bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR Ligands in the Local Treatment of Established Intracerebral Murine Gliomas
Authors: Grauer Oliver M. , Molling Johan W. , Bennink Erik , Toonen Liza W. J. , Sutmuller Roger P. M. , Nierkens Stefan , Adema Gosse J.
Published: April 2014

Local TLR stimulation is an attractive approach to induce antitumor immunity. In this study, we compared various TLR ligands for their ability to affect murine GL261 cells in vitro and to eradicate established intracerebral murine gliomas in vivo. Our data show that GL261 cells express TLR2, TLR3, and TLR4 and respond to the corresponding TLR ligands with increasing MHC class I expression and inducing IL-6 secretion in vitro, while TLR5, TLR7, and TLR9 are essentially absent. Remarkably, CpG-oligonucleotides (CpG-ODN, TLR9) appeared to inhibit GL261 cell proliferation in a cell-type specific, but CpG-motif and TLR9-independent manner. A single intratumoral injection of CpG-ODN most effectively inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice. Intratumoral injection of Pam3Cys-SK4 (TLR1/2) or R848 (TLR7) also produced a significant survival benefit, whereas poly(I:C) (TLR3) or purified LPS (TLR4) stimulation alone was not effective. Additional studies using TLR9+/+ wild-type and TLR9−/− knockout mice revealed that the efficacy of local CpG-ODN treatment in vivo required TLR9 expression on nontumor cells. Additional experiments demonstrated increased frequencies of tumor-infiltrating IFN-γ producing CD4+ and CD8+ effector T cells and a marked increase in the ratio of CD4+ effector T cells to CD4+FoxP3+ regulatory T cells upon CpG-ODN treatment. Surviving CpG-ODN treated mice were also protected from a subsequent tumor challenge without further addition of CpG-ODN. In summary, this study underlines the potency of local TLR treatment in antiglioma therapy and demonstrates that local CpG-ODN treatment most effectively restores antitumor immunity in a therapeutic murine glioma model.
IFN-β Production by TLR4-Stimulated Innate Immune Cells Is Negatively Regulated by GSK3-β
Authors: Wang Huizhi , Garcia Carlos A. , Rehani Kunal , Cekic Caglar , Alard Pascale , Kinane Denis F. , Mitchell Thomas , Martin Michael
Published: April 2014

TLR 4 stimulation of innate immune cells induces a MyD88-independent signaling pathway that leads to the production of IFN-β. In this study, we demonstrate glycogen synthase kinase 3-β (GSK3-β) plays a fundamental role in this process. Suppression of GSK3-β activity by either pharmacological inhibition, small interfering RNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-β mutant enhanced IFN-β production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-β mutant severely attenuated IFN-β production. GSK3-β was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. Small interfering RNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-β inhibition to augment IFN-β, demonstrating that the ability of GSK3 to control IFN-β production was due to its ability to regulate c-Jun levels. The ability of GSK3 inhibition to control IFN-β production was confirmed in vivo as mice treated with a GSK3 inhibitor exhibited enhanced systemic levels of IFN-β upon LPS challenge. These findings identify a novel regulatory pathway controlling IFN-β production by TLR4-stimulated innate immune cells.
Ion Channels Modulating Mouse Dendritic Cell Functions
Authors: Matzner Nicole , Zemtsova Irina M. , Xuan Nguyen Thi , Duszenko Michael , Shumilina Ekaterina , Lang Florian
Published: April 2014

Ca2+-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca2+]i upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca2+]i, which was due to Ca2+ release from intracellular stores and influx of extracellular Ca2+ across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca2+ release-activated Ca2+ channels (CRAC). These currents were highly selective for Ca2+, exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca2+-dependent inactivation. In addition, the LPS-induced increase of [Ca2+]i was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K+ (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-α and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the ICRAC inhibitor SKF-96365. Taken together, our results demonstrate that Ca2+ influx in LPS-stimulated DCs occurs via Ca2+ release-activated Ca2+ channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.
Suppression of Murine Allergic Airway Disease by IL-2:Anti-IL-2 Monoclonal Antibody-Induced Regulatory T Cells
Authors: Wilson Mark S. , Pesce John T. , Ramalingam Thirumalai R. , Thompson Robert W. , Cheever Allen , Wynn Thomas A.
Published: April 2014

Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airway inflammation and eosinophilia while suppressing IL-5 and eotaxin-1 production. Mucus production, airway hyperresponsiveness to methacholine, and parenchymal tissue inflammation were also dramatically reduced following IL-2:anti-IL-2 treatment. The suppression in allergic airway disease was associated with a marked expansion of Tregs (IL-10+CD4+CD25+ and Foxp3+CD4+CD25+) in the tissues, with a corresponding decrease in effector T cell responses. The ability of IL-2:anti-IL-2 complexes to suppress airway inflammation was dependent on Treg-derived IL-10, as IL-10+/+, but not IL-10−/− Tregs, were capable of mediating the suppression. Furthermore, a therapeutic protocol using a model of established airway allergy highlighted the ability of IL-2:anti-IL-2 complexes to expand Tregs and prevent successive airway inflammation and airway hyperresponsiveness. This study suggests that endogenous Treg therapy may be a useful tool to combat the rising incidence of allergic airway disease.
CD4+ and CD8+ T Cells Exhibit Differential Requirements for CCR7-Mediated Antigen Transport during Influenza Infection
Authors: Heer Alex K. , Harris Nicola L. , Kopf Manfred , Marsland Benjamin J.
Published: April 2014

Upon encounter of viral Ags in an inflammatory environment, dendritic cells up-regulate costimulatory molecules and the chemokine receptor CCR7, with the latter being pivotal for their migration to the lymph node. By utilizing mice deficient in CCR7, we have examined the requirement of dendritic cell-mediated Ag transport from the lung to the draining lymph node for the induction of anti-influenza immune responses in vivo. We found that CCR7-mediated migration of dendritic cells was more crucial for CD8+ T cell than CD4+ T cell responses. While no specific CD8+ T cell response could be detected in the airways or lymphoid tissues during the primary infection, prolonged infection in CCR7-deficient mice did result in a sustained inflammatory chemokine profile, which led to nonspecific CD8+ T cell recruitment to the airways. The recruitment of influenza-specific CD4+ T cells to the airways was also below levels of detection in the absence of CCR7 signaling, although a small influenza-specific CD4+ T cell population was detectable in the draining lymph node, which was sufficient for the generation of class-switched anti-influenza Abs and a normal CD4+ T cell memory population. Overall, our data show that CCR7-mediated active Ag transport is differentially required for CD4+ and CD8+ T cell expansion during influenza infection.
A Novel C1q Family Member of Amphioxus Was Revealed to Have a Partial Function of Vertebrate C1q Molecule
Authors: Yu Yanhong , Huang Huiqing , Wang Yan , Yu Yingcai , Yuan Shaochun , Huang Shengfeng , Pan Minming , Feng Kaixia , Xu Anlong
Published: April 2014

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and adaptive immunities. Its globular signature domain is also found in a variety of noncomplement protein that can be grouped together as a C1q family. In this study, we have cloned and identified a novel C1q family member in cephalochordate amphioxus and named it as AmphiC1q1. The high transcriptional levels of this gene were detected during all stages of embryonic development, and the section in situ hybridization demonstrated that AmphiC1q1 was mainly expressed in the ovary, intestine, and nerve system of mature individuals. The transcript of AmphiC1q1 was up-regulated by LPS and Gram-negative bacteria, but hardly by lipoteichoic acid and Staphylococcus aureus. The recombinant AmphiC1q1 protein could not bind with N-acetyl-glucosamine and did not possess hemagglutinating activity, indicating that AmphiC1q1 could not act as its lamprey homologue. But both the full-length protein and its truncated globular domain of C1q protein could interact with LPS. Moreover, recombinant AmphiC1q1 protein could inhibit collagen-induced platelet aggregation, but the truncated globular C1q domain protein would not, indicating that the blocking activity of AmphiC1q1 protein was via the collagen region of the protein. Our study on the primitive form of C1q family in protochordate will shed a light on understanding the gradual functional evolution of C1q family and eventual formation of mammalian homologues.
Hyaluronan Induces Cell Death in Activated T Cells through CD44
Authors: Ruffell Brian , Johnson Pauline
Published: April 2014

In the immune system, leukocyte activation induces CD44 to bind hyaluronan, a component of the extracellular matrix. Here we used gain and loss of hyaluronan-binding mutants of CD44 to examine the consequence of hyaluronan binding in T cells. Jurkat T cells transfected with CD44 mutated at S180, which prevented the addition of chondroitin sulfate, displayed constitutively high levels of hyaluronan binding. These cells were more susceptible to activation-induced cell death, whereas cells expressing a CD44 mutant unable to bind hyaluronan (R41A) were resistant to cell death. In TCR or PMA activated Jurkat T cells, hyaluronan induced rapid cell death. This depended on the level of hyaluronan binding by the cell, and the amount and size of hyaluronan. High molecular mass hyaluronan had the greatest effect and cell death occurred independently of Fas and caspase activation. In splenic T cells, high hyaluronan binding occurred in a subpopulation of cells undergoing activation-induced cell death. In addition, hyaluronan induced cell death in ∼10% of reactivated splenic T cells when Fas-dependent apoptosis was prevented by Ab blocking or in Fas negative MRL/lpr T cells. This demonstrates that hyaluronan can induce cell death in activated, high hyaluronan binding T cells via a Fas-independent mechanism.
Histoplasma capsulatumCyclophilin A Mediates Attachment to Dendritic Cell VLA-5
Authors: Gomez Francisco J. , Pilcher-Roberts Robyn , Alborzi Arash , Newman Simon L.
Published: April 2014

Histoplasma capsulatum (Hc) is a pathogenic fungus that replicates in macrophages (Mφ). In dendritic cells (DC), Hc is killed and fungal Ags are processed and presented to T cells. DC recognize Hc yeasts via the VLA-5 receptor, whereas Mφ recognize yeasts via CD18. To identify ligand(s) on Hc recognized by DC, VLA-5 was used to probe a Far Western blot of a yeast freeze/thaw extract (F/TE) that inhibited Hc binding to DC. VLA-5 recognized a 20-kDa protein, identified as cyclophilin A (CypA), and CypA was present on the surface of Hc yeasts. rCypA inhibited the attachment of Hc to DC, but not to Mφ. Silencing of Hc CypA by RNA interference reduced yeast binding to DC by 65–85%, but had no effect on binding to Mφ. However, F/TE from CypA-silenced yeasts still inhibited binding of wild-type Hc to DC, and F/TE from wild-type yeasts depleted of CypA also inhibited yeast binding to DC. rCypA did not further inhibit the binding of CypA-silenced yeasts to DC. Polystyrene beads coated with rCypA or fibronectin bound to DC and Mφ and to Chinese hamster ovary cells transfected with VLA-5. Binding of rCypA-coated beads, but not fibronectin-coated beads, was inhibited by rCypA. These data demonstrate that CypA serves as a ligand for DC VLA-5, that binding of CypA to VLA-5 is at a site different from FN, and that there is at least one other ligand on the surface of Hc yeasts that mediates binding of Hc to DC.
Cutting Edge: Viral Infection Breaks NK Cell Tolerance to “Missing Self”
Authors: Sun Joseph C. , Lanier Lewis L.
Published: April 2014

NK cells attack cells lacking MHC class I, yet MHC class I-deficient mice have normal numbers of NK cells with intact, albeit diminished, functions. Moreover, wild-type NK cells are tolerant of MHC class I-deficient cells in mixed bone marrow chimeras. In this study, we investigated how the absence of MHC class I affects NK cells. NK cells from β2-microglobulin-deficient (B2m−/−) and wild-type mice exhibit similar phenotypic and functional characteristics. Both B2m−/− and wild-type Ly49H+ NK cells proliferated robustly and produced IFN-γ after infection with mouse CMV. NK cells in mixed wild-type:B2m−/− chimeric mice were initially tolerant of MHC class I-deficient host cells. However, this tolerance was gradually lost over time and after mouse CMV infection was rapidly broken, with a pronounced rejection of host B2m−/− hematopoietic cells. Thus, although NK cells can be held in check against “missing self,” acute inflammation driven by infection can rapidly break established self-tolerance.
Cutting Edge: Selective Blockade of LIGHT-Lymphotoxin β Receptor Signaling Protects Mice from Experimental Cerebral Malaria Caused by Plasmodium berghei ANKA
Authors: Randall Louise M. , Amante Fiona H. , Zhou Yonghong , Stanley Amanda C. , Haque Ashraful , Rivera Fabian , Pfeffer Klaus , Scheu Stefanie , Hill Geoff R. , Tamada Koji , Engwerda Christian R.
Published: April 2014

Studies in experimental cerebral malaria (ECM) in mice have identified T cells and TNF family members as critical mediators of pathology. In this study we report a role for LIGHT-lymphotoxin β Receptor (LTβR) signaling in the development of ECM and control of parasite growth. Specific blockade of LIGHT-LTβR, but not LIGHT-herpesvirus entry mediator interactions, abrogated the accumulation of parasites and the recruitment of pathogenic CD8+ T cells and monocytes to the brain during infection without affecting early activation of CD4+ T cells, CD8+ T cells, or NK cells. Importantly, blockade of LIGHT-LTβR signaling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process Ag during infection, as well as reduced systemic cytokine levels when control mice displayed severe ECM symptoms. In summary, we have discovered a novel pathogenic role for LIGHT and LTβR in ECM, identifying this TNF family receptor-ligand interaction as an important immune regulator during experimental malaria.
Cutting Edge: Members of the Staphylococcus aureus Extracellular Fibrinogen-Binding Protein Family Inhibit the Interaction of C3d with Complement Receptor 2
Authors: Ricklin Daniel , Ricklin-Lichtsteiner Salome K. , Markiewski Maciej M. , Geisbrecht Brian V. , Lambris John D.
Published: April 2014

Staphylococcus aureus expresses a highly diversified arsenal of immune evasion proteins, many of which target the complement system. The extracellular fibrinogen-binding protein (Efb) and the Efb homologous protein (Ehp) have previously been demonstrated to bind to C3 and inhibit complement activation and amplification. In this study we present the first evidence that Efb and Ehp are also capable of inhibiting the interaction of C3d with complement receptor 2 (CR2), which plays an important role in B cell activation and maturation. The C-terminal domain of Efb efficiently blocked this interaction both in surface plasmon resonance-based competition studies and cellular assays and prevented the CR2-mediated stimulation of B cells. Furthermore, analyses of the available structural data were consistent with a molecular mechanism that reflects both steric and electrostatic effects on the C3d-CR2 interaction. Our study therefore suggests that S. aureus may disrupt both the innate and adaptive immune responses with a single protein module.
Cutting Edge: Migration of Langerhans Dendritic Cells Is Impaired in Autoimmune Dermatitis
Authors: Eriksson Anna U. , Singh Ram Raj
Published: April 2014

Tissue-resident dendritic cells, such as Langerhans cells (LC), normally carry Ags from tissues to lymph nodes to induce immunity to tissue Ags. In this study, we report that LC are reduced in the skin-draining lymph nodes of MRL-Faslpr/lpr and MRL-Fas+/+ mice that develop T cell-mediated autoimmune skin inflammation as compared with MHC-matched healthy strains. This deficiency of LC in skin-draining lymph nodes is due to a profound impairment of LC migration, resulting in the accumulation of activated LC in the skin. Such a defect in LC migration develops before the onset of skin lesions and correlates with the onset and severity of dermatitis. The reduced, rather than increased, migration of LC from skin to skin-draining lymph nodes represents a novel functional abnormality of LC in autoimmune dermatitis.
Enhancement in Specific CD8+ T Cell Recognition of EphA2+ Tumors In Vitro and In Vivo after Treatment with Ligand Agonists
Authors: Wesa Amy K. , Herrem Christopher J. , Mandic Maja , Taylor Jennifer L. , Vasquez Cecilia , Kawabe Mayumi , Tatsumi Tomohide , Leibowitz Michael S. , Finke James H. , Bukowski Ronald M. , Bruckheimer Elizabeth , Kinch Michael S. , Storkus Walter J.
Published: April 2014

The EphA2 receptor tyrosine kinase is an attractive therapeutic target that is commonly overexpressed on solid tumors, with the degree of overexpression associated with disease progression, metastatic potential, and poor prognosis. Agonistic mAbs or ligand (ephrinA1)-Fc fusion protein are capable of inducing EphA2 internalization and degradation, thereby (at least transiently) eliminating the influence of this oncoprotein. We and others have also shown that EphA2 contains multiple peptide epitopes that can be recognized by effector CD4+ and CD8+ T cells isolated from tumor-bearing patients. Herein, we show that “agonist” reagents that trigger the proteasome-dependent degradation of tumor cell EphA2 result in the improved presentation of peptides derived from (both the extracellular and intracellular domains of) EphA2 in MHC class I complexes expressed on the tumor cell membrane for at least 48 h, as manifested by increased recognition by EphA2-specific CD8+ T cells in vitro. We also observed that while delivery of ephrinA1-Fc fusion protein or agonist mAb into EphA2+ tumor lesions promotes EphA2 degradation in situ, this single administration of agent does not dramatically alter tumor progression in a humanized SCID model. However, when combined with the adoptive transfer of normally nontherapeutic (human) anti-EphA2 CD8+ CTL, this dual-agent regimen results in complete tumor eradication. These results suggest that strategies targeting the conditional proteasome-mediated destruction of tumor cell EphA2 may enable EphA2-specific CD8+ T cells (of modest functional avidity) to realize improved therapeutic potential.
Self-Antigen Prevents CD8 T Cell Effector Differentiation by CD134 and CD137 Dual Costimulation
Authors: Bandyopadhyay Suman , Long Meixiao , Qui Harry Z. , Hagymasi Adam T. , Slaiby Aaron M. , Mihalyo Marianne A. , Aguila Hector L. , Mittler Robert S. , Vella Anthony T. , Adler Adam J.
Published: April 2014

We compared how CD4 vs CD8 cells attain the capacity to express the effector cytokine IFN-γ under both immunogenic and tolerogenic conditions. Although the Ifng gene locus was epigenetically repressed in naive Ag-inexperienced CD4 cells, it had already undergone partial remodeling toward a transcriptionally competent configuration in naive CD8 cells. After TCR stimulation, CD8 cells fully remodeled the Ifng locus and gained the capacity to express high levels of IFN-γ more rapidly than CD4 cells. Enforced dual costimulation through OX40 and 4-1BB redirected CD8 cells encountering soluble exogenous peptide to expand and differentiate into IFN-γ and TNF-α double-producing effectors rather than becoming tolerant. Despite this and the stronger tendency of CD8 compared with CD4 cells to differentiate into IFN-γ-expressing effectors, when parenchymal self-Ag was the source of tolerizing Ag, enforced dual costimulation selectively boosted expansion but did not push effector differentiation in CD8 cells while both expansion and effector differentiation were dramatically boosted in CD4 cells. Notably, enforced dual costimulation was able to push effector differentiation in CD8 cells encountering cognate parenchymal self-Ag when CD4 cells were simultaneously engaged. Thus, the ability of enforced OX40 plus 4-1BB dual costimulation to redirect CD8 cells to undergo effector differentiation was unexpectedly influenced by the source of tolerizing Ag and help was selectively required to facilitate CD8 cell effector differentiation when the tolerizing Ag derived from self.
Rapid Deletion of Antigen-Specific CD4+ T Cells following Infection Represents a Strategy of Immune Evasion and Persistence for Anaplasma marginale
Authors: Han Sushan , Norimine Junzo , Palmer Guy H. , Mwangi Waithaka , Lahmers Kevin K. , Brown Wendy C.
Published: April 2014

Acquired T cell immunity is central for protection against infection. However, the immunological consequences of exposing memory T cells to high Ag loads during acute and persistent infection with systemic pathogens are poorly understood. We investigated this by using infection with Anaplasma marginale, a ruminant pathogen that replicates to levels of 109 bacteria per ml of blood during acute infection and maintains mean bacteremia levels of 106 per ml during long-term persistent infection. We established that immunization-induced Ag-specific peripheral blood CD4+ T cell responses were rapidly and permanently lost following infection. To determine whether these T cells were anergic, sequestered in the spleen, or physically deleted from peripheral blood, CD4+ T lymphocytes from the peripheral blood specific for the major surface protein (MSP) 1a T cell epitope were enumerated by DRB3*1101 tetramer staining and FACS analysis throughout the course of immunization and challenge. Immunization induced significant epitope-specific T lymphocyte responses that rapidly declined near peak bacteremia to background levels. Concomitantly, the mean frequency of tetramer+CD4+ cells decreased rapidly from 0.025% before challenge to a preimmunization level of 0.0003% of CD4+ T cells. Low frequencies of tetramer+CD4+ T cells in spleen, liver, and inguinal lymph nodes sampled 9–12 wk postchallenge were consistent with undetectable or unsustainable Ag-specific responses and the lack of T cell sequestration. Thus, infection of cattle with A. marginale leads to the rapid loss of Ag-specific T cells and immunologic memory, which may be a strategy for this pathogen to modulate the immune response and persist.
MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional Endosomes and Is Influenced by Melanosomes
Authors: Robila Valentina , Ostankovitch Marina , Altrich-VanLith Michelle L. , Theos Alexander C. , Drover Sheila , Marks Michael S. , Restifo Nicholas , Engelhard Victor H.
Published: April 2014

Many human solid tumors express MHC class II (MHC-II) molecules, and proteins normally localized to melanosomes give rise to MHC-II-restricted epitopes in melanoma. However, the pathways by which this response occurs have not been defined. We analyzed the processing of one such epitope, gp10044–59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1high/MHC-II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044–59 presentation. By depletion of the AP-2 adaptor protein using small interfering RNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044–59 epitope production. The gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC-II molecules. The gp10044–59 presentation is dramatically reduced, and processing occurs entirely in early endosomes or stage I melanosomes. This occurrence suggests that melanosomes are inefficient Ag-processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC-II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition.
TREM-2 Mediated Signaling Induces Antigen Uptake and Retention in Mature Myeloid Dendritic Cells
Authors: Radhakrishnan Suresh , Arneson Laura N. , Upshaw Jadee L. , Howe Charles L. , Felts Sara J. , Colonna Marco , Leibson Paul J. , Rodriguez Moses , Pease Larry R.
Published: April 2014

Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-γ1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.
The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages
Authors: Abe Akira , Kelly Robert , Kollmeyer Jessica , Hiraoka Miki , Lu Ye , Shayman James A.
Published: April 2014

Macrophages have long been known to secrete a Phospholipase A2 with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A2 (LPLA2) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA2. LPLA2 is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA2 produced by HEK293 cells was applied to LPLA2-deficient (LPLA2−/−) mouse alveolar macrophages. The uptake of exogenous LPLA2 into LPLA2−/− alveolar macrophages occurred in a concentration-dependent manner. The LPLA2 taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of α-methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA2 is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA2−/− alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA2 rescued the LPLA2−/− alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA2 revealed that the enzymatic activity of LPLA2 was required for the phospholipid reduction. These studies identify LPLA2 as a high m.w.-secreted Phospholipase A2.
Nod2-Dependent Th2 Polarization of Antigen-Specific Immunity
Authors: Magalhaes Joao Gamelas , Fritz Jörg H. , Le Bourhis Lionel , Sellge Gernot , Travassos Leonardo H. , Selvanantham Thirumahal , Girardin Stephen E. , Gommerman Jennifer L. , Philpott Dana J.
Published: April 2014

While a number of microbial-associated molecular patterns have been known for decades to act as adjuvants, the mechanisms and the signaling pathways underlying their action have remained elusive. Here, we examined the unfolding of the adaptive immune response induced by Nod2 in vivo upon activation by its specific ligand, muramyl dipeptide, a component of peptidoglycan. Our findings demonstrate that this bacterial sensor triggers a potent Ag-specific immune response with a Th2-type polarization profile, characterized by the induction of IL-4 and IL-5 by T cells and IgG1 Ab responses. Nod2 was also found to be critical for the induction of both Th1- and Th2-type responses following costimulation with TLR agonists. Importantly, the synergistic responses to Nod2 and TLR agonists seen in vivo were recapitulated by dendritic cells in vitro, suggesting that these cells likely play a central role in the integration of Nod2- and TLR-dependent signals for driving the adaptive immune response. Taken together, our results identify Nod2 as a critical mediator of microbial-induced potentiation and polarization of Ag-dependent immunity. Moreover, these findings affect our understanding of Crohn’s diseases pathogenesis, where lack of Nod2-dependent Th2 signaling in a subset of these patients might explain heightened Th1-mediated inflammation at the level of the intestinal mucosa.
OX40 Drives Protective Vaccinia Virus-Specific CD8 T Cells
Authors: Salek-Ardakani Shahram , Moutaftsi Magdalini , Crotty Shane , Sette Alessandro , Croft Michael
Published: April 2014

Vaccinia virus (VACV) affords long-lasting protection against variola virus, the agent of smallpox. VACV-reactive CD8 T cells contribute to protection but their molecular control is unknown. We show that the TNFR molecule OX40 (CD134) controls primary VACV-specific CD8 T cell expansion and antiviral cytokine production and dictates development of strong memory to both dominant and subdominant VACV epitopes. Using adoptive transfer of OX40-deficient CD8 TCR-transgenic T cells responding to Ag in the context of VACV infection, we found that this reflects a direct action of OX40 expressed by CD8 T cells. Furthermore, CD8 T cells that can protect against lethal VACV challenge do not develop in mice deficient in OX40. Thus, OX40, which has been found to play little if any role in the generation of CD8 T cells to several viruses, including lymphocytic choriomeningitis virus and influenza, plays a dominant role in shaping the CD8 T cell response to VACV. These data suggest that unique costimulatory pathways might control alternate antiviral CD8 responses, demonstrating the plasticity of the immune response in utilizing different mechanisms to achieve similar ultimate goals.
Persistence of Lung CD8 T Cell Oligoclonal Expansions upon Smoking Cessation in a Mouse Model of Cigarette Smoke-Induced Emphysema
Authors: Motz Gregory T. , Eppert Bryan L. , Sun Guangyun , Wesselkamper Scott C. , Linke Michael J. , Deka Ranjan , Borchers Michael T.
Published: April 2014

The role of adaptive immunity in the development or progression of chronic obstructive pulmonary disease (COPD) remains undefined. Recently, the presence of autoantibodies and autoreactive T cells has been demonstrated in COPD patients. In addition, oligoclonal expansions of lung T cells have been observed in COPD patients, but the overlapping incidence of infections, tumors, and cigarette smoke exposure obscures the antigenic stimulus. We analyzed the TCR Vβ repertoire of CD4 and CD8 T cells purified from the lungs and spleens of mice chronically exposed to cigarette smoke. In a mouse model of COPD, we demonstrate that chronic cigarette smoke exposure causes oligoclonal expansions of T cells isolated from the lungs, but not spleens. TCR Vβ repertoire analyses revealed oligoclonal expansions predominantly occurred in lung CD8 T cells, with preferential usage of Vβ7, Vβ9, Vβ13, and Vβ14. Using nucleotide sequence analysis based on Jβ analyses, we demonstrate selection of CDR3 amino acid motifs, which strongly suggests Ag-driven oligoclonal T cell expansion. Analysis of the lung TCR Vβ repertoire of mice with cigarette smoke-induced emphysema, which had undergone smoking cessation for 6 mo, revealed that oligoclonal expansions persisted. This study formally demonstrates that chronic cigarette smoke exposure, alone, causes a persistent adaptive T cell immune response. These findings have important implications for therapeutic approaches in the treatment of COPD, and provide insight into potential mechanisms involved in disease pathogenesis.
Multidrug Resistance-Associated Transporter 2 Regulates Mucosal Inflammation by Facilitating the Synthesis of Hepoxilin A3
Authors: Pazos Michael , Siccardi Dario , Mumy Karen L. , Bien Jeffrey D. , Louie Steve , Shi Hai Ning , Gronert Karsten , Mrsny Randall J. , McCormick Beth A.
Published: April 2014

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A3, an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A3 is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A3 is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A3 synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A3 synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.
TLR9-Activating DNA Up-Regulates ZAP70 via Sustained PKB Induction in IgM+ B Cells
Authors: Bekeredjian-Ding Isabelle , Doster Anne , Schiller Martin , Heyder Petra , Lorenz Hanns-Martin , Schraven Burkhart , Bommhardt Ursula , Heeg Klaus
Published: April 2014

In the past, ZAP70 was considered a T cell-specific kinase, and its aberrant expression in B-CLL cells was interpreted as a sign of malignant transformation and dedifferentiation. It was only recently that ZAP70 was detected in normal human B cells. In this study, we show that TLR9-activated B cells resemble B-cell chronic lymphocytic leukemia cells with regard to CD5, CD23, CD25, and heat shock protein 90 expression. Furthermore, stimulatory CpG and GpC DNA oligonucleotides target CD27+IgM+ and CD27−IgM+ B cells (but not IgM− B cells) and enhance ZAP70 expression predominantly in the IgM+CD27+ B cell subset. ZAP70 is induced via activation of TLR-7 or -9 in a MyD88-dependent manner, depends on protein kinase B (PKB)/mammalian target of rapamycin signaling and is rapamycin sensitive. Furthermore, ZAP70 expression levels correlate with induction of cyclin A2, prolonged B cell proliferation, and sustained induction of PKB. These events are not observed upon CD40 ligation. However, this deficit can be overcome by the expression of constitutively active PKB, given that CD40 ligation of PKB-transgenic B cells induces B cell proliferation and ZAP70 expression. These results highlight a major difference between CD40- and TLR-7/9-mediated B cell activation and suggest that ZAP70 expression levels in B cells give an estimate of the proliferative potential and the associated PKB availability.
Induction of Innate Immune Response through TLR2 and Dectin 1 Prevents Type 1 Diabetes
Authors: Karumuthil-Melethil Subha , Perez Nicolas , Li Ruobing , Vasu Chenthamarakshan
Published: April 2014

Studies have suggested a correlation between the decline in infectious diseases and increase in the incidence of type 1 diabetes (T1D) in developed countries. Pathogens influence the disease outcome through innate immune receptors such as TLRs. Here we report the effect of ligation of TLR2 and dectin 1 on APCs and the influence of innate immune response induced through these receptors on T1D. Exposure of APCs of NOD mice to zymosan, a fungal cell wall component that interacts with TLR2 and dectin 1, resulted in the release of significant amounts of IL-10, TGF-β1, IL-2, and TNF-α. Treatment of pre- and early hyperglycemic mice with zymosan resulted in suppression of insulitis, leading to a significant delay in hyperglycemia. T cells from zymosan-treated mice showed reduced ability to induce diabetes in NOD-Scid mice compared with control T cells. Zymosan treatment induced suppression of T1D was associated with an increase in the L-selectinhigh T cell frequencies and enhanced suppressor function of CD4+CD25+ T regulatory cells. Further, activation by anti-CD3-Ab induced larger amounts of TGF-β1 and/or IL-10 production by CD4+CD25+ and CD4+CD25− T cells from zymosan-treated mice. These results show that innate immune response through TLR2 and dectin 1 results in suppressor cytokine production by APCs and promotes the regulatory function of T cells. Our study demonstrates the possible involvement of signaling through innate immune receptors such as TLR2 and dectin 1 in reduced T1D incidence under the conditions of low hygiene, and the potential of targeting them for treating T1D.
Gradual Decline in Malaria-Specific Memory T Cell Responses Leads to Failure to Maintain Long-Term Protective Immunity to Plasmodium chabaudi AS Despite Persistence of B Cell Memory and Circulating Antibody
Authors: Freitas do Rosário Ana Paula , Muxel Sandra Márcia , Rodríguez-Málaga Sérgio Marcelo , Sardinha Luiz Roberto , Zago Cláudia Augusta , Castillo-Méndez Sheyla Inés , Álvarez José Maria , D'Império Lima Maria Regina
Published: April 2014

The mechanisms responsible for the generation and maintenance of immunological memory to Plasmodium are poorly understood and the reasons why protective immunity in humans is so difficult to achieve and rapidly lost remain a matter for debate. A possible explanation for the difficulty in building up an efficient immune response against this parasite is the massive T cell apoptosis resulting from exposure to high-dose parasite Ag. To determine the immunological mechanisms required for long-term protection against P. chabaudi malaria and the consequences of high and low acute phase parasite loads for acquisition of protective immunity, we performed a detailed analysis of T and B cell compartments over a period of 200 days following untreated and drug-treated infections in female C57BL/6 mice. By comparing several immunological parameters with the capacity to control a secondary parasite challenge, we concluded that loss of full protective immunity is not determined by acute phase parasite load nor by serum levels of specific IgG2a and IgG1 Abs, but appears to be a consequence of the progressive decline in memory T cell response to parasites, which occurs similarly in untreated and drug-treated mice with time after infection. Furthermore, by analyzing adoptive transfer experiments, we confirmed the major role of CD4+ T cells for guaranteeing long-term full protection against P. chabaudi malaria.
Antigenic Experience Dictates Functional Role of Glycogen Synthase Kinase-3 in Human CD4+ T Cell Responses
Authors: Garcia Carlos A. , Benakanakere Manjunatha R. , Alard Pascale , Kosiewicz Michelle M. , Kinane Denis F. , Martin Michael
Published: April 2014

Signals induced by the TCR and CD28 costimulatory pathway have been shown to lead to the inactivation of the constitutively active enzyme, glycogen synthase kinase-3 (GSK3), which has been implicated in the regulation of IL-2 and T cell proliferation. However, it is unknown whether GSK3 plays a similar role in naive and memory CD4+ T cell responses. Here we demonstrate a divergence in the dependency on the inactivation of GSK3 in the proliferative responses of human naive and memory CD4+ T cells. We find that although CD28 costimulation increases the frequency of phospho-GSK3 inactivation in TCR-stimulated naive and memory CD4+ T cells, memory cells are less reliant on GSK3 inactivation for their proliferative responses. Rather we find that GSK3β plays a previously unrecognized role in the selective regulation of the IL-10 recall response by human memory CD4+ T cells. Furthermore, GSK3β-inactivated memory CD4+ T cells acquired the capacity to suppress the bystander proliferation of CD4+ T cells in an IL-10-dependent, cell contact-independent manner. Our findings reveal a dichotomy present in the function of GSK3 in distinct human CD4+ T cell populations.
Binding of the Long Pentraxin PTX3 to Factor H: Interacting Domains and Function in the Regulation of Complement Activation
Authors: Deban Livija , Jarva Hanna , Lehtinen Markus J. , Bottazzi Barbara , Bastone Antonio , Doni Andrea , Jokiranta T. Sakari , Mantovani Alberto , Meri Seppo
Published: April 2014

The long pentraxin PTX3 is a multifunctional soluble molecule involved in inflammation and innate immunity. As an acute phase protein, PTX3 binds to the classical pathway complement protein C1q, limits tissue damage in inflammatory conditions by regulating apoptotic cell clearance, and plays a role in the phagocytosis of selected pathogens. This study was designed to investigate the interaction of PTX3 with factor H (FH), the main soluble alternative pathway regulatory protein. We report that PTX3 binds FH with an apparent Kd of 1.1 × 10−7 M, and define two binding sites for PTX3 on FH. The primary binding site is located on FH domains 19–20, which interact with the N-terminal domain of PTX3, while a secondary binding site on domain 7 binds the glycosylated PTX3 pentraxin domain. The FH Y402H polymorphism, which affects binding to the short pentraxin CRP, did not affect binding to PTX3. Surface-bound PTX3 enhances FH recruitment and iC3b deposition and PTX3-bound FH retains its activity as a cofactor for factor I-mediated C3b cleavage. Thus, our findings identify PTX3 as a unique FH ligand in that it can bind both of the two hot-spots of FH, namely SCR7 and SCR19–20 and indicate that PTX3 participates in the localization of functionally active FH.
Decoy Receptor 3 Expression in AsPC-1 Human Pancreatic Adenocarcinoma Cells via the Phosphatidylinositol 3-Kinase-, Akt-, and NF-κB-Dependent Pathway
Authors: Chen Pei-Hsuan , Yang Chia-Ron
Published: April 2014

Many cancers develop different means of escaping destruction by the immune system, such as resistance to Fas ligand (FasL)-Fas interaction-mediated apoptotic signals. Decoy receptor 3 (DcR3), a soluble receptor for FasL, is highly expressed in cancer cells and plays a significant role in immune suppression and tumor progression. However, how DcR3 expression is modulated is unclear. In this study, immunoprecipitation and ELISA using human pancreatic cancer cells showed the presence of high levels of DcR3 protein in AsPC-1 cells, but not in PANC-1 cells. Treatment with herbimycin A (a tyrosine kinase inhibitor), LY294002 or wortmannin (PI3K inhibitors), pyrrolidine dithiocarbamate (an NF-κB inhibitor), or AG1024 (an insulin-like growth factor-1 inhibitor) significantly reduced endogenous DcR3 levels in AsPC-1 cells. Furthermore, transfection of AsPC-1 cells with Akt or IκBα dominant-negative plasmids also markedly reduced DcR3 levels. In contrast, 48-h transfection of PANC-1 cells with a constitutively active Akt induced DcR3 expression. Flow cytometry assays indicated that apoptosis was not seen in AsPC-1 cells incubated with soluble FasL or membrane-bound FasL, but was seen when DcR3 small interfering RNA-transfected AsPC-1 cells underwent the same treatment. In addition, PANC-1 cell incubation with conditioned medium from AsPC-1 cells transfected with dominant-negative Akt or IκBα plasmids or DcR3 small interfering RNA showed increased soluble FasL-mediated apoptosis compared with the control group. Our results show that insulin-like growth factor-1-induced activation of the PI3K/Akt/NF-κB signaling pathway is involved in the modulation of endogenous DcR3 expression in AsPC-1 cells, and that reducing endogenous DcR3 levels increases FasL-induced apoptosis of human pancreatic cancer cells.
Dendritic Cells Expressing Plasmacytoid Marker PDCA-1 Are Trojan Horses during Toxoplasma gondii Infection
Authors: Bierly Allison L. , Shufesky William J. , Sukhumavasi Woraporn , Morelli Adrian E. , Denkers Eric Y.
Published: April 2014

Plasmacytoid dendritic cells (pDCs) play a key role in the innate immune response to viral infection, due largely to their ability to produce large quantities of type I IFNs. These cells are also notable for their ability to differentiate into conventional dendritic cells after appropriate stimulation. Here, we show that a splenic population of murine CD11c+ cells expressing pDC markers Gr-1, B220, and PDCA-1 is preferentially parasitized after infection with the virulent RH strain of Toxoplasma gondii. Although these markers are closely associated with pDCs, the population we identified was unusual because the cells express CD11b and higher than expected levels of CD11c. By adoptive transfer of CD45.1-positive cells into CD45.2 congenic mice, we show that CD11c+Gr-1+ cells migrate from the peritoneal cavity to the spleen. During infection, these cells accumulate in the marginal zone region. Recruitment of infected CD11c+Gr-1+ cells to the spleen is partially dependent upon signaling through chemokine receptor CCR2. Intracellular cytokine staining demonstrates that infected, but not noninfected, splenic CD11c+Gr-1+ dendritic cells are suppressed in their ability to respond to ex vivo TLR stimulation. We hypothesize that Toxoplasma exploits pDCs as Trojan horses, targeting them for early infection, suppressing their cytokine effector function, and using them for dissemination within the host.