bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of the Transcription Factor Nrf2 Promotes Pro-Oxidative Dendritic Cells That Stimulate Th2-Like Immunoresponsiveness upon Activation by Ambient Particulate Matter
Authors: Williams Marc A. , Rangasamy Tirumalai , Bauer Stephen M. , Killedar Smruti , Karp Matthew , Kensler Thomas W. , Yamamoto Masayuki , Breysse Patrick , Biswal Shyam , Georas Steve N.
Published: April 2014

Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2−/− DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2−/− DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2+/+ DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2+/+ DCs, coincubation of Nrf2−/− DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.
Differential Susceptibility of Allogeneic Targets to Indirect CD4 Immunity Generates Split Tolerance
Authors: Chan William F. N. , Razavy Haide , Anderson Colin C.
Published: April 2014

CD4 T cells frequently help to activate CD8 T and B cells that effect transplant rejection. However, CD4 T cells alone can reject transplants, either directly or indirectly. The relative effectiveness of indirect CD4 immunity in rejecting different types of allogeneic grafts is unknown. To address this, we used a TCR transgenic mouse model in which indirect CD4 alloimmunity alone can be studied. We challenged transgenic recipients with hematopoietic cells and shortly thereafter skin transplants that could only be rejected indirectly, and observed Ag-specific indirect donor B cell and skin rejection, but not T cell elimination, reflecting a state of split tolerance. Deficiency of indirect CD4 alloimmunity in donor T cell rejection was also apparent when acute indirect rejection of donor islets occurred despite generation and maintenance of mixed T cell chimerism, due to migration of the few passenger T cells into recipient circulation. Although passenger lymphocytes delayed indirect islet rejection, they enhanced rejection by a full repertoire capable of both direct and indirect reactivity. Interestingly, the persistence of chimerism was associated with the eventual development of tolerance, as demonstrated by acceptance of donor skin grafts given late to hematopoietic cell recipients, and hyporesponsiveness of transgenic T cells from islet recipients in vitro. Mechanistically, tolerance was recessive and associated with progressive down-regulation of CD4. Collectively, our data indicate that indirect CD4 immunity is not equally destructive toward different types of allogeneic grafts, the deficiency of which generates split tolerance. The futility of these responses can convert immunity into tolerance.
Proinflammatory S100 Proteins Regulate the Accumulation of Myeloid-Derived Suppressor Cells
Authors: Sinha Pratima , Okoro Chinonyerem , Foell Dirk , Freeze Hudson H. , Ostrand-Rosenberg Suzanne , Srikrishna Geetha
Published: April 2014

Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1β, IL-6, and PGE2 induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1highCD11bhighF4/80−CD80+IL4Rα+/−Arginase+ MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-κB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-κB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.
Protein Kinase C-θ Is Required for Efficient Positive Selection
Authors: Morley Sharon Celeste , Weber K. Scott , Kao Henry , Allen Paul M.
Published: April 2014

Protein kinase C-θ (PKCθ) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCθ in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCθ in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCθ in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCθ−/− mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCθ−/− thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCθ−/− mice. Inefficient positive selection of n3.L2 PKCθ−/− CD4 single-positive cells resulted from “weaker” signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCθ−/− mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCθ−/− mice. These findings now place PKCθ as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.
Protein Kinase C δ Localizes to Secretory Lysosomes in CD8+ CTL and Directly Mediates TCR Signals Leading to Granule Exocytosis-Mediated Cytotoxicity
Authors: Ma Jennifer S. Y. , Haydar Tarik F. , Radoja Saša
Published: April 2014

Lytic granule exocytosis is the major effector function used by CD8+ CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C δ (PKC δ) selectively regulates TCR-induced lytic granule polarization in mouse CD8+ CTL. To better understand how PKC δ facilitates granule movement, here we studied dynamics of intracellular localization of PKC δ in living CD8+ CTL. Strikingly, we found that PKC δ localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC δ becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC δ, providing the first example of colocalization of a kinase with secretory lysosomes in CD8+ CTL and demonstrating that PKC δ directly transduces TCR signals leading to polarized granule secretion.
Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL21
Authors: Byers Melissa A. , Calloway Psachal A. , Shannon Laurie , Cunningham Heather D. , Smith Sarah , Li Fang , Fassold Brian C. , Vines Charlotte M.
Published: April 2014

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 ± 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 ± 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2−/−/arrestin 3−/− murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.
Induction of IL-17+ T Cell Trafficking and Development by IFN-γ: Mechanism and Pathological Relevance in Psoriasis
Authors: Kryczek Ilona , Bruce Allen T. , Gudjonsson Johann E. , Johnston Andrew , Aphale Abhishek , Vatan Linhua , Szeliga Wojciech , Wang Yin , Liu Yan , Welling Theodore H. , Elder James T. , Zou Weiping
Published: April 2014

Th1 and Th17 T cells are often colocalized in pathological environments, yet Th1-derived IFN-γ inhibits Th17 cell development in vitro. We explored the physiologic basis of this paradox in humans. In this study, we demonstrate increased the number of CD4+ and CD8+ IL-17+ T cells in skin lesions of psoriasis. Furthermore, we show that myeloid APCs potently support induction of IL-17+ T cells, and that this activity is greatly increased in psoriasis. We tested stimuli that might account for this activity. Th1 cells and IFN-γ are increased in psoriatic blood and lesional skin. We show that IFN-γ programs myeloid APCs to induce human IL-17+ T cells via IL-1 and IL-23. IFN-γ also stimulates APC production of CCL20, supporting migration of IL-17+ T cells, and synergizes with IL-17 in the production of human β-defensin 2, an antimicrobial and chemotactic protein highly overexpressed by psoriatic keratinocytes. This study reveals a novel mechanistic interaction between Th1 and IL-17+ T cells, challenges the view that Th1 cells suppress Th17 development through IFN-γ, and suggests that Th1 and IL-17+ T cells may collaboratively contribute to human autoimmune diseases.
Transplantation Survival Is Maintained by Granzyme B+ Regulatory Cells and Adaptive Regulatory T Cells
Authors: Gondek David C. , DeVries Victor , Nowak Elizabeth C. , Lu Li-Fan , Bennett Kathryn A. , Scott Zachary A. , Noelle Randolph J.
Published: April 2014

Granzyme B (GZB) has been implicated as an effector mechanism in regulatory T cells (Treg) suppression. In a model of Treg-dependent graft tolerance, it is shown that GZB- deficient mice are unable to establish long-term tolerance. Moreover, mice overexpressing the inhibitor of GZB, serine protease inhibitor 6, are also resistant to tolerization to alloantigen. Graft survival was shorter in bone marrow-mixed chimeras reconstituted with GZB-deficient Treg as compared with wild-type Treg. Whereas there was no difference in graft survival in mixed chimeras reconstituted with wild-type, perforin-deficient, or Fas ligand-deficient Treg. Finally, data also show that if alloreactive effectors cannot express FoxP3 and be induced to convert in the presence of competent Treg, then graft tolerance is lost. Our data are the first in vivo data to implicate GZB expression by Treg in sustaining long-lived graft survival.
Two Distinct T Cell Subsets, CD4+ and CD8+CD60+, and Their Cytokines Are Required for In Vitro Induction of Human Ragweed-Specific Memory IgE Responses
Authors: Smith-Norowitz Tamar A. , Silverberg Jonathan , Norowitz Kevin B. , Bluth Martin H. , Chice Seto , Joks Rauno , Nowakowski Maja , Durkin Helen G.
Published: April 2014

CD8+CD60+ T cells (80–98% CD45RO+; 20% CD23+) are significantly increased in the blood of serum IgE+ ragweed-sensitized (RS) compared with serum IgE-nonatopic humans (p = 0.001). CD8+CD60+ T cells of the RS patients produced IL-2, IL-4, IL-10, IL-12, IFN-α. and IFN-γ, but not IL-6 or IL-13. When their PBMC were cultured with ragweed Ag (RA), peak IgE responses occurred on day 10; none was induced with non-cross-reacting or without Ag; nonatopic PBMC did not respond to any stimulant. When either CD4+ or CD8+CD60+ T cells were depleted from RS PBMC before culture with RA, no IgE responses were induced. If purified CD4+ T cells or low numbers of CD8+CD60+ T cells were added back to the depleted PBMC, IgE responses were restored. However, higher numbers of CD8+CD60+ T cells totally suppressed IgE responses. Total suppression also was obtained when RS PBMC were cultured with RA and either anti-IL-2, IL-4, IL-10, IL-12, IFN-γ (all concentrations), or IFN-α (low concentrations), but not anti-IL-6 or IL-13. Higher concentrations of anti-IFN-α potentiated IgE responses.
Impact of Mammalian Target of Rapamycin Inhibition on Lymphoid Homing and Tolerogenic Function of Nanoparticle-Labeled Dendritic Cells following Allogeneic Hematopoietic Cell Transplantation
Authors: Reichardt Wilfried , Dürr Christoph , von Elverfeldt Dominik , Jüttner Eva , Gerlach Ulrike V. , Yamada Mayumi , Smith Benjie , Negrin Robert S. , Zeiser Robert
Published: April 2014

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-γR-mediated endocytosis. Locally injected luc+ DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.
Definition of APC Presentation of Phosphoantigen (E)-4-Hydroxy-3-methyl-but-2-enyl Pyrophosphate to Vγ2Vδ2 TCR
Authors: Wei Huiyong , Huang Dan , Lai Xiaomin , Chen Meiling , Zhong Weihua , Wang Richard , Chen Zheng W.
Published: April 2014

Although microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) can activate primate Vγ2Vδ2 T cells, molecular mechanisms by which HMBPP interacts with Vγ2Vδ2 T cells remain poorly characterized. Here, we developed soluble, tetrameric Vγ2Vδ2 TCR of rhesus macaques to define HMBPP/APC interaction with Vγ2Vδ2 TCR. While exogenous HMBPP was associated with APC membrane in an appreciable affinity, the membrane-associated HMBPP readily bound to the Vγ2Vδ2 TCR tetramer. The Vγ2Vδ2 TCR tetramer was shown to bind stably to HMBPP presented on membrane by various APC cell lines from humans and nonhuman primates but not those from mouse, rat, or pig. The Vγ2Vδ2 TCR tetramer also bound to the membrane-associated HMBPP on primary monocytes, B cells and T cells. Consistently, endogenous phosphoantigen produced in Mycobacterium-infected dendritic cells was transported and presented on membrane, and bound stably to the Vγ2Vδ2 TCR tetramer. The capability of APC to present HMBPP for recognition by Vγ2Vδ2 TCR was diminished after protease treatment of APC. Thus, our studies elucidated an affinity HMBPP-APC association conferring stable binding to the Vγ2Vδ2 TCR tetramer and the protease-sensitive nature of phosphoantigen presentation. The findings defined APC presentation of phosphoantigen HMBPP to Vγ2Vδ2 TCR.
Bone Marrow Precursor Cells from Aged Mice Generate CD4 T Cells That Function Well in Primary and Memory Responses
Authors: Eaton Sheri M. , Maue Alexander C. , Swain Susan L. , Haynes Laura
Published: April 2014

Understanding how aging impacts the function of memory CD4 T cells is critical for designing effective vaccines. Our studies show that immunological memory generated during youth functions well into old age, whereas that generated later in life functions poorly. This is the result of declines in the function of naive CD4 T cells from aged individuals and contributes to reduced efficacy of vaccines in the elderly. To begin to identify the cause of this defect, we examined the function of memory T cells generated from bone marrow precursor cells (BMPC) from young or aged mice in young hosts. In two different models, memory cells derived from young and aged BMPC exhibit good ex vivo and in vivo function. Importantly, memory CD4 T cells generated from aged BMPC exhibit potent cognate helper function for humoral responses, which are critical for effective immunization. These results indicate that there are no apparent age-related intrinsic defects in BMPC with regards to generation of functional memory T cells.
T Cell-Dendritic Cell Immunological Synapses Contain TCR-dependent CD28-CD80 Clusters That Recruit Protein Kinase Cθ
Authors: Tseng Su-Yi , Waite Janelle C. , Liu Mengling , Vardhana Santosha , Dustin Michael L.
Published: April 2014

Short-lived TCR microclusters and a longer-lived protein kinase Cθ-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCθ, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca2+ signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.
Disruption of Allergenic Activity of the Major Grass Pollen Allergen Phl p 2 by Reassembly as a Mosaic Protein
Authors: Mothes-Luksch Nadine , Stumvoll Sabine , Linhart Birgit , Focke Margit , Krauth Marie-Therese , Hauswirth Alexander , Valent Peter , Verdino Petra , Pavkov Tea , Keller Walter , Grote Monika , Valenta Rudolf
Published: April 2014

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients’ IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.
Large Scale Mass Spectrometric Profiling of Peptides Eluted from HLA Molecules Reveals N-Terminal-Extended Peptide Motifs
Authors: Escobar Hernando , Crockett David K. , Reyes-Vargas Eduardo , Baena Andres , Rockwood Alan L. , Jensen Peter E. , Delgado Julio C.
Published: April 2014

The majority of >2000 HLA class I molecules can be clustered according to overlapping peptide binding specificities or motifs recognized by CD8+ T cells. HLA class I motifs are classified based on the specificity of residues located in the P2 and the C-terminal positions of the peptide. However, it has been suggested that other positions might be relevant for peptide binding to HLA class I molecules and therefore be used for further characterization of HLA class I motifs. In this study we performed large-scale sequencing of endogenous peptides eluted from K562 cells (HLA class I null) made to express a single HLA molecule from HLA-B*3501, -B*3502, -B*3503, -B*3504, -B*3506, or -B*3508. Using sequence data from >1,000 peptides, we characterized novel peptide motifs that include dominant anchor residues extending to all positions in the peptide. The length distribution of HLA-B35-bound peptides included peptides of up to 15 residues. Remarkably, we determined that some peptides longer than 11 residues represented N-terminal-extended peptides containing an appropriate HLA-B35 peptide motif. These results provide evidence for the occurrence of endogenous N-terminal-extended peptide-HLA class I configurations. In addition, these results expand the knowledge about the identity of anchor positions in HLA class I-associated peptides that can be used for characterization of HLA class I motifs.
The Pseudomonas Toxin Pyocyanin Inhibits the Dual Oxidase-Based Antimicrobial System as It Imposes Oxidative Stress on Airway Epithelial Cells
Authors: Rada Balázs , Lekstrom Kristen , Damian Sorin , Dupuy Corinne , Leto Thomas L.
Published: April 2014

The dual oxidase-thiocyanate-lactoperoxidase (Duox/SCN−/LPO) system generates the microbicidal oxidant hypothiocyanite in the airway surface liquid by using LPO, thiocyanate, and Duox-derived hydrogen peroxide released from the apical surface of the airway epithelium. This system is effective against several microorganisms that infect airways of cystic fibrosis and other immunocompromised patients. We show herein that exposure of airway epithelial cells to Pseudomonas aeruginosa obtained from long-term cultures inhibits Duox1-dependent hydrogen peroxide release, suggesting that some microbial factor suppresses Duox activity. These inhibitory effects are not seen with the pyocyanin-deficient P. aeruginosa strain PA14 Phz1/2. We show that purified pyocyanin, a redox-active virulence factor produced by P. aeruginosa, inhibits human airway cell Duox activity by depleting intracellular stores of NADPH, as it generates intracellular superoxide. Long-term exposure of human airway (primary normal human bronchial and NCI-H292) cells to pyocyanin also blocks induction of Duox1 by Th2 cytokines (IL-4, IL-13), which was prevented by the antioxidants glutathione and N-acetylcysteine. Furthermore, we showed that low concentrations of pyocyanin blocked killing of wild-type P. aeruginosa by the Duox/SCN−/LPO system on primary normal human bronchial epithelial cells. Thus, pyocyanin can subvert Pseudomonas killing by the Duox-based system as it imposes oxidative stress on the host. We also show that lactoperoxidase can oxidize pyocyanin, thereby diminishing its cytotoxicity. These data establish a novel role for pyocyanin in the survival of P. aeruginosa in human airways through competitive redox-based reactions between the pathogen and host.
The Varicellovirus UL49.5 Protein Blocks the Transporter Associated with Antigen Processing (TAP) by Inhibiting Essential Conformational Transitions in the 6+6 Transmembrane TAP Core Complex
Authors: Verweij Marieke C. , Koppers-Lalic Danijela , Loch Sandra , Klauschies Florian , de la Salle Henri , Quinten Edwin , Lehner Paul J. , Mulder Arend , Knittler Michael R. , Tampé Robert , Koch Joachim , Ressing Maaike E. , Wiertz Emmanuel J. H. J.
Published: April 2014

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHC class I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptide complexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitor of TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation of peptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it is unknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complex has not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAP was defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required for efficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6+6 transmembrane core complex of TAP was shown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptide transport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be independent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5 acts directly on the 6+6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required for peptide transport.
CD8 T Cells Utilize TRAIL to Control Influenza Virus Infection
Authors: Brincks Erik L. , Katewa Arna , Kucaba Tamara A. , Griffith Thomas S. , Legge Kevin L.
Published: April 2014

Elimination of influenza virus-infected cells during primary influenza virus infections is thought to be mediated by CD8+ T cells though perforin- and FasL-mediated mechanisms. However, recent studies suggest that CD8+ T cells can also utilize TRAIL to kill virally infected cells. Therefore, we herein examined the importance of TRAIL to influenza-specific CD8+ T cell immunity and to the control of influenza virus infections. Our results show that TRAIL deficiency increases influenza-associated morbidity and influenza virus titers, and that these changes in disease severity are coupled to decreased influenza-specific CD8+ T cell cytotoxicity in TRAIL−/− mice, a decrease that occurs despite equivalent numbers of pulmonary influenza-specific CD8+ T cells. Furthermore, TRAIL expression occurs selectively on influenza-specific CD8+ T cells, and high TRAIL receptor (DR5) expression occurs selectively on influenza virus-infected pulmonary epithelial cells. Finally, we show that adoptive transfer of TRAIL+/+ but not TRAIL−/− CD8+ effector T cells alters the mortality associated with lethal dose influenza virus infections. Collectively, our results suggest that TRAIL is an important component of immunity to influenza infections and that TRAIL deficiency decreases CD8+ T cell-mediated cytotoxicity, leading to more severe influenza infections.
Infection of Myeloid Dendritic Cells withListeria monocytogenesLeads to the Suppression of T Cell Function by Multiple Inhibitory Mechanisms
Authors: Popov Alexey , Driesen Julia , Abdullah Zeinab , Wickenhauser Claudia , Beyer Marc , Debey-Pascher Svenja , Saric Tomo , Kummer Silke , Takikawa Osamu , Domann Eugen , Chakraborty Trinad , Krönke Martin , Utermöhlen Olaf , Schultze Joachim L.
Published: April 2014

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.
Calcium/Calmodulin-Dependent Protein Kinase (CaMK) IV Mediates Nucleocytoplasmic Shuttling and Release of HMGB1 during Lipopolysaccharide Stimulation of Macrophages
Authors: Zhang Xianghong , Wheeler David , Tang Ying , Guo Lanping , Shapiro Richard A. , Ribar Thomas J. , Means Anthony R. , Billiar Timothy R. , Angus Derek C. , Rosengart Matthew R.
Published: April 2014

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mφ) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mφ were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mφ isolated from CaMKIV+/+ and CaMKIV−/− mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mφ treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV+/+ Mφ showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV−/− Mφ. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.
Secretogranin III Directs Secretory Vesicle Biogenesis in Mast Cells in a Manner Dependent upon Interaction with Chromogranin A
Authors: Prasad Prerna , Yanagihara Angel A. , Small-Howard Andrea L. , Turner Helen , Stokes Alexander J.
Published: April 2014

Mast cells are granular immunocytes that reside in the body’s barrier tissues. These cells orchestrate inflammatory responses. Proinflammatory mediators are stored in granular structures within the mast cell cytosol. Control of mast cell granule exocytosis is a major therapeutic goal for allergic and inflammatory diseases. However, the proteins that control granule biogenesis and abundance in mast cells have not been elucidated. In neuroendocrine cells, whose dense core granules are strikingly similar to mast cell granules, granin proteins regulate granulogenesis. Our studies suggest that the Secretogranin III (SgIII) protein is involved in secretory granule biogenesis in mast cells. SgIII is abundant in mast cells, and is organized into vesicular structures. Our results show that over-expression of SgIII in mast cells is sufficient to cause an expansion of a granular compartment in these cells. These novel granules store inflammatory mediators that are released in response to physiological stimuli, indicating that they function as bona fide secretory vesicles. In mast cells, as in neuroendocrine cells, we show that SgIII is complexed with Chromogranin A (CgA). CgA is granulogenic when complexed with SgIII. Our data show that a novel non-granulogenic truncation mutant of SgIII (1–210) lacks the ability to interact with CgA. Thus, in mast cells, a CgA-SgIII complex may play a key role in secretory granule biogenesis. SgIII function in mast cells is unlikely to be limited to its partnership with CgA, as our interaction trap analysis suggests that SgIII has multiple binding partners, including the mast cell ion channel TRPA1.
Lipopolysaccharide-Induced Expression of Matrix Metalloproteinases in Human Monocytes Is Suppressed by IFN-γ via Superinduction of ATF-3 and Suppression of AP-1
Authors: Ho Hao H. , Antoniv Taras T. , Ji Jong-Dae , Ivashkiv Lionel B.
Published: April 2014

Matrix metalloproteinases (MMPs) are induced during inflammatory responses and are important for immune regulation, angiogenesis, wound healing, and tissue remodeling. Expression of MMPs needs to be tightly controlled to avoid excessive tissue damage. In this study, we investigated the regulation of MMP expression by inflammatory factors in primary human monocytes and macrophages. IFN-γ, which augments inflammatory cytokine production in response to macrophage-activating factors such as TLR ligands, instead broadly suppressed TLR-induced MMP expression. Inhibition of MMP expression was dependent on STAT1 and required de novo protein synthesis. IFN-γ strongly enhanced TLR-induced expression of the transcriptional repressor activating transcription factor (ATF-3) in a STAT1-dependent manner, which correlated with recruitment of ATF-3 to the endogenous MMP-1 promoter as detected by chromatin immunoprecipitation assays. RNA interference experiments further supported a role for ATF-3 in suppression of MMP-1 expression. In addition, IFN-γ suppressed DNA binding by AP-1 transcription factors that are known to promote MMP expression and a combination of supershift, RNA interference and overexpression experiments implicated AP-1 family member Fra-1 in the regulation of MMP-1 expression. These results define an IFN-γ-mediated homeostatic loop that limits the potential for tissue damage associated with inflammation, and identify transcriptional factors that regulate MMP expression in myeloid cells in inflammatory settings.
Alterations of the Medullary Epithelial Compartment in the Aire-Deficient Thymus: Implications for Programs of Thymic Epithelial Differentiation
Authors: Dooley James , Erickson Matthew , Farr Andrew G.
Published: April 2014

A widely held model of thymic epithelial differentiation is based on patterns of keratin expression, where a K8+K5+ progenitor gives rise to K8+K5/K14− cortical thymic epithelium (CTEC), and medullary thymic epithelium (MTEC) are K8−K5+K14+. The thymic phenotype of p63-deficient mice indicates that p63 is an important regulator of proximal stages of thymic epithelial differentiation. In this study, we have examined several features of the thymic medullary compartment in wild-type and Aire-deficient thymi in an effort to integrate the proapoptotic activity of Aire with these different perspectives of TE differentiation. Patterns of keratin and p63 expression by MTEC described here are difficult to reconcile with postmitotic MTEC that express a K8−K14+ phenotype and suggest that the patterns of p63 and keratin expression reflecting differentiation programs of other epithelial tissues provide a useful framework for revising models of TE differentiation. Alterations of the Aire−/− MTEC compartment included reduced expression of p63, increased frequency of MTEC expressing truncated Aire protein, and shifts in the pattern of keratin expression and epithelial morphology. These data suggest a scenario where cellular targets of Aire-mediated apoptosis are postmitotic MTEC that have not yet completed their terminal differentiation program. According to this view, the minor population of globular K8+K14−/low MTEC observed in the Aire+/+ thymus and significantly expanded in the Aire−/− thymic medulla represent end-stage, terminally differentiated MTEC. These Aire-dependent alterations of the MTEC compartment suggest that the activity of Aire is not neutral with respect to the program of MTEC differentiation.
Differential Pathways Govern CD4+CD28− T Cell Proinflammatory and Effector Responses in Patients with Coronary Artery Disease
Authors: Zal Behnam , Kaski Juan C. , Akiyu Julius P. , Cole Della , Arno Gavin , Poloniecki Jan , Madrigal Alejandro , Dodi Anthony , Baboonian Christina
Published: April 2014

Patients with acute coronary syndromes experience circulatory and intraplaque expansion of an aggressive and unusual CD4+ lymphocyte subpopulation lacking the CD28 receptor. These CD4+CD28− cells produce IFN-γ and perforin, and are thought to play an important role in coronary atheromatous plaque destabilization. Aberrant expression of killer Ig-like receptors (KIRs) in CD4+CD28− cells is broadly thought to be responsible for their cytotoxicity, but the mechanisms involved remain poorly defined. We therefore sought to investigate the mechanism and regulation of CD4+CD28− cell functionality using T cell clones (n = 536) established from patients with coronary artery disease (n = 12) and healthy volunteers (n = 3). Our functional studies demonstrated that KIR2DS2 specifically interacted with MHC class I-presenting human heat shock protein 60 (hHSP60) inducing cytotoxicity. Further investigations revealed the novel finding that hHSP60 stimulation of TCR alone could not induce a cytotoxic response, and that this response was specific and KIR dependent. Analysis of CD4+CD28−2DS2+ clones (n = 162) showed that not all were hHSP60 cytotoxic; albeit, their prevalence correlated with coronary disease status (p = 0.017). A higher proportion of clones responded to hHSP60 by IFN-γ compared with perforin (p = 0.008). In this study, for the first time, we define the differential regulatory pathways involved in CD4+CD28− cell proinflammatory and effector responses. We describe in this study that, contrary to previous reports, CD4+CD28− cell recognition and killing can be specific and discriminate. These results, in addition to contributing to the understanding of CD4+CD28− cell functionality, may have implications for the monitoring and management of coronary artery disease progression.
PD-1-Dependent Mechanisms Maintain Peripheral Tolerance of Donor-Reactive CD8+ T Cells to Transplanted Tissue
Authors: Koehn Brent H. , Ford Mandy L. , Ferrer Ivana R. , Borom Keshawna , Gangappa Shivaprakash , Kirk Allan D. , Larsen Christian P.
Published: April 2014

Peripheral mechanisms of self-tolerance often depend on the quiescent state of the immune system. To what degree such mechanisms can be engaged in the enhancement of allograft survival is unclear. To examine the role of the PD-1 pathway in the maintenance of graft survival following blockade of costimulatory pathways, we used a single-Ag mismatch model of graft rejection where we could track the donor-specific cells as they developed endogenously and emerged from the thymus. We found that graft-specific T cells arising under physiologic developmental conditions at low frequency were actively deleted at the time of transplantation under combined CD28/CD40L blockade. However, this deletion was incomplete, and donor-specific cells that failed to undergo deletion up-regulated expression of PD-1. Furthermore, blockade of PD-1 signaling on these cells via in vivo treatment with anti-PD-1 mAb resulted in rapid expansion of donor-specific T cells and graft loss. These results suggest that the PD-1 pathway was engaged in the continued regulation of the low-frequency graft-specific immune response and thus in maintenance of graft survival.
IFN-γ-Dependent Recruitment of Mature CD27high NK Cells to Lymph Nodes Primed by Dendritic Cells
Authors: Watt Sally V. , Andrews Daniel M. , Takeda Kazuyoshi , Smyth Mark J. , Hayakawa Yoshihiro
Published: April 2014

NK cells have been proposed to be an initial source of IFN-γ that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27high NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27high NK cell subset in the draining LN was dependent on host IFN-γ and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN-γ-dependent mechanism. Thus, our results identify that CD27high NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN-γ in coordinating with DC migration.
c-FLIP Protects Mature T Lymphocytes from TCR-Mediated Killing
Authors: Zhang Nu , Hopkins Kaycie , He You-Wen
Published: April 2014

Although c-FLIP has been identified as an important player in the extrinsic (death receptor-induced) apoptosis pathway, its endogenous function in mature T lymphocytes remains undefined. c-FLIP may inhibit or promote T cell death as previous data demonstrate that the c-FLIPL isoform can promote or inhibit caspase 8 activation while the c-FLIPS isoform promotes or inhibits T cell death when overexpressed. Although the c-FLIPR isoform inhibits cell death in cell lines, its function in T cells remains unknown. To investigate the function of c-FLIP in mature T cells, we have generated several genetic mouse models with c-FLIP or its individual isoforms deleted in mature T cells. Surprisingly, we found that c-FLIP protects mature T cells not only from apoptosis induced by the death receptors Fas and TNFR but also from TCR-mediated and spontaneous apoptosis. Thus, c-FLIP plays an essential role in protecting mature T cells from a death signal induced through the TCR itself and is required for naive T cell survival. Our results demonstrate that c-FLIP functions beyond the extrinsic death pathway.
Normal Development and Activation but Altered Cytokine Production of Fyn-Deficient CD4+ T Cells
Authors: Mamchak Alusha A. , Sullivan Brandon M. , Hou Baidong , Lee Linda M. , Gilden Julia K. , Krummel Matthew F. , Locksley Richard M. , DeFranco Anthony L.
Published: April 2014

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4+ T cell responses. In Fyn-deficient mice, CD4+ T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10+CD4+ T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323–339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10+ Fyn-deficient CD4+ T cells, was virtually indistinguishable from the response of DO11.10+ wild-type CD4+ T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4+ Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10+ Fyn-deficient CD4+ T cells were more potent cytokine-producing cells than DO11.10+ wild-type CD4+ T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4+ T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4+ T cells.