bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Requirement for CD28/CTLA-4-CD80/CD86 Interactions in Drug-Induced Type 1 and Type 2 Immune Responses to Trinitrophenyl-Ovalbumin
Authors: Nierkens Stefan , Aalbers Marloes , Bol Marianne , Bleumink Rob , van Kooten Peter , Boon Louis , Pieters Raymond
Published: April 2014

The use of mAbs to abrogate costimulatory interactions has attracted much attention with regard to prevention and modulation of adverse (auto)immune-like reactions. However, the role of costimulatory molecules and possible therapeutic use of Ab-treatment in drug-induced immunostimulation is poorly elucidated. In the present studies, we show that CD28/CTLA-4-CD80/CD86 costimulatory interactions differently regulate drug-induced type 1 and type 2 responses to an identical bystander Ag, TNP-OVA, in BALB/c mice using the reporter Ag popliteal lymph node assay. The antirheumatic drug D-Penicillamine, which may induce lupus-like side-effects, stimulated type 2 responses against TNP-OVA, characterized by the production of IL-4 and TNP-specific IgG1 and IgE. These responses were abrogated in CD80/CD86-deficient mice and in wild-type mice that were treated with anti-CD80 and anti-CD86, or CTLA-4-Ig. Anti-CTLA-4 intensively enhanced the D-Penicillamine-induced effects. In contrast, the type 1 response (IFN-γ, TNF-α, IgG2a) to TNP-OVA induced by the diabetogen streptozotocin still developed in the absence of CD80/CD86 costimulatory signaling. In addition, it was demonstrated that coadministration of anti-CD80 and anti-CD86 mAbs slightly enhanced streptozotocin-induced type 1 responses, whereas the CTLA-4-Ig fusion protein completely abrogated this response. In conclusion, different drugs may stimulate distinct types of immune responses against an identical bystander Ag, which are completely dependent on (type 2) or independent of (type 1) the CD28/CTLA-4-CD80/CD86 pathway. Importantly, the effects of treatment with anti-CD80/CD86 mAbs and CTLA-4-Ig may be considerably different in responses induced by distinct drugs.
IL-4 Receptor Signaling in Clara Cells Is Required for Allergen-Induced Mucus Production
Authors: Kuperman Douglas A. , Huang Xiaozhu , Nguyenvu Louis , Hölscher Christoph , Brombacher Frank , Erle David J.
Published: April 2014

Excessive mucus production is an important pathological feature of asthma. The Th2 cytokines IL-4 and IL-13 have both been implicated in allergen-induced mucus production, inflammation, and airway hyperreactivity. Both of these cytokines use receptors that contain the IL-4Rα subunit, and these receptors are expressed on many cell types in the lung. It has been difficult to determine whether allergen-induced mucus production is strictly dependent on direct effects of IL-4 and IL-13 on epithelial cells or whether other independent mechanisms exist. To address this question, we used a cell type-specific inducible gene-targeting strategy to selectively disrupt the IL-4Rα gene in Clara cells, an airway epithelial cell population that gives rise to mucus-producing goblet cells. Clara cell-specific IL-4Rα-deficient mice and control mice developed similar elevations in serum IgE levels, airway inflammatory cell numbers, Th2 cytokine production, and airway reactivity following OVA sensitization and challenge. However, compared with control mice, Clara cell-specific IL-4Rα-deficient mice were nearly completely protected from allergen-induced mucus production. Because only IL-13 and IL-4 are thought to signal via IL-4Rα, we conclude that direct effects of IL-4 and/or IL-13 on Clara cells are required for allergen-induced mucus production in the airway epithelium.
The Influence of CD1d in Postselection NKT Cell Maturation and Homeostasis
Authors: McNab Finlay W. , Berzins Stuart P. , Pellicci Daniel G. , Kyparissoudis Konstantinos , Field Kenneth , Smyth Mark J. , Godfrey Dale I.
Published: April 2014

After being positively selected on CD1d-expressing thymocytes, NKT cells undergo a series of developmental changes that can take place inside or outside the thymus. We asked whether CD1d continues to play a role in late-stage NKT cell development and, in particular, during the functionally significant acquisition of NK1.1 that is indicative of NKT cell maturity. We report that CD1d is indeed crucial for this step, because immature NK1.1− NKT cells fail to fully mature when transferred to a CD1d-deficient environment. Surprisingly, however, the lack of CD1d did not greatly affect the long-term survival of NKT cells, and they continued to express CD69 and slowly proliferate. This directly contradicts the currently held view that these phenomena are caused by autoreactivity directed against CD1d/TCR-restricted self-Ags. Our findings demonstrate an ongoing role for TCR-mediated signaling throughout NKT cell development, but the characteristic semiactivated basal state of NKT cells is controlled by CD1d-independent factors or is intrinsic to the cells themselves.
RORγt Recruits Steroid Receptor Coactivators to Ensure Thymocyte Survival
Authors: Xie Huimin , Sadim Maureen S. , Sun Zuoming
Published: April 2014

Thymocytes undergo apoptosis unless a functional TCR is assembled. Steroid receptor coactivators (SRCs) regulate nuclear receptor-mediated transcription by associated histone acetyltransferase activity. However, it has been a challenge to demonstrate the in vivo function of SRCs due to the overlapping functions among different members of SRCs. In this study, we show that recruitment of SRCs is required for thymic-specific retinoic acid-related orphan receptor γ (RORγ)t-regulated thymocyte survival in vivo. An activation function 2 domain, identified at the carboxyl terminus of RORγt, is responsible for recruiting SRCs. A mutation in the activation function domain (Y479F) of RORγt disrupted the interaction with SRCs and abolished RORγt-mediated trans-activation but not its ability to inhibit transcription. Transgenes encoding the wild-type RORγt, but not the mutant, restored thymocyte survival in RORγ null mice. Our results thus clearly demonstrate that RORγt recruits SRCs to impose a gene expression pattern required to expand the life span of thymocytes in vivo, which increases the opportunities for assembling a functional TCR.
The H-2Kk MHC Peptide-Binding Groove Anchors the Backbone of an Octameric Antigenic Peptide in an Unprecedented Mode
Authors: Kellenberger Christine , Roussel Alain , Malissen Bernard
Published: April 2014

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2b and H-2d haplotypes. In contrast, there is a dearth of structural data regarding H-2k-encoded molecules. Therefore, the structures of H-2Kk complexed to an octameric peptide from influenza A virus (HA259–266) and to a nonameric peptide from SV40 (SV40560–568) have been determined by x-ray crystallography at 2.5 and 3.0 Å resolutions, respectively. The structure of the H-2Kk-HA259–266 complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2Kk groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2Kk peptide-binding groove. As a result, the Cα atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2Kb-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2Kk, the backbone of the SV40560–568 nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2Ld. This structural convergence occurs despite the totally different architectures of the H-2Ld and H-2Kk peptide-binding grooves. Therefore, these two H-2Kk-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.
CTL Recognition of a Bulged Viral Peptide Involves Biased TCR Selection
Authors: Miles John J. , Elhassen Diah , Borg Natalie A. , Silins Sharon L. , Tynan Fleur E. , Burrows Jacqueline M. , Purcell Anthony W. , Kjer-Nielsen Lars , Rossjohn Jamie , Burrows Scott R. , McCluskey James
Published: April 2014

MHC class I molecules generally present peptides of 8–10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related αβ TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.
Leukotrienes Play a Role in the Control of Parasite Burden in Murine Strongyloidiasis
Authors: Machado Eleuza R. , Ueta Marlene T. , Lourenço Elaine V. , Anibal Fernanda F. , Sorgi Carlos Artério , Soares Edson G. , Roque-Barreira Maria C. , Medeiros Alexandra I. , Faccioli Lúcia H.
Published: April 2014

It is clear that leukotrienes mediate inflammatory response; new aspects of leukotriene function have recently been described. In this study, we demonstrate that leukotrienes are key chemical mediators in the control of parasite burdens in mice infected with Strongyloides venezuelensis. High leukotriene levels were detected in the lungs and small intestines of Swiss mice. In infected Swiss mice treated with MK886, a leukotriene synthesis inhibitor, numbers of adult worms, and eggs/g/feces were greater than in infected-only animals. The MK886 treatment inhibited leukotriene B4 production in the lungs and small intestines, albeit on different postinfection days. Similarly, parasite burdens and eggs/g/feces were greater in 5-lipoxygenase−/− mice than in wild-type animals. These observation were confirmed by histopathological study of the duodena. We subsequently observed significant lower numbers of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid of Swiss mice treated with MK886. In the lung parenchyma of infected animals, MK886 significantly inhibited synthesis of IL-5 at the beginning of infection, whereas levels of IL-12 increased progressively throughout the postinfection period. However, levels of leukotriene C4, PGE2, TNF-α, IL-3, IL-4, IFN-γ, and IL-10 were comparable between the treated and untreated groups. Nevertheless, IgE and IgG1 (but not IgG2a) synthesis was also significantly inhibited by MK886 administration. Therefore, in S. venezuelensis-infected mice, adult worm and egg burdens are leukotriene dependent. These findings indicate potential immunostimulatory strategies involving leukotriene administration, and may serve as an alert to physicians treating Strongyloides stercoralis-infected patients presenting asthma-like symptoms because use of 5-lipoxygenase inhibitors may worsen the infection.
Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils
Authors: Voyich Jovanka M. , Braughton Kevin R. , Sturdevant Daniel E. , Whitney Adeline R. , Saïd-Salim Battouli , Porcella Stephen F. , Long R. Daniel , Dorward David W. , Gardner Donald J. , Kreiswirth Barry N. , Musser James M. , DeLeo Frank R.
Published: April 2014

Polymorphonuclear leukocytes (PMNs, or neutrophils) are critical for human innate immunity and kill most invading bacteria. However, pathogens such as Staphylococcus aureus avoid destruction by PMNs to survive, thereby causing human infections. The molecular mechanisms used by pathogens to circumvent killing by the immune system remain largely undefined. To that end, we studied S. aureus pathogenesis and bacteria-PMN interactions using strains originally isolated from individuals with community-acquired (CA) and hospital-acquired infections. Compared with strains from hospital infections (COL and MRSA252), strain MW2 and a methicillin-susceptible relative, MnCop, were significantly more virulent in a mouse model of S. aureus infection, and caused the greatest level of pathology in major vital organs. Although phagocytosis of each strain triggered production of reactive oxygen species and granule-phagosome fusion, those from CA infections were significantly more resistant to killing by human PMNs and caused greater host cell lysis. Microarray analysis of the strains during neutrophil phagocytosis identified genes comprising a global S. aureus response to human innate host defense. Genes involved in capsule synthesis, gene regulation, oxidative stress, and virulence, were up-regulated following ingestion of the pathogen. Notably, phagocytosis of strains from CA infections induced changes in gene expression not observed in the other strains, including up-regulation of genes encoding virulence factors and hypothetical proteins. Our studies reveal a gene transcription program in a prominent human pathogen that likely contributes to evasion of innate host defense.
Critical Roles for Both STAT1-Dependent and STAT1-Independent Pathways in the Control of Primary Dengue Virus Infection in Mice
Authors: Shresta Sujan , Sharar Kristin L. , Prigozhin Daniil M. , Snider Heidi M. , Beatty P. Robert , Harris Eva
Published: April 2014

Dengue virus (DEN), a flavivirus, causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, the most common mosquito-borne viral illnesses in humans worldwide. In this study, using STAT1−/− mice bearing two different mutant stat1 alleles in the 129/Sv/Ev background, we demonstrate that IFNR-dependent control of primary DEN infection involves both STAT1-dependent and STAT1-independent mechanisms. The STAT1 pathway is necessary for clearing the initial viral load, whereas the STAT1-independent pathway controls later viral burden and prevents DEN disease in mice. The STAT1-independent responses in mice with primary DEN infection included the early activation of B and NK cells as well as the up-regulation of MHC class I molecules on macrophages and dendritic cells. Infection of bone marrow-derived dendritic cell cultures with either DEN or Sindbis virus, another positive-strand RNA virus, confirmed the early vs late natures of the STAT1-dependent and STAT1-independent pathways. Collectively, these data begin to define the nature of the STAT1-dependent vs the STAT1-independent pathway in vivo.
Interferon-γ Inhibits T84 Epithelial Cell Migration by Redirecting Transcytosis of β1 Integrin from the Migrating Leading Edge
Authors: Tong Qiao , Vassilieva Elena V. , Ivanov Andrei I. , Wang Zili , Brown Gregory T. , Parkos Charles A. , Nusrat Asma
Published: April 2014

Intestinal inflammation is associated with epithelial damage and formation of mucosal wounds. Epithelial cells migration is required for wound closure. In inflammatory status, migrating epithelial cells are exposed to proinflammatory cytokines such as IFN-γ. However, influence of such cytokines on intestinal epithelial wound closure remains unknown. The present study was designed to investigate the effect of IFN-γ on migration of model T84 intestinal epithelial cells and recovery of epithelial wounds. IFN-γ significantly inhibited rate of T84 cell migration and closure of epithelial wounds. This effect was accompanied by the formation of large aberrant lamellipodia at the leading edge as well as significant decrease in the number of β1 integrin containing focal adhesions. IFN-γ exposure increased endocytosis of β1 integrin and shifted its accumulation from early/recycling endosomes at the leading edge to a yet unidentified compartment at the cell base. This redirection in β1 integrin transcytosis was inhibited by depolymerization of microtubules with nocodazole and was unaffected by stabilization of microtubules with docetaxel. These results suggest that IFN-γ attenuates epithelial wound closure by microtubule-dependent redirection of β1 integrin transcytosis from the leading edge of migrating cells thereby inhibiting adequate turnover of focal adhesion complexes and cell migration.
Transcriptional Induction of Matrix Metalloproteinase-9 in the Chondrocyte and Synoviocyte Cells Is Regulated via a Novel Mechanism: Evidence for Functional Cooperation between Serum Amyloid A-Activating Factor-1 and AP-1
Authors: Ray Alpana , Bal B. Sonny , Ray Bimal K.
Published: April 2014

Increased expression of matrix metalloproteinase-9 (MMP-9) by IL-1β and TNF-α is regarded as a key factor in the degradation of cartilage during arthritis. However, the underlying molecular mechanism of this induction process especially in the cells of the joint capsule remains elusive. Chondrocytes and synoviocytes, the resident cells of joint capsule, markedly increase transcription of MMP-9 in response to IL-1β- and TNF-α-mediated stimulation. Using progressively deleted and mutant promoter constructs of MMP-9, we show that serum amyloid A-activating factor (SAF)-1, a novel transcription factor, and the AP-1 family of proteins cooperatively regulate cytokine-mediated induction of MMP-9 in the resident cells of the joint capsule. In the MMP-9 promoter, SAF-1 and AP-1 DNA-binding elements are present in close proximity with only 14 nucleotides apart. SAF-1 DNA-binding activity is increased in both cytokine-stimulated cells as well as in osteoarthritic cartilage tissues. Although overexpression of SAF-1 could increase expression of the MMP-9 promoter and endogenous MMP-9 gelatinolytic activity, for maximal induction of MMP-9 gene concurrent participation of SAF-1 and AP-1 is required. Mutation of either one of these two elements resulted in a severe reduction in cytokine responsiveness of MMP-9 promoter and compromised the transactivation potential of both SAF-1 and AP-1. Simultaneous requirement for two distinct DNA-binding elements suggests that SAF-1 and AP-1 function in a mutually beneficial manner acting as essential coactivators to drive cytokine-mediated transcriptional activation of MMP-9.
Regulation of Lipopolysaccharide-Induced Lung Inflammation by Plasminogen Activator Inhibitor-1 through a JNK-Mediated Pathway
Authors: Arndt Patrick G. , Young Scott K. , Worthen G. Scott
Published: April 2014

The neutrophil is of undoubted importance in lung inflammation after exposure to LPS. We have shown recently that systemic inhibition of JNK decreased neutrophil recruitment to the lung after exposure to LPS, although the mechanisms underlying this inhibition are incompletely understood. As plasminogen activator inhibitor-1 (PAI-1) accentuates cell migration, with JNK activation recently shown to up-regulate PAI-1 expression, this suggested that systemic JNK inhibition may down-regulate LPS-induced pulmonary neutrophil recruitment through a decrease in PAI-1 expression. We show in this study that exposure of mice to aerosolized LPS increased PAI-1 expression in the lung and alveolar compartment, which was decreased by pretreatment with the JNK inhibitor SP600125. Exogenous, intratracheally administered PAI-1 prevented the inhibition of pulmonary neutrophil recruitment in the setting of systemic JNK inhibition, thereby suggesting a role for PAI-1 in the JNK-mediated pathway regulating LPS-induced neutrophil recruitment. In addition, PAI-1−/− mice had a decrease in neutrophil recruitment to the alveolar compartment after exposure to LPS, compared with wild-type controls, further suggesting a role for PAI-1 in LPS-induced lung inflammation. An increase in the intravascular level of KC is a likely mechanism for the inhibition of pulmonary neutrophil recruitment after LPS exposure in the setting of decreased PAI-1 expression, as systemic KC levels after exposure to LPS were increased in PAI-1-deficient mice and in mice pretreated with SP600125, with augmentation of intravascular KC levels inhibiting neutrophil recruitment to the lung after exposure to LPS.
The Urokinase/Urokinase Receptor System Mediates the IgG Immune Complex-Induced Inflammation in Lung
Authors: Shushakova Nelli , Eden Gabriele , Dangers Marc , Zwirner Joerg , Menne Jan , Gueler Faikah , Luft Friedrich C. , Haller Hermann , Dumler Inna
Published: April 2014

Immune complex (IC) deposition induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by the cell surface-specific receptor (uPAR), a binding partner for the urokinase-type plasminogen activator (uPA). We assessed the role of the uPA/uPAR system in IC-induced inflammation using the pulmonary reverse passive Arthus reaction in mice lacking uPA and uPAR compared with their corresponding wild-type controls. Both uPA-deficient C57BL/6J (uPA−/−) and uPAR-deficient mice on a mixed C57BL/6J (75%) × 129 (25%) background (uPAR−/−) demonstrated a marked reduction of the inflammatory response due to decreased production of proinflammatory mediators TNF-α and Glu-Leu-Arg (ELR)-CXC chemokine MIP-2. In uPAR−/− animals, the reduction of inflammatory response was more pronounced because of decreased migratory capacity of polymorphonuclear leukocytes. We show that the uPA/uPAR system is activated in lung of wild-type mice, particularly in resident alveolar macrophages (AM), early in IC-induced alveolitis. This activation is necessary for an adequate C5a anaphylatoxin receptor signaling on AM that, in turn, modulates the functional balance of the activating/inhibitory IgG FcγRs responsible for proinflammatory mediator release. These data provide the first evidence that the uPA/uPAR plays an important immunoregulatory role in the initiation of the reverse passive Arthus reaction in the lung by setting the threshold for C5a anaphylatoxin receptor/FcγR activation on AM. The findings indicate an important link between the uPA/uPAR system and the two main components involved in the IC inflammation, namely, complement and FcγRs.
Uterine NK Cells Mediate Inflammation-Induced Fetal Demise in IL-10-Null Mice
Authors: Murphy Shaun P. , Fast Loren D. , Hanna Nazeeh N. , Sharma Surendra
Published: April 2014

Specialized NK cells are recruited in high numbers to the mammalian embryo implantation sites, yet remain pregnancy compatible. It is not well understood whether uterine NK (uNK) cells become adversely activated and mediate fetal demise, a common complication of early pregnancy. In this study we show that mating of IL-10−/− mice resulted in fetal resorption or intrauterine growth restriction in response to very low doses of LPS. Pregnancy in congenic wild-type mice was normal even at 10-fold higher LPS doses. Fetal resorption in IL-10−/− mice was associated with a significant increase in uNK cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells, TNF-α neutralization, or IL-10 administration rescued pregnancy in LPS-treated IL-10−/− animals. Our results identify an immune mechanism of fetal demise involving IL-10 deficiency, NK cells, and inflammation. These results may provide insight into adverse pregnancy outcomes in humans.
Mast Cell Survival and Activation by IgE in the Absence of Antigen: A Consideration of the Biologic Mechanisms and Relevance
Authors: Kawakami Toshiaki , Kitaura Jiro
Published: April 2014

Mast cells are not only major effector cells in allergy and host defense against parasites and bacteria but also important cellular components in other immune responses. Recent studies on the effects of monomeric IgE on mast cell survival and activation have made an impact on our view of the IgE binding to its high-affinity receptors, FcεRI. Traditionally, IgE binding to FcεRI has been considered as a passive action of “sensitization” before receptor aggregation by Ag. However, recent studies indicate that at high concentrations some monoclonal IgEs have effects on mast cells similar to or identical to those induced by IgE+Ag stimulation. These effects may be due to induction of FcεRI aggregation by these IgEs in the absence of Ag. This review will synthesize recent findings of the heterogeneity of IgEs in their ability to induce survival and activation events, their mechanisms, the potential in vivo significance of IgE-FcεRI interactions, and the implications of the mouse studies to human diseases.
Cutting Edge: Bacterial Modulation of Epithelial Signaling via Changes in Neddylation of Cullin-1
Authors: Collier-Hyams Lauren S. , Sloane Valerie , Batten Brigid C. , Neish Andrew S.
Published: April 2014

The human enteric flora plays a significant role in intestinal health and disease. Certain enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination. IκB-α ubiquitination is catalyzed by the E3-SCFβTrCP ubiquitin ligase, which is itself regulated via covalent modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Neddylation is a biochemical event associated with diverse cellular processes related to cell signaling, however, physiological regulation of cullin neddylation has not been described in mammalian systems. We report that interaction of nonpathogenic bacteria with epithelial cells resulted in a rapid loss of neddylated Cul-1 and consequent repression of the NF-κB pathway. This observation may explain the ability of intestinal bacterial communities to influence diverse eukaryotic processes in general and inflammatory tolerance of the mammalian intestinal epithelia specifically.
Regulated Costimulation in the Thymus Is Critical for T Cell Development: Dysregulated CD28 Costimulation Can Bypass the Pre-TCR Checkpoint
Authors: Williams Joy A. , Hathcock Karen S. , Klug David , Harada Yohsuke , Choudhury Baishakhi , Allison James P. , Abe Ryo , Hodes Richard J.
Published: April 2014

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4−CD8− cells initiate expression of TCRβ. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRβ expression manifest in wild-type thymocytes, occurring even in Rag− or CD3ε− knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the “β” checkpoint for pre-TCR/TCR-dependent thymic differentiation.
The Mannich Base NC1153 Promotes Long-Term Allograft Survival and Spares the Recipient from Multiple Toxicities
Authors: Stepkowski Stanislaw M. , Kao Judy , Wang Mou-Er , Tejpal Neelam , Podder Hemangshu , Furian Lucrezia , Dimmock Jonathan , Jha Amitabh , Das Umashankar , Kahan Barry D. , Kirken Robert A.
Published: April 2014

JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-α-driven NF-κB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.
Virus-Induced Inhibition of CD1d1-Mediated Antigen Presentation: Reciprocal Regulation by p38 and ERK
Authors: Renukaradhya Gourapura J. , Webb Tonya J. Roberts , Khan Masood A. , Lin Yin Ling , Du Wenjun , Gervay-Hague Jacquelyn , Brutkiewicz Randy R.
Published: April 2014

A critical component of the host’s innate immune response involves lipid Ag presentation by CD1d molecules to NK T cells. In this study we used murine CD1d1-transfected L (L-CD1) cells to study the effect of viruses on CD1d-mediated Ag presentation to NKT cells and found that an infection with vesicular stomatitis and vaccinia (but not lymphocytic choriomeningitis) virus inhibited murine CD1d1-mediated Ag presentation. This was under the reciprocal control of the MAPKs, p38 and ERK, and was due to changes in the intracellular trafficking of CD1d1. The reciprocal regulation of CD1d1-mediated Ag presentation by MAPK suggests that the targeting of these pathways is a novel means of immune evasion by viruses.
An Organized Medullary Epithelial Structure in the Normal Thymus Expresses Molecules of Respiratory Epithelium and Resembles the Epithelial Thymic Rudiment of Nude Mice
Authors: Dooley James , Erickson Matt , Farr Andrew G.
Published: April 2014

The expression of tissue-specific Ags (TSA) within the thymic environment has emerged as an important contribution to the establishment of self-tolerance. The mechanistic basis for this property is poorly understood. One model has proposed stochastic derepression of gene expression by mature medullary epithelial cells, whereas another model has suggested that this property of thymic epithelial cells reflects transcriptional activity during their differentiation. Most of the analyses of thymic TSA expression have been done with populations of dissociated thymic epithelial cells; therefore, there is little information regarding the spatial pattern of TSA expression within the thymus. We have evaluated a subset of thymic epithelial cells in the murine thymus that display several unique features. First, within the normal thymus, they form cysts that express several TSA of respiratory epithelium and exhibit some morphological features consistent with respiratory epithelium. These cells also display a phenotypic profile that has been proposed for immature thymic epithelium. The cystic epithelia in the normal thymus and in the nude thymic rudiment are phenotypically very similar, suggesting that they may have a similar developmental program. The coordinated expression of respiratory TSA by an organized subset of thymic epithelial cells and the phenotypic resemblance of these cells to progenitor cells seem consistent with a developmental basis for TSA expression by thymic epithelial cells. Finally, epitopes that define thymic epithelial heterogeneity are reciprocally expressed by respiratory epithelium, which raises interesting questions regarding the developmental relationship of different endodermal derivatives.
αEβ7 (CD103) Expression Identifies a Highly Active, Tonsil-Resident Effector-Memory CTL Population
Authors: Woodberry Tonia , Suscovich Todd J. , Henry Leah M. , August Meredith , Waring Michael T. , Kaur Amitinder , Hess Christoph , Kutok Jeffery L. , Aster Jon C. , Wang Frederick , Scadden David T. , Brander Christian
Published: April 2014

The characterization of antiviral CTL responses has largely been limited to assessing Ag-specific immune responses in the peripheral blood. Consequently, there is an incomplete understanding of the cellular immune responses at mucosal sites where many viruses enter and initially replicate and how the Ag specificity and activation status of CTL derived from these mucosal sites may differ from that of blood-derived CTL. In this study, we show that EBV-specific CTL responses in the tonsils are of comparable specificity and breadth but of a significantly higher magnitude compared with responses in the peripheral blood. EBV-specific, tonsil-resident, but not PBMC-derived, T cells expressed the integrin/activation marker CD103 (αEβ7), consistent with the detection of its ligand, E-cadherin, on tonsillar squamous cells. These CD8-positive, CD103-positive, tonsil-derived CTL were largely CCR7- and CD45RA- negative effector-memory cells and responded to lower Ag concentrations in in vitro assays than their CD103-negative PBMC-derived counterparts. Thus, EBV-specific CTL in the tonsil, a crucial site for EBV entry and replication, are of greater magnitude and phenotypically distinct from CTL in the peripheral blood and may be important for effective control of this orally transmitted virus.
Stepwise Development of Committed Progenitors in the Bone Marrow That Generate Functional T Cells in the Absence of the Thymus
Authors: García-Ojeda Marcos E. , Dejbakhsh-Jones Sussan , Chatterjea-Matthes Devavani , Mukhopadhyay Aditi , BitMansour Andrew , Weissman Irving L. , Brown Janice M. Y. , Strober Samuel
Published: April 2014

We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCRβ gene; express a variety of genes associated with commitment to the T cell lineage, including GATA-3, T cell-specific factor-1, Cβ, and Id2; and show a surface marker pattern (CD44+CD25−CD24+CD5−) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors in the marrow have rearranged the TCR gene loci, express Vα and Vβ genes as well as CD3ε, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the αβ T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN-γ after stimulation in vitro, and protected the mice against lethal infection with murine CMV. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway in athymic nu/nu mice.
Gs Protein-Coupled Adenosine Receptor Signaling and Lytic Function of Activated NK Cells
Authors: Raskovalova Tatiana , Huang Xiaojun , Sitkovsky Michail , Zacharia Lefteris C. , Jackson Edwin K. , Gorelik Elieser
Published: April 2014

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1−/− and AdoRA3−/− mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.
IL-7 Receptor Expression Levels Do Not Identify CD8+ Memory T Lymphocyte Precursors following Peptide Immunization
Authors: Lacombe Marie-Hélène , Hardy Marie-Pierre , Rooney Julie , Labrecque Nathalie
Published: April 2014

Identification of the mechanisms underlying the survival of effector T cells and their differentiation into memory T lymphocytes are critically important to understanding memory development. Because cytokines regulate proliferation, differentiation, and survival of T lymphocytes, we hypothesized that cytokine signaling dictates the fate of effector T cells. To follow cytokine receptor expression during T cell responses, we transferred murine TCR transgenic T cells into naive recipients followed by immunization with peptide emulsified in adjuvant or pulsed on dendritic cells. Our findings did not correlate IL-7R α-chain and IL-2R β-chain expression on effector CD8+ cells with the generation of memory T lymphocytes. However, we could correlate the extent of IL-7Rα expression down-regulation on effector T cells with the level of inflammation generated by the immunization. Furthermore, our findings showed that the maintenance of a high level of IL-7R expression by effector T cells at the peak of the response does not preclude their death. This suggests that maintenance of IL-7R expression is not sufficient to prevent T cell contraction. Thus, our results indicate that expression of the IL-7R is not always a good marker for identifying precursors of memory T cells among effectors and that selective expression of the IL-7R by effector T cells should not be used to predict the success of vaccination.
IFN-γ Controls the Generation/Activation of CD4+CD25+ Regulatory T Cells in Antitumor Immune Response
Authors: Nishikawa Hiroyoshi , Kato Takuma , Tawara Isao , Ikeda Hiroaki , Kuribayashi Kagemasa , Allen Paul M. , Schreiber Robert D. , Old Lloyd J. , Shiku Hiroshi
Published: April 2014

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+CD25+ T cells and CD4+CD25− T cells, respectively. In the present study, IFN-γ was found to abrogate the generation/activation of CD4+CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+CD25+ T cells from these IFN-γ-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-γ produced by CD8+ T cells was shown in experiments demonstrating that CD4+CD25+ T cells cotransferred with CD8+ T cells from IFN-γ−/− mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-γ regulates the generation/activation of CD4+CD25+ regulatory T cells.
Down-Regulation of Basophil Function by Human CD200 and Human Herpesvirus-8 CD200
Authors: Shiratori Ikuo , Yamaguchi Masao , Suzukawa Maho , Yamamoto Kazuhiko , Lanier Lewis L. , Saito Takashi , Arase Hisashi
Published: April 2014

Human and rodent CD200 are recognized by the inhibitory CD200R, and these molecules play an important role in the regulation of the immune system. Several viruses, such as human herpesvirus-6 (HHV-6), HHV-7, and HHV-8, possess a CD200 homologue, suggesting that these viruses regulate the immune response via CD200R. In this study, we analyzed the effect of human CD200 and the viral CD200 homologues on human CD200R-expressing cells. We found that human CD200R is predominantly expressed on basophils in amounts higher than on other human peripheral blood leukocytes. Furthermore, the viral CD200 homologues as well as human CD200 were recognized by human CD200R, and the activation of basophils was down-regulated by these CD200 proteins. These results suggested that CD200R is an important regulatory molecule of basophil activation. In addition, the presence of CD200 homologues on several viruses suggests a potentially unique relationship between basophil function and viral infection.
Binding of IgG-Opsonized Particles to FcγR Is an Active Stage of Phagocytosis That Involves Receptor Clustering and Phosphorylation
Authors: Sobota Andrzej , Strzelecka-Kiliszek Agnieszka , Gładkowska Ewelina , Yoshida Kiyotsugu , Mrozińska Kazimiera , Kwiatkowska Katarzyna
Published: April 2014

FcγR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of FcγR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant FcγRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type FcγRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both FcγRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control FcγR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted FcγRIIA upon binding of immune complexes. We found that a lack of FcγRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of FcγR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.
Reduction of Runx1 Transcription Factor Activity Up-Regulates Fas and Bim Expression and Enhances the Apoptotic Sensitivity of Double Positive Thymocytes
Authors: Abe Natsumi , Kohu Kazuyoshi , Ohmori Hidetaka , Hayashi Keitaro , Watanabe Toshio , Hozumi Katsuto , Sato Takehito , Habu Sonoko , Satake Masanobu
Published: April 2014

The death or survival of double positive (DP) thymocytes is determined by the strength of their TCR signaling. Of the three Runx family proteins, the DP cells only express the Runx1 transcription factor. We introduced and expressed in murine thymocytes the Runt domain of Runx1, which antagonizes the activity of endogenous Runx1. The Runt transgenic DP thymocytes expressed higher levels of the proapoptotic molecules Fas and Bim compared with the wild-type cells. Furthermore, the Runt transgenic cells were more susceptible to apoptosis induced by the artificial cross-linking of the TCR by the anti-CD3 Ab. This susceptibility was partially abrogated by the lpr/lpr background. In addition, Runx1:HY-TCR-double transgenic DP thymocytes were resistant to the apoptosis induced by the endogenously presented HY Ag. We propose that Runx1 functions to suppress the apoptotic sensitivity of DP thymocytes in the context of TCR signaling.
Synergy between Two Active Sites of Human Complement Receptor Type 1 (CD35) in Complement Regulation: Implications for the Structure of the Classical Pathway C3 Convertase and Generation of More Potent Inhibitors
Authors: Krych-Goldberg Malgorzata , Hauhart Richard E. , Porzukowiak Tina , Atkinson John P.
Published: April 2014

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1–3) and two copies of site 2 (CCPs 8–10 and CCPs 15–17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1–7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.