IL-12 Enhances IL-4 Production by Invariant NKT Cells: Relevance for T Cell-Mediated Hepatitis
Authors: Zhu Ren , Diem Séverine , Araujo Luiza M. , Aumeunier Aude , Denizeau Jordan , Philadelphe Emilie , Damotte Diane , Samson Michel , Gourdy Pierre , Dy Michel , Schneider Elke , Herbelin André
Published: April 2014

IL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokine profile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model, in which iNKT cells, IL-12, IL-4, and IFN-γ are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interacts directly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential for disease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 than their wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenous mediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast to conventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to disease induction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreased iNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 can enhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticity of this subset.
Altering Effects of Antigenic Variations in HIV-1 on Antiviral Effectiveness of HIV-Specific CTLs
Authors: Ueno Takamasa , Idegami Yuka , Motozono Chihiro , Oka Shinichi , Takiguchi Masafumi
Published: April 2014

The mutational escape of HIV-1 from established CTL responses is becoming evident. However, it is not yet clear whether antigenic variations of HIV-1 may have an additional effect on the differential antiviral effectiveness of HIV-specific CTLs. Herein, we characterized HIV-specific CTL responses toward Pol, Env, and Nef optimal epitopes presented by HLA-B*35 during a chronic phase of HIV-1 infection. We found CTL escape variants within Pol and Nef epitopes that affected recognition by TCRs, although there was no mutation within the Env epitope. An analysis of peptide-HLA tetrameric complexes revealed that CD8 T cells exclusively specific for the Nef variant were generated following domination by the variant viruses. The variant-specific cells were capable of killing target cells and producing antiviral cytokines but showed impaired Ag-specific proliferation ex vivo, whereas wild-type specific cells had potent activities. Moreover, clonotypic CD8 T cells specific for the Pol variant showed diminished proliferation, whereas Env-specific ones had no functional heterogeneity. Taken together, our data indicate that antigenic variations that abolished TCR recognition not only resulted in escape from established CTL responses but also eventually generated another subset of variant-specific CTLs having decreased antiviral activity, causing an additional negative effect on antiviral immune responses during a chronic HIV infection.
CD200 Expression on Tumor Cells Suppresses Antitumor Immunity: New Approaches to Cancer Immunotherapy
Authors: Kretz-Rommel Anke , Qin Fenghua , Dakappagari Naveen , Ravey E. Prenn , McWhirter John , Oltean Daniela , Frederickson Shana , Maruyama Toshiaki , Wild Martha A. , Nolan Mary-Jean , Wu Dayang , Springhorn Jeremy , Bowdish Katherine S.
Published: April 2014

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.
Macrophages and Dendritic Cells Use Different Axl/Mertk/Tyro3 Receptors in Clearance of Apoptotic Cells
Authors: Seitz Heather M. , Camenisch Todd D. , Lemke Greg , Earp H. Shelton , Matsushima Glenn K.
Published: April 2014

The clearance of apoptotic cells is important for regulating tissue homeostasis, inflammation, and autoimmune responses. The absence of receptor tyrosine kinases (Axl, Mertk, and Tyro3) results in widespread accumulation of apoptotic cells and autoantibody production in mice. In this report, we examine the function of the three family members in apoptotic cell clearance by different phagocytic cell types. Mertk elimination nearly abolished macrophage apoptotic cell phagocytosis; elimination of Axl, Tyro3, or both, reduced macrophage phagocytosis by approximately half, indicating that these also play a role. In contrast, apoptotic cell clearance in splenic and bone marrow-derived dendritic cells (DCs) is prolonged compared with macrophages and relied primarily on Axl and Tyro3. The slower ingestion may be due to lower DC expression of Axl and Tyro3 or absence of GAS6 expression, a known ligand for this receptor family. In vivo, phagocytosis of apoptotic material by retinal epithelial cells required Mertk. Unlike macrophages, there did not appear to be any role for Axl or Tyro3 in retinal homeostasis. Likewise, clearance of apoptotic thymocytes in vivo was dramatically reduced in mertkkd mice, but was normal in axl/tyro3−/− mice. Thus, cell and organ type specificity is clearly delineated, with DCs relying on Axl and Tyro3, retina and thymus requiring Mertk, and macrophages exhibiting an interaction that involves all three family members. Surprisingly, in macrophages, tyrosine phosphorylation of Mertk in response to apoptotic cells is markedly diminished from axl/tyro3−/− mice, suggesting that the interactions of these receptors by heterodimerization may be important in some cells.
Plasmid DNA Vaccine-Elicited Cellular Immune Responses Limit In Vivo Vaccine Antigen Expression through Fas-Mediated Apoptosis
Authors: Greenland John R. , Geiben Ralf , Ghosh Sharmistha , Pastor William A. , Letvin Norman L.
Published: April 2014

Particularly potent cellular or humoral immune responses are needed to confer protection in animal models against such pathogens as HIV/SIV, Mycobacterium tuberculosis, and malarial parasites. Persistent, high-level vaccine Ag expression may be required for eliciting such potent and durable immune responses. Although plasmid DNA immunogens are being explored as potential vaccines for protection against these pathogens, little is known about host factors that restrict long-term plasmid DNA vaccine Ag expression in vivo. We observed rapid damping of transgene expression from a plasmid DNA immunogen in wild-type, but not in T cell-deficient mice. This damping of Ag expression was temporally associated with the emergence of Ag-specific cellular immune responses. A requirement for Fas and the appearance of apoptotic nuclei at the site of vaccine inoculation suggest that T cells induce Fas-mediated apoptosis of plasmid DNA vaccine Ag-expressing cells. These studies demonstrate that high levels of in vivo Ag expression are associated with high-frequency cellular immune responses that in turn rapidly down-regulate vaccine Ag expression in vivo. These findings argue that it may not be possible to maintain persistent, high-level production of vaccine Ag in vivo to drive persistent immune responses as long as vaccine Ag production can be limited by host immune responses.
Different Evolutionary Histories of the Two Classical Class I Genes BF1 and BF2 Illustrate Drift and Selection within the Stable MHC Haplotypes of Chickens
Authors: Shaw Iain , Powell Timothy J. , Marston Denise A. , Baker Ken , van Hateren Andrew , Riegert Patricia , Wiles Michael V. , Milne Sarah , Beck Stephan , Kaufman Jim
Published: April 2014

Compared with the MHC of typical mammals, the chicken MHC (BF/BL region) of the B12 haplotype is smaller, simpler, and rearranged, with two classical class I genes of which only one is highly expressed. In this study, we describe the development of long-distance PCR to amplify some or all of each class I gene separately, allowing us to make the following points. First, six other haplotypes have the same genomic organization as B12, with a poorly expressed (minor) BF1 gene between DMB2 and TAP2 and a well-expressed (major) BF2 gene between TAP2 and C4. Second, the expression of the BF1 gene is crippled in three different ways in these haplotypes: enhancer A deletion (B12, B19), enhancer A divergence and transcription start site deletion (B2, B4, B21), and insertion/rearrangement leading to pseudogenes (B14, B15). Third, the three kinds of alterations in the BF1 gene correspond to dendrograms of the BF1 and poorly expressed class II B (BLB1) genes reflecting mostly neutral changes, while the dendrograms of the BF2 and well-expressed class II (BLB2) genes each have completely different topologies reflecting selection. The common pattern for the poorly expressed genes reflects the fact the BF/BL region undergoes little recombination and allows us to propose a pattern of descent for these chicken MHC haplotypes from a common ancestor. Taken together, these data explain how stable MHC haplotypes predominantly express a single class I molecule, which in turn leads to striking associations of the chicken MHC with resistance to infectious pathogens and response to vaccines.
Coronin Function Is Required for Chemotaxis and Phagocytosis in Human Neutrophils
Authors: Yan Ming , Di Ciano-Oliveira Caterina , Grinstein Sergio , Trimble William S.
Published: April 2014

Coronins are a family of conserved actin-associated proteins that have been implicated in a variety of cellular processes dependent on actin rearrangements. In this study, we show that in primary human neutrophils, coronins-1–4 and -7 are expressed. Coronin-1 accumulates at the leading edge of migrating neutrophils and at the nascent phagosome. Inhibition of coronin function by transduction of a dominant-negative form of the protein leads to inhibition of chemotaxis and a reduction in neutrophil spreading and adhesion. This inhibition appears to correlate with changes in the distribution of F-actin structures within the cell. In addition, phagocytosis is inhibited, but neither secretion nor activation of the NADPH oxidase appears to be affected. Together, these results show that coronins are required for actin-dependent changes in cell morphology that lead to migration and phagocytosis.
The Host Immune Regulator Factor H Interacts via Two Contact Sites with the PspC Protein of Streptococcus pneumoniae and Mediates Adhesion to Host Epithelial Cells
Authors: Hammerschmidt Sven , Agarwal Vaibhav , Kunert Anja , Haelbich Steffi , Skerka Christine , Zipfel Peter F.
Published: April 2014

Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to host cells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, which is composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, we identified within the factor H protein two separate PspC binding regions, which were localized to SCR8–11 and SCR19–20, by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. A detailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within the sequences of SCR8–11 and SCR19–20. In addition, the factor H binding site was mapped within the pneumococcal PspC protein to a 121-aa-long stretch positioned in the N terminus (residues 38–158). Factor H attached to the surface of pneumococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion was specific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition of factor H by pneumococci via PspC occurs via two contact sites located in SCR8–11 and SCR19–20, and factor H attached to the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells.
Cross-Regulation of Carbon Monoxide and the Adenosine A2a Receptor in Macrophages
Authors: Haschemi Arvand , Wagner Oswald , Marculescu Rodrig , Wegiel Barbara , Robson Simon C. , Gagliani Nicola , Gallo David , Chen Jiang-Fan , Bach Fritz H. , Otterbein Leo E.
Published: April 2014

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5′-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-α production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-α. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.
Interactions between CD47 and Thrombospondin Reduce Inflammation
Authors: Lamy Laurence , Foussat Arnaud , Brown Eric J. , Bornstein Paul , Ticchioni Michel , Bernard Alain
Published: April 2014

CD47 on the surface of T cells was shown in vitro to mediate either T cell activation or, in the presence of high amounts of thrombospondin (TSP), T cell apoptosis. We report here that CD47-deficient mice, as well as TSP-1 or TSP-2-deficient mice, sustain oxazolone-induced inflammation for more than four days, whereas wild-type mice reduce the inflammation within 48 h. We observe that prolonged inflammation in CD47-, TSP-1-, or TSP-2-deficient mice is accompanied by a local deficiency of T cell apoptosis. Finally, we show that upon activation normal T cells increase the expression of the proapoptotic Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein) and undergo CD47-mediated apoptosis. This finding is consistent with our previous demonstration of a physical interaction between BNIP3 and CD47 that inhibits BNIP3 degradation by the proteasome, sensitizing T cells to CD47-induced apoptosis. Overall, these results reveal an important role in vivo for this new CD47/BNIP3 pathway in limiting inflammation by controlling the number of activated T cells.
NAD+ Released during Inflammation Participates in T Cell Homeostasis by Inducing ART2-Mediated Death of Naive T Cells In Vivo
Authors: Adriouch Sahil , Hubert Sandra , Pechberty Severine , Koch-Nolte Friedrich , Haag Friedrich , Seman Michel
Published: April 2014

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD+ onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD+-induced T cell death (NICD). Yet, the origin of extracellular NAD+ and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD+ into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD+ glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD+ used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38−/− mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD+ abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38−/− mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD+ released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.
Cutting Edge: Antioxidative Properties of Myeloid Dendritic Cells: Protection of T Cells and NK Cells from Oxygen Radical-Induced Inactivation and Apoptosis
Authors: Thorén Fredrik B. , Betten Åsa , Romero Ana I. , Hellstrand Kristoffer
Published: April 2014

Dendritic cells (DCs) communicate with nonadaptive and adaptive lymphocytes on multiple levels. Efficient DC-lymphocyte interactions require that lymphocytes remain viable and functional also under conditions of oxidative stress, such as in microbial infection or in the malignant microenvironment. For this study, we exposed human T and NK cells to oxidants delivered either by autologous phagocytes or in the form of exogenous hydrogen peroxide. In accordance with earlier studies, these lymphocytes became dysfunctional and subsequently apoptotic. The presence of myeloid DCs efficiently rescued T cells (CD4+ and CD8+) and NK cells from oxidant-induced inactivation and apoptosis. The mechanism of the myeloid DC-mediated lymphocyte protection was, at least in part, explained by the capacity of the myeloid DCs to neutralize extracellular oxygen radicals, which, in turn, was reversible upon coincubation with a catalase inhibitor. Our results are suggestive of a novel aspect of DC-lymphocyte interaction that may have implications for lymphocyte function in inflamed tissue.
Role of Phosphatidylinositol-3 Kinase in Transcriptional Regulation of TLR-Induced IL-12 and IL-10 by Fcγ Receptor Ligation in Murine Macrophages
Authors: Polumuri Swamy Kumar , Toshchakov Vladimir Y. , Vogel Stefanie N.
Published: April 2014

Ligation of FcγR concurrent with LPS stimulation of murine macrophages results in decreased IL-12 and increased IL-10 production. Because PI3K deficiency has been associated with increased IL-12, we hypothesized that PI3K was central to the anti-inflammatory effect of FcγR ligation on TLR-induced IL-12. FcγR ligation of macrophages increased pAKT, a correlate of PI3K activity, above levels induced by TLR4 or TLR2 agonists. This increase was blocked by PI3K inhibitors, wortmannin or LY294002, as was the effect of FcγR ligation on TLR-induced IL-12 and IL-10. LPS-induced binding of NF-κB to the IL-12 p40 promoter NF-κB-binding site was not affected by FcγR ligation at 1 h; however, by 4 h, NF-κB binding was markedly inhibited, confirmed in situ by chromatin immunoprecipitation analysis. This effect was wortmannin sensitive. Although TLR-induced IκBα degradation was not affected by FcγR ligation, IκBα accumulated in the nuclei of cells treated with LPS and FcγR ligation for 4 h, and was blocked by PI3K inhibitors. LPS-induced IFN regulatory factor-8/IFN consensus sequence-binding protein mRNA, and an IFN regulatory factor-8-dependent gene, Nos2, were inhibited by concurrent FcγR ligation, and this was also reversed by wortmannin. Thus, FcγR ligation modulates LPS-induced IL-12 via multiple PI3K-sensitive pathways that affect production, accumulation, and binding of key DNA-binding proteins required for IL-12 induction.
IL-5-Induced Hypereosinophilia Suppresses the Antigen-Induced Immune Response via a TGF-β-Dependent Mechanism
Authors: Nakagome Kazuyuki , Dohi Makoto , Okunishi Katsuhide , Tanaka Ryoichi , Kouro Taku , Kano Mitsunobu R. , Miyazono Kohei , Miyazaki Jun-ichi , Takatsu Kiyoshi , Yamamoto Kazuhiko
Published: April 2014

Although eosinophils play an essential role in allergic inflammation, their role has recently been under controversy. Epidemic studies suggest that hypereosinophilia induced by parasite infection could suppress subsequent Ag sensitization, although the mechanism has not been fully clarified. In this study, we investigated whether eosinophils could suppress the Ag-specific immune response in the airway. BALB/c mice were sensitized and airway challenged with OVA. Systemic hypereosinophilia was induced by delivery of an IL-5-producing plasmid. IL-5 gene delivery suppressed the Ag-specific proliferation and cytokine production of CD4+ T cells in the spleen. IL-5 gene delivery before OVA sensitization significantly suppressed airway eosinophilia and hyperresponsiveness provoked by subsequent OVA airway challenge, while delivery during the OVA challenge did not suppress them. This IL-5-induced immune suppression was abolished in eosinophil-ablated mice, suggesting an essential role of eosinophils. IL-5 treatment increased the production of TGF-β1 in the spleen, and we demonstrated that the main cellular source of TGF-β1 production was eosinophils, using eosinophil-ablated mice and depletion study. TGF-β1, but not IL-5 itself, suppressed the Ag-specific immune response of CD4+ T cells in vitro. Furthermore, IL-5 treatment enhanced phosphorylation of Smad2 in CD4+ T cells. Finally, a TGF-β type I receptor kinase inhibitor restored this IL-5-induced immune suppression both in vitro and in vivo. These results suggest that IL-5-induced hypereosinophilia could suppress sensitization to Ag via a TGF-β-dependent mechanism, thus suppressed allergic airway inflammation. Therefore, hypereosinophilia could reveal an immunosuppressive effect in the early stage of Ag-induced immune response.
IL-10 Controls Ultraviolet-Induced Carcinogenesis in Mice
Authors: Loser Karin , Apelt Jenny , Voskort Maik , Mohaupt Mariette , Balkow Sandra , Schwarz Thomas , Grabbe Stephan , Beissert Stefan
Published: April 2014

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10+/+, IL-10+/−, and IL-10−/− mice were chronically irradiated with UV. IL-10+/+ and IL-10+/− mice developed skin cancer to similar extents, whereas IL-10−/− mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10−/− mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10−/− mice showed impaired suppressor function when cocultured with conventional CD4+CD25− T cells. CD4+CD25− T cells from IL-10−/− mice produced increased amounts of IFN-γ and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10−/− mice, suggesting strong Th1-drived immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10−/− mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10−/− animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.
Gliadin Regulates the NK-Dendritic Cell Cross-Talk by HLA-E Surface Stabilization
Authors: Terrazzano Giuseppe , Sica Michela , Gianfrani Carmen , Mazzarella Giuseppe , Maurano Francesco , De Giulio Beatrice , de Saint-Mezard Sophie , Zanzi Delia , Maiuri Luigi , Londei Marco , Jabri Bana , Troncone Riccardo , Auricchio Salvatore , Zappacosta Serafino , Carbone Ennio
Published: April 2014

We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-γ production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The α and ω fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin α increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.