bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

AbstractShort-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

Abstract
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

Abstract
The majority of &gt;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 &gt;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

Abstract
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

Abstract
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

Abstract
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.