bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR2- and TLR4-Mediated Signals Determine Attenuation or Augmentation of Inflammation by Acute Alcohol in Monocytes
Authors: Oak Shilpa , Mandrekar Pranoti , Catalano Donna , Kodys Karen , Szabo Gyongyi
Published: April 2014

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-α protein and mRNA levels and NF-κB activation. In contrast, acute alcohol augmented TNF-α production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- plus TLR4-stimulated monocytes by alcohol. This was supported by decreased IRAK-1:TRAF6 association in TLR4 induced but sustained presence of IRAK-1:TRAF6 complexes in TLR2- plus TLR4-stimulated monocytes after alcohol treatment. Phosphorylation of MAPKs such as ERK1/2 was selectively inhibited by acute alcohol in TLR4-stimulated cells. In contrast, JNK phosphorylation as well as AP-1 nuclear binding were augmented by acute alcohol in the presence of combined TLR4 and TLR2 stimulation. Consistent with this result, the JNK inhibitor prevented alcohol-induced augmentation of TNF-α production. These results suggest that acute alcohol attenuates TLR4-induced inflammation via inhibition of IRAK-1 and ERK1/2 kinases and increases in IRAK-monocyte levels in monocytes. Conversely, in the presence of TLR2 and TLR4 ligands, acute alcohol augments inflammatory responses via IRAK-1 activation and JNK phosphorylation. Thus, the complexity of TLR-mediated signals may determine attenuation or augmentation of inflammatory responses by acute alcohol.
The Envelope Protein of a Human Endogenous Retrovirus-W Family Activates Innate Immunity through CD14/TLR4 and Promotes Th1-Like Responses
Authors: Rolland Alexandre , Jouvin-Marche Evelyne , Viret Christophe , Faure Mathias , Perron Hervé , Marche Patrice N.
Published: April 2014

Multiple sclerosis-associated retroviral element (MSRV) is a retroviral element, the sequence of which served to define the W family of human endogenous retroviruses. MSRV viral particles display proinflammatory activities both in vitro in human mononuclear cell cultures and in vivo in a humanized SCID mice model. To understand the molecular basis of such properties, we have investigated the inflammatory potential of the surface unit of the MSRV envelope protein (ENV-SU), the fraction that is poised to naturally interact with host cells. We report in this study that MSRV ENV-SU induces, in a specific manner, human monocytes to produce major proinflammatory cytokines through engagement of CD14 and TLR4, which are pattern recognition receptors of primary importance in innate immunity. ENV-SU could also trigger a maturation process in human dendritic cells. Finally, ENV-SU endowed dendritic cells with the capacity to support a Th1-like type of Th cell differentiation. The data are discussed in the context of immune responses and chronic proinflammatory disorders.
TLR2 Transmodulates Monocyte Adhesion and Transmigration via Rac1- and PI3K-Mediated Inside-Out Signaling in Response toPorphyromonas gingivalisFimbriae
Authors: Harokopakis Evlambia , Albzreh Mohamad H. , Martin Michael H. , Hajishengallis George
Published: April 2014

We present evidence for a novel TLR2 function in transmodulating the adhesive activities of human monocytes in response to the fimbriae of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis. Monocyte recruitment into the subendothelium is a crucial step in atherosclerosis, and we investigated the role of P. gingivalis fimbriae in stimulating monocyte adhesion to endothelial cells and transendothelial migration. Fimbriae induced CD11b/CD18-dependent adhesion of human monocytes or mouse macrophages to endothelial receptor ICAM-1; these activities were inhibited by TLR2 blockade or deficiency or by pharmacological inhibitors of PI3K. Moreover, this inducible adhesive activity was sensitive to the action of Clostridium difficile toxin B, but was not affected by Clostridium botulinum C3 exoenzyme, pertussis toxin, or cholera toxin. Accordingly, we subsequently showed through the use of dominant negative signaling mutants of small GTPases, that Rac1 mediates the ability of fimbria-stimulated monocytes to bind ICAM-1. A dominant negative mutant of Rac1 also inhibited the lipid kinase activity of PI3K suggesting that Rac1 acts upstream of PI3K in this proadhesive pathway. Furthermore, fimbriae stimulated monocyte adhesion to HUVEC and transmigration across HUVEC monolayers; both activities required TLR2 and Rac1 signaling and were dependent upon ICAM-1 and the high-affinity state of CD11b/CD18. P. gingivalis-stimulated monocytes displayed enhanced transendothelial migration compared with monocytes stimulated with nonfimbriated isogenic mutants. Thus, P. gingivalis fimbriae activate a novel proadhesive pathway in human monocytes, involving TLR2, Rac1, PI3K, and CD11b/CD18, which may constitute a mechanistic basis linking P. gingivalis to inflammatory atherosclerotic processes.
Lovastatin Enhances Clearance of Apoptotic Cells (Efferocytosis) with Implications for Chronic Obstructive Pulmonary Disease
Authors: Morimoto Konosuke , Janssen William J. , Fessler Michael B. , McPhillips Kathleen A. , Borges Valeria M. , Bowler Russell P. , Xiao Yi-Qun , Kench Jennifer A. , Henson Peter M. , Vandivier R. William
Published: April 2014

Statins are potent, cholesterol-lowering agents with newly appreciated, broad anti-inflammatory properties, largely based upon their ability to block the prenylation of Rho GTPases, including RhoA. Because phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, which is inhibited by RhoA, we sought to determine whether statins enhanced efferocytosis. The effect of lovastatin on efferocytosis was investigated in primary human macrophages, in the murine lung, and in human alveolar macrophages taken from patients with chronic obstructive pulmonary disease. In this study, we show that lovastatin increased efferocytosis in vitro in an 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase-dependent manner. Lovastatin acted by inhibiting both geranylgeranylation and farnesylation, and not by altering expression of key uptake receptors or by increasing binding of apoptotic cells to phagocytes. Lovastatin appeared to exert its positive effect on efferocytosis by inhibiting RhoA, because it 1) decreased membrane localization of RhoA, to a greater extent than Rac-1, and 2) prevented impaired efferocytosis by lysophosphatidic acid, a potent inducer of RhoA. Finally, lovastatin increased efferocytosis in the naive murine lung and ex vivo in chronic obstructive pulmonary disease alveolar macrophages in an HMG-CoA reductase-dependent manner. These findings indicate that statins enhance efferocytosis in vitro and in vivo, and suggest that they may play an important therapeutic role in diseases where efferocytosis is impaired and inflammation is dysregulated.
Regional Differences in Blood-Brain Barrier Permeability Changes and Inflammation in the Apathogenic Clearance of Virus from the Central Nervous System
Authors: Phares Timothy W. , Kean Rhonda B. , Mikheeva Tatiana , Hooper D. Craig
Published: April 2014

The loss of blood-brain barrier (BBB) integrity in CNS inflammatory responses triggered by infection and autoimmunity has generally been associated with the development of neurological signs. In the present study, we demonstrate that the clearance of the attenuated rabies virus CVS-F3 from the CNS is an exception; increased BBB permeability and CNS inflammation occurs in the absence of neurological sequelae. We speculate that regionalization of the CNS inflammatory response contributes to its lack of pathogenicity. Despite virus replication and the expression of several chemokines and IL-6 in both regions being similar, the up-regulation of MIP-1β, TNF-α, IFN-γ, and ICAM-1 and the loss of BBB integrity was more extensive in the cerebellum than in the cerebral cortex. The accumulation of CD4- and CD19-positive cells was higher in the cerebellum than the cerebral cortex. Elevated CD19 levels were paralleled by κ-L chain expression levels. The timing of BBB permeability changes, κ-L chain expression in CNS tissues, and Ab production in the periphery suggest that the in situ production of virus-neutralizing Ab may be more important in virus clearance than the infiltration of circulating Ab. The data indicate that, with the possible exception of CD8 T cells, the effectors of rabies virus clearance are more commonly targeted to the cerebellum. This is likely the result of differences in the capacity of the tissues of the cerebellum and cerebral cortex to mediate the events required for BBB permeability changes and cell invasion during virus infection.
CD4 T Cells Play Major Effector Role and CD8 T Cells Initiating Role in Spontaneous Autoimmune Myocarditis of HLA-DQ8 Transgenic IAb Knockout Nonobese Diabetic Mice
Authors: Hayward Sarah L. , Bautista-Lopez Norma , Suzuki Kunimasa , Atrazhev Alexey , Dickie Peter , Elliott John F.
Published: April 2014

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab0; DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown. In the present study, we first investigated whether NOD/Ab0 mice transgenic for a different DQ allele, DQ6, (DQA1*0102, DQB1*0602) would also develop myocarditis. NOD.DQ6/Ab0 animals showed no cardiac pathology, implying that DQ8 is specifically required for the myocarditis phenotype. To further characterize the cellular immune mechanisms, we established crosses of our NOD.DQ8/Ab0 animals with Rag1 knockout (Rag10), Ig H chain knockout (IgH0), and β2-microglobulin knockout (β2m0) lines. Adoptive transfer of purified CD4 T cells from NOD.DQ8/Ab0 mice with complete heart block (an indication of advanced myocarditis) into younger NOD.DQ8/Ab0 Rag10 animals induced cardiac pathology in all recipients, whereas adoptive transfer of purified CD8 T cells or B lymphocytes had no effect. Despite the absence of B lymphocytes, NOD.DQ8/Ab0IgH0 animals still developed complete heart block, whereas NOD.DQ8/Ab0β2m0 mice (which lack CD8 T cells) failed to develop any cardiac pathology. CD8 T cells (and possibly NK cells) seem to be necessary to initiate disease, whereas once initiated, CD4 T cells alone can orchestrate the cardiac pathology, likely through their capacity to recruit and activate macrophages. Understanding the cellular immune mechanisms causing spontaneous myocarditis/IDCM in this relevant animal model will facilitate the development and testing of new therapies for this devastating disease.
Modulation by IL-2 of CD70 and CD27 Expression on CD8+ T Cells: Importance for the Therapeutic Effectiveness of Cell Transfer Immunotherapy
Authors: Huang Jianping , Kerstann Keith W. , Ahmadzadeh Mojgan , Li Yong F. , El-Gamil Mona , Rosenberg Steven A. , Robbins Paul F.
Published: April 2014

Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated CD8+ T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000 IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell surface CD27 expression on CD8+ cells. Withdrawal of IL-2 from activated CD8+ T cells that had been maintained in IL-2 resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70+CD8+ T cells that up-regulated IL-2 receptor expression but did not occur in CD70−CD8+ T cells. Blocking CD70 resulted in a significant reduction of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered to melanoma patients indicated that the size of the pool of CD27+CD8+ T cells in bulk TILs was highly associated (p = 0.004) with the ability of these TILs to mediate tumor regression following adoptive transfer.
IL-23 Is Increased in Dendritic Cells in Multiple Sclerosis and Down-Regulation of IL-23 by Antisense Oligos Increases Dendritic Cell IL-10 Production
Authors: Vaknin-Dembinsky Adi , Balashov Konstantin , Weiner Howard L.
Published: April 2014

IL-23 is a heterodimeric cytokine comprising a p19 subunit associated with the IL-12/23p40 subunit. Like IL-12, IL-23 is expressed predominantly by activated dendritic cells (DCs) and phagocytic cells, and both cytokines induce IFN-γ secretion by T cells. The induction of experimental autoimmune encephalitis, the animal model of multiple sclerosis (MS), occurs in mice lacking IL-12, but not in mice with targeted disruption of IL-23 or both IL-12 and IL-23. Thus, IL-23 expression in DCs may play an important role in the pathogenesis of human autoimmune diseases such as MS. We quantified the expression of IL-23 in monocyte-derived DCs in MS patients and healthy donors and found that DCs from MS patients secrete elevated amounts of IL-23 and express increased levels of IL-23p19 mRNA. Consistent with this abnormality, we found increased IL-17 production by T cells from MS patients. We then transfected monocyte-derived DCs from healthy donors with antisense oligonucleotides specific for the IL-23p19 and IL-12p35 genes and found potent suppression of gene expression and blockade of bioactive IL-23 and IL-12 production without affecting cellular viability or DCs maturation. Inhibition of IL-23 and IL-12 was associated with increased IL-10 and decreased TNF-α production. Furthermore, transfected DCs were poor allostimulators in the MLR. Our results demonstrate that an abnormal Th1 bias in DCs from MS patients related to IL-23 exists, and that antisense oligonucleotides specific to IL-23 can be used for immune modulation by targeting DC gene expression.
Molecular Characterization of the Anti-Idiotypic Immune Response of a Relapse-Free Neuroblastoma Patient following Antibody Therapy: A Possible Vaccine against Tumors of Neuroectodermal Origin?
Authors: Uttenreuther-Fischer Martina M. , Krüger Jörg A. , Fischer Peter
Published: April 2014

Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis. According to Jerne’s network theory, a subset of anti-idiotypic Abs (Ab2β) carries an “internal image” of the Ag and induces Abs (Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative germline genes ranged between 94.90 and 100% for VH and between 93.90 and 99.60% for VL. An overall high rate of replacement mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and GK8, inhibited binding of ch14.18 to GD2 just as the patient’s serum did. GK8 alone inhibited >80% of the patient’s anti-idiotypic serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag GD2. GK8 may be useful as a tumor vaccine for CD3-positive tumors.
Intracellular TLR Signaling: A Structural Perspective on Human Disease
Authors: Lasker Michael V. , Nair Satish K.
Published: April 2014

TLRs are crucial sensors of microbial infection. Maintaining structural integrity of TLR signaling components is essential for subsequent immunological protection. Alterations to the structure of these signaling molecules are often associated with profound clinical outcomes and susceptibility to various infectious diseases. These changes in structure are sometimes the result of a single nucleotide polymorphism (SNP). Numerous SNPs have been found in components of the TLR signaling pathway. Recently, the medical consequences and effects on TLR signaling of several of these SNPs have been elucidated. In addition, there have been numerous structures solved that are important to our understanding of the TLR signaling pathway at the molecular level. The scope of this review is to tie together current structural, biochemical, and genetic information of TLR signaling.
Interleukin-21 Enhances NK Cell Activation in Response to Antibody-Coated Targets
Authors: Roda Julie M. , Parihar Robin , Lehman Amy , Mani Aruna , Tridandapani Susheela , Carson William E.
Published: April 2014

NK cells express an activating FcR (FcγRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-γ secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-γ. Increased secretion of TNF-α and the chemokines IL-8, MIP-1α, and RANTES was also observed under these conditions. NK cell IFN-γ production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-γ by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-γ. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.
Kynurenine Pathway Enzymes in Dendritic Cells Initiate Tolerogenesis in the Absence of Functional IDO
Authors: Belladonna Maria L. , Grohmann Ursula , Guidetti Paolo , Volpi Claudia , Bianchi Roberta , Fioretti Maria C. , Schwarcz Robert , Fallarino Francesca , Puccetti Paolo
Published: April 2014

Dendritic cell (DC) tryptophan catabolism has emerged in recent years as a major mechanism of peripheral tolerance. However, there are features of this mechanism, initiated by IDO, that are still unclear, including the role of enzymes that are downstream of IDO in the kynurenine pathway and the role of the associated production of kynurenines. In this study, we provide evidence that 1) murine DCs express all enzymes necessary for synthesis of the downstream product of tryptophan breakdown, quinolinate; 2) IFN-γ enhances transcriptional expression of all of these enzymes, although posttranslational inactivation of IDO may prevent metabolic steps that are subsequent and consequent to IDO; 3) overcoming the IDO-dependent blockade by provision of a downstream quinolinate precursor activates the pathway and leads to the onset of suppressive properties; and 4) tolerogenic DCs can confer suppressive ability on otherwise immunogenic DCs across a Transwell in an IDO-dependent fashion. Altogether, these data indicate that kynurenine pathway enzymes downstream of IDO can initiate tolerogenesis by DCs independently of tryptophan deprivation. The paracrine production of kynurenines might be one mechanism used by IDO-competent cells to convert DCs lacking functional IDO to a tolerogenic phenotype within an IFN-γ-rich environment.
Self-Antigen Maintains the Innate Antibacterial Function of Self-Specific CD8 T Cells In Vivo
Authors: Dhanji Salim , Chow Michael T. , Teh Hung-Sia
Published: April 2014

Self-specific CD8 T cells, which are selected by high-affinity interactions with self-Ags, develop into a lineage distinct from conventional CD8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. In this study, we show that these self-specific cells have the ability to produce large amounts of IFN-γ in response to infection with Listeria monocytogenes in a bystander fashion. The rapid production of IFN-γ is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific CD8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific CD8 T cells are also more efficient in the production of IFN-γ and TNF-α, thus providing more cytokine-dependent protection against bacterial infection when compared with NK cells. These findings illustrate that self-reactive CD8 T cells can play an important innate function in the early defense against bacterial infection.
Flotillin-1 Regulates IgE Receptor-Mediated Signaling in Rat Basophilic Leukemia (RBL-2H3) Cells
Authors: Kato Naoto , Nakanishi Mamoru , Hirashima Naohide
Published: April 2014

Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca2+ mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)2 rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca2+ mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the γ-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.
A Modified Tyrosinase-Related Protein 2 Epitope Generates High-Affinity Tumor-Specific T Cells but Does Not Mediate Therapeutic Efficacy in an Intradermal Tumor Model
Authors: McWilliams Jennifer A. , McGurran Sean M. , Dow Steven W. , Slansky Jill E. , Kedl Ross M.
Published: April 2014

The generation of tumor-specific T cells is hampered by the presentation of poorly immunogenic tumor-specific epitopes by the tumor. Here, we demonstrate that, although CD8+ T cells specific for the self/tumor Ag tyrosinase-related protein 2 (TRP2) are readily detected in tumor-bearing hosts, vaccination of either tumor-bearing or naive mice with an epitope derived from TRP2 fails to generate significant numbers of tetramer-staining TRP2-specific T cells or antitumor immunity. We identified an altered peptide epitope, called deltaV, which elicits T cell responses that are cross-reactive to the wild-type TRP2 epitope. Immunization with deltaV generates T cells with increased affinity for TRP2 compared with immunization with the wild-type TRP2 epitope, although TRP2 immunization often generates a greater number of TRP2-specific T cells based on intracellular IFN-γ analysis. Despite generating higher affinity responses, deltaV immunization alone fails to provide any greater therapeutic efficacy against tumor growth than TRP2 immunization. This lack of tumor protection is most likely a result of both the deletion of high affinity and functional tolerance induction of lower affinity TRP2-specific T cells. Our data contribute to a growing literature demonstrating the ability of variant peptide epitopes to generate higher affinity T cell responses against tumor-specific Ags. However, consistent with most clinical data, simple generation of higher affinity T cells is insufficient to mediate tumor immunity.
Tumor Escape Mutants Develop within an Immune-Privileged Environment in the Absence of T Cell Selection
Authors: Chen Peter W. , Uno Toshihiko , Ksander Bruce R.
Published: April 2014

The establishment of tumor escape mutants, which can be driven by innate and/or adaptive immune effector cells, presents a significant obstacle in the development of successful tumor immunotherapies. Our study documents that tumors growing within an immune-privileged site within the eye develop a tumor escape phenotype in the absence of selective T cell pressure. P815 tumor cells that are recovered from progressively growing tumors within the anterior chamber of the eye escape elimination when injected into the flanks of a second group of syngeneic DBA/2 mice that were previously immunized against P815 tumor cells. The escape phenotype of eye-derived P815 tumors was stable and permanent when the tumor cells were cultured in vitro. Eye-derived tumor cells recovered from the anterior chamber of CB-17 SCID mice also escaped elimination when injected into the flanks of immunized mice, demonstrating that selective pressure by tumor Ag-specific T cells did not contribute to the development of the escape phenotype. In vitro studies demonstrated that eye-derived tumor cells were not lysed by specific CTL and were unable to restimulate primed Ag-specific T cells. Immune escape of eye-derived tumor cells was not due to down-regulation of either MHC class I or ICAM-1. Our data demonstrate that the immune-privileged environment within the eye induces a tumor escape phenotype that is not driven by selective T cell pressure. We predict that immune escape within the eye is driven by the unique ocular environment that permanently alters gene expression in eye-derived tumor cells.
Leptin Selectively Augments Thymopoiesis in Leptin Deficiency and Lipopolysaccharide-Induced Thymic Atrophy
Authors: Hick Ryan W. , Gruver Amanda L. , Ventevogel Melissa S. , Haynes Barton F. , Sempowski Gregory D.
Published: April 2014

The thymus is a lymphoid organ that selects T cells for release to the peripheral immune system. Unfortunately, thymopoiesis is highly susceptible to damage by physiologic stressors and can contribute to immune deficiencies that occur in a variety of clinical settings. No treatment is currently available to protect the thymus from stress-induced involution. Leptin-deficient (ob/ob) mice have severe thymic atrophy and this finding suggests that this hormone is required for normal thymopoiesis. In this study, the ability of leptin to promote thymopoiesis in wild-type C57BL/6 and BALB/c mice, as well as in leptin-deficient (ob/ob) and endotoxin-stressed (Escherichia coli LPS) mice, was determined. Leptin administration induced weight loss and stimulated thymopoiesis in ob/ob mice, but did not stimulate thymopoiesis in wild-type C57BL/6 nor BALB/c mice. In endotoxin-stressed mice, however, leptin prevented LPS-induced thymus weight loss and stimulated TCRα gene rearrangement. Coadministration of leptin with LPS blunted endotoxin-induced systemic corticosterone response and production of proinflammatory cytokines. Thus, leptin has a selective thymostimulatory role in settings of leptin deficiency and endotoxin administration, and may be useful for protecting the thymus from damage and augmenting T cell reconstitution in these clinical states.
Cutting Edge: The Silent Chemokine Receptor D6 Is Required for Generating T Cell Responses That Mediate Experimental Autoimmune Encephalomyelitis
Authors: Liu LiPing , Graham Gerard J. , Damodaran Anita , Hu Taofang , Lira Sergio A. , Sasse Margaret , Canasto-Chibuque Claudia , Cook Donald N. , Ransohoff Richard M.
Published: April 2014

D6, a promiscuous nonsignaling chemokine binding molecule expressed on the lymphatic endothelium, internalizes and degrades CC chemokines, and D6−/− mice demonstrated increased cutaneous inflammation following topical phorbol ester or CFA injection. We report that D6−/− mice were unexpectedly resistant to the induction of experimental autoimmune encephalomyelitis due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35–55 in CFA, D6−/− mice showed reduced spinal cord inflammation and demyelination with lower incidence and severity of experimental autoimmune encephalomyelitis attacks as compared with D6+/+ littermates. In adoptive transfer studies, MOG-primed D6+/− T cells equally mediated disease in D6+/+ or D6−/− mice, whereas cells from D6−/− mice transferred disease poorly to D6+/− recipients. Lymph node cells from MOG-primed D6−/− mice showed weak proliferative responses and made reduced IFN-γ but normal IL-5. CD11c+ dendritic cells accumulated abnormally in cutaneous immunization sites of D6−/− mice. Surprisingly, D6, a “silent” chemokine receptor, supports immune response generation.
Increased Level and Longevity of Protective Immune Responses Induced by DNA Vaccine Expressing the HIV-1 Env Glycoprotein when Combined withIL-21andIL-15Gene Delivery
Authors: Bolesta Elizabeth , Kowalczyk Aleksandra , Wierzbicki Andrzej , Eppolito Cheryl , Kaneko Yutaro , Takiguchi Masafumi , Stamatatos Leonidas , Shrikant Protul A. , Kozbor Danuta
Published: April 2014

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140ΔCFIHXB2/89.6 vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env121–129 epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.
Prolonged Cell Cycle Transit Is a Defining and Developmentally Conserved Hemopoietic Stem Cell Property
Authors: Nygren Jens M. , Bryder David , Jacobsen Sten Eirik W.
Published: April 2014

Adult mouse hemopoietic stem cells (HSCs) are typically quiescent and enter and progress through the cell cycle rarely in steady-state bone marrow, but their rate of proliferation can be dramatically enhanced on demand. We have studied the cell cycle kinetics of HSCs in the developing fetal liver at a stage when they expand extensively. Despite that 100% of fetal liver HSCs divide within a 48-h period, their average cell cycle transit time (10.6 h) is twice that of their downstream progenitors, translating into a prolonged G1 transit and a period of relative quiescence (G0). In agreement with their prolonged G1 transit when compared with hemopoietic progenitors, competitive transplantation experiments demonstrate that fetal HSCs are highly enriched in G1 but also functional in S-G2-M. This observation combined with experimental data demonstrating that adult HSCs forced to expand ex vivo also sustain a uniquely prolonged cell cycle and G1 transit, demonstrate at least in part why purified HSCs at any state of development or condition are highly enriched in the G0-G1 phases of the cell cycle. We propose that a uniquely prolonged cell cycle transit is a defining stem cell property, likely to be critical for their maintenance and self-renewal throughout development.
Cutting Edge: STAT1 and T-bet Play Distinct Roles in Determining Outcome of Visceral Leishmaniasis Caused byLeishmania donovani
Authors: Rosas Lucia E. , Snider Heidi M. , Barbi Joseph , Satoskar Anjali A. , Lugo-Villarino Geanncarlo , Keiser Tracy , Papenfuss Tracy , Durbin Joan E. , Radzioch Danuta , Glimcher Laurie H. , Satoskar Abhay R.
Published: April 2014

T-bet and STAT1 regulate IFN-γ gene transcription in CD4+ T cells, which mediate protection against Leishmania. Here we show that T-bet and STAT1 are required for the induction of an efficient Th1 response during Leishmania donovani infection, but they play distinct roles in determining disease outcome. Both STAT1−/− and T-bet−/− mice failed to mount a Th1 response, but STAT1−/− mice were highly resistant to L. donovani and developed less immunopathology, whereas T-bet−/− mice were highly susceptible and eventually developed liver inflammation. Adoptive cell transfer studies showed that RAG2−/− recipients receiving STAT1+/+ or STAT1−/− T cells developed comparable liver pathology, but those receiving STAT1−/− T cells were significantly more susceptible to infection. These unexpected findings reveal distinct roles for T-bet and STAT1 in mediating host immunity and liver pathology during visceral leishmaniasis.
Tight Linkage between Translation and MHC Class I Peptide Ligand Generation Implies Specialized Antigen Processing for Defective Ribosomal Products
Authors: Qian Shu-Bing , Reits Eric , Neefjes Jacques , Deslich Jeanne M. , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2014

There is mounting evidence that MHC class I peptide ligands are predominantly generated from defective ribosomal products and other classes of polypeptides degraded rapidly (t1/2 < 10 min) following their synthesis. The most direct evidence supporting this conclusion is the rapid inhibition of peptide ligand generation following cycloheximide-mediated inhibition of protein synthesis. In this study, we show that this linkage is due to depleting the pool of rapidly degraded proteins, and not to interference with other protein synthesis-dependent processes. Our findings indicate that in the model systems used in this study, MHC class I peptides are preferentially generated from rapidly degraded polypeptides relative to slowly degraded proteins. This conclusion is supported by the properties of peptide presentation from slowly degraded (t1/2 = 4 h) defective ribosomal products generated artificially by incorporation of the amino acid analog canavanine into a model viral Ag. We propose that specialized machinery exists to link protein synthesis with class I peptide ligand generation to enable the rapid detection of viral gene expression.
IL-18 Bridges Innate and Adaptive Immunity through IFN-γ and the CD134 Pathway
Authors: Maxwell Joseph R. , Yadav Rajwardhan , Rossi Robert J. , Ruby Carl E. , Weinberg Andrew D. , Aguila Hector L. , Vella Anthony T.
Published: April 2014

IL-18 induces inflammation resulting in either enhanced protection from pathogens or exacerbation of autoimmunity, and T cells are profoundly activated during these responses. How IL-18 influences T cell activation is unknown, but this study in mice shows that IL-18 boosted Ag-specific T cell clonal expansion of effector T cells and induced a subpopulation of IFN-γ superproducing T cells. Commitment to IFN-γ production through IL-18 was independent of NK cells and IL-12 but dependent on host-derived IFN-γ. To determine how expansion of these effectors occurred, IL-18 was shown to induce OX40L on dendritic cells, whereas peptide stimulation induced CD134 (OX40) on specific T cells. CD134 blockade inhibited T cell effector expansion thereby reducing the number of IFN-γ superproducers by 12-fold. Thus, independent of IL-12, IL-18 impacts T cell immunity throughout lymphoid and nonlymphoid tissue by bridging the innate and adaptive arms of the immune system through IFN-γ and the CD134 costimulatory pathway.
Accumulation of CD8+ T Cells in Advanced-Stage Tumors and Delay of Disease Progression following Secondary Immunization against an Immunorecessive Epitope
Authors: Ryan Christina M. , Schell Todd D.
Published: April 2014

Self-reactive T cells that survive the process of positive and negative selection during thymocyte development represent potential effector cells against tumors that express these same self-Ags. We have previously shown that CD8+ T lymphocytes (TCD8) specific for an immunorecessive epitope, designated epitope V, from the SV40 large T Ag (Tag) escape thymic deletion in line SV11 Tag-transgenic mice. In contrast, these mice are tolerant to the three most dominant Tag epitopes. The majority of the residual epitope V-specific TCD8 have a low avidity for the target epitope, but a prime/boost regimen can expand higher avidity clones in vivo. Whether higher avidity TCD8 targeting this epitope are affected by Tag-expressing tumors in the periphery or can be recruited for control of tumor progression remains unknown. In the current study, we determined the fate of naive TCR-transgenic TCD8 specific for Tag epitope V (TCR-V cells) following transfer into SV11 mice bearing advanced-stage choroid plexus tumors. The results indicate that TCR-V cells are rapidly triggered by the endogenous Tag and acquire effector function, but fail to accumulate within the tumors. Primary immunization enhanced TCR-V cell frequency in the periphery and promoted entry into the brain, but a subsequent booster immunization caused a dramatic accumulation of TCR-V T cells within the tumors and inhibited tumor progression. These results indicate that epitope V provides a target for CD8+ T cells against spontaneous tumors in vivo, and suggests that epitopes with similar properties can be harnessed for tumor immunotherapy.
Cutting Edge: Impaired Glycosphingolipid Trafficking and NKT Cell Development in Mice Lacking Niemann-Pick Type C1 Protein
Authors: Sagiv Yuval , Hudspeth Kelly , Mattner Jochen , Schrantz Nicolas , Stern Randi K. , Zhou Dapeng , Savage Paul B. , Teyton Luc , Bendelac Albert
Published: April 2014

Niemann-Pick Type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Vα14-Jα18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent α-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Vα14-Jα18 NKT cells.
CD44 Differentially Activates Mouse NK T Cells and Conventional T Cells
Authors: Larkin Jonathan , Renukaradhya Gourapura J. , Sriram Venkataraman , Du Wenjun , Gervay-Hague Jacquelyn , Brutkiewicz Randy R.
Published: April 2014

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.
Cutting Edge: Dendritic Cells Are Essential for In Vivo IL-12 Production and Development of Resistance againstToxoplasma gondiiInfection in Mice
Authors: Liu Cheng-Hu , Fan Yu-ting , Dias Alexandra , Esper Lisia , Corn Radiah A. , Bafica Andre , Machado Fabiana S. , Aliberti Julio
Published: April 2014

A powerful IFN-γ response is triggered upon infection with the protozoan parasite, Toxoplasma gondii. Several cell populations, including dendritic cells (DCs), macrophages, and neutrophils, produce IL-12, a key cytokine for IFN-γ induction. However, it is still unclear which of the above cell populations is its main source. Diphtheria toxin (DT) injection causes transient DC depletion in a transgenic mouse expressing Simian DT receptors under the control of the CD11c promoter, allowing us to investigate the role of DCs in IL-12 production. T. gondii-inoculated DT-treated and control groups were monitored for IL-12 levels and survival. We show in this study that DC depletion abolished IL-12 production and led to mortality. Furthermore, replenishment with wild-type, but not MyD88- or IL-12p35-deficient, DCs rescued IL-12 production, IFN-γ-induction, and resistance to infection in DC-depleted mice. Taken together, the results presented in this study indicate that DCs constitute the major IL-12-producing cell population in vivo during T. gondii infection.
IFN-γ and Its Receptor Subunit IFNGR1 Are Recruited to the IFN-γ-Activated Sequence Element at the Promoter Site of IFN-γ-Activated Genes: Evidence of Transactivational Activity in IFNGR1
Authors: Ahmed Chulbul M. I. , Johnson Howard M.
Published: April 2014

We have shown previously that IFN-γ and one of its receptor subunits, IFNGR1, are translocated to the nucleus, together with STAT1α as one macromolecular complex, via the classical importin-dependent pathway. In this study, we have identified the nuclear targets of IFN-γ and IFNGR1. By chromatin immunoprecipitation followed by PCR, IFN-γ, its receptor subunit IFNGR1, and STAT1α were found to be associated with the IFN-γ-activated sequence (GAS) in the promoter of two of the genes stimulated by IFN-γ. Immunoprecipitated chromatin also showed the association of the IFN-γ, IFNGR1, and STAT1α on the same DNA sequence. Examination of nuclear extracts from WISH cells treated with IFN-γ revealed the specific binding of IFN-γ, IFNGR1, and STAT1α to biotinylated GAS nucleotide sequence. Association of IFN-γ, IFNGR1, and STAT1α with the GAS promoter was also demonstrated by EMSA. Transfection with a GAS-luciferase gene together with the IFNGR1 and nonsecreted IFN-γ resulted in enhanced reporter activity. In addition, IFNGR1 fused to the yeast GAL4 DNA binding domain resulted in enhanced transcription from a GAL4 response element, suggesting the presence of a trans activation domain in IFNGR1. Our observations put IFN-γ and its receptor subunit, IFNGR1, in direct contact with the promoter region of IFN-γ-activated genes with associated increased activity, thus suggesting a transcriptional/cotranscriptional role for IFN-γ/IFNGR1 as well as a possible role in determining the specificity of IFN-γ action.
Trends in Antibody Sequence Changes during the Somatic Hypermutation Process
Authors: Clark Louis A. , Ganesan Skanth , Papp Sarah , van Vlijmen Herman W. T.
Published: April 2014

Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are ∼4% for core residues, ∼5% for surface residues, and ∼12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.