bluuebunny/crossref_metadata_2025 huggingface dataset:
β-Catenin Expression Enhances IL-7 Receptor Signaling in Thymocytes during Positive Selection
Authors: Yu Qing , Xu Mai , Sen Jyoti Misra
Published: April 2014

Differentiation of CD4+CD8+ double-positive thymocytes into CD8+ single-positive (SP) thymocytes is regulated by TCR and cytokine receptor signals. Previously, we have shown that expression of stabilized β-catenin, the major transcriptional cofactor of T cell factor, results in increase in both CD4SP and CD8SP thymocytes with a preferential effect on CD8SP thymocytes. In this report, using mice expressing stabilized β-catenin and mice with T cell specific deletion of β-catenin, we show that β-catenin expression augments IL-7Rα-chain expression and down-regulates suppressor of cytokine signaling-1 expression in thymocytes undergoing positive selection. Consequently, β-catenin expression augments IL-7R signaling in thymocytes during positive selection and promotes the development of CD8SP thymocytes.
TLR4 Polymorphisms Mediate Impaired Responses to Respiratory Syncytial Virus and Lipopolysaccharide
Authors: Tulic Meri K. , Hurrelbrink Robert J. , Prêle Cecilia M. , Laing Ingrid A. , Upham John W. , Le Souef Peter , Sly Peter D. , Holt Patrick G.
Published: April 2014

Severe bronchiolitis following respiratory syncytial virus (RSV) infection occurs in only a small subset of infected infants and the basis for variations in disease severity is not understood. Innate immune responses to RSV are mediated by TLR-4, and the 299Gly and 399Ile alleles of the TLR4 gene have been linked epidemiologically with increased severity of RSV disease in children. We hypothesized that cellular immune responses to RSV mediated by these variant forms of the receptor are defective relative to responses mediated via the common form of the receptor. Human bronchial epithelial cells were transfected with TLR4 constructs encoding the common TLR4 gene sequence (299Asp/399Thr), or the 299Gly or 399Ile alleles, and cytokine responses to in vitro RSV challenge were analyzed in the different transfected cells. Follow-up studies compared RSV-induced responses in PBMC from children expressing these same TLR4 genotypes. Human bronchial epithelial expressing 299Gly or 399Ile displayed normal levels of intracellular TLR4 but failed to efficiently translocate the receptor to the cell surface. This was associated with reduced NF-κB signaling post-TLR4 engagement, reduced production of IFNs, IL-8, IL-10, IL-12p35, IL-18, and CCL8, and the absence of acute-phase TNF-α. These findings were mirrored by blunted PBMC responses to RSV in children expressing the same TLR4 variants. Compromised first-line defense against RSV at the airway-epithelial surface of children expressing these TLR4 variants may thus confer increased susceptibility to severe infections with this virus.
Memory Generation and Maintenance of CD8+ T Cell Function during Viral Persistence
Authors: Cush Stephanie S. , Anderson Kathleen M. , Ravneberg David H. , Weslow-Schmidt Janet L. , Flaño Emilio
Published: April 2014

During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agent in the presence of persistent Ag. By using a γ-herpesvirus (γHV) infection model, we demonstrate that a small number of virus-specific central-memory CD8+ T cells develop early during infection, and that virus-specific CD8+ T cells maintain functional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response, we show generation of CD8+ memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeostatic cytokine receptors (IL-7α, IL-2/IL-15β). During long-term persistent infection, central-memory cells constitute 20–50% of the virus-specific CD8+ T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functional analyses demonstrate that during viral persistence: 1) CD8+ T cells maintain TCR affinity for peptide/MHC complexes, 2) the functional avidity of CD8+ T cells measured as the capacity to produce IFN-γ is preserved intact, and 3) virus-specific CD8+ T cells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8+ T cells are capable of mediating a protective recall response against the establishment of γHV68 splenic latency. These observations provide evidence that functional CD8+ memory T cells can be generated and maintained during low-load γHV68 persistence.
Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses
Authors: McCall Matthew B. B. , Netea Mihai G. , Hermsen Cornelus C. , Jansen Trees , Jacobs Liesbeth , Golenbock Douglas , van der Ven André J. A. M. , Sauerwein Robert W.
Published: April 2014

TLRs are a major group of pattern recognition receptors that are crucial in initiating innate immune responses and are capable of recognizing Plasmodium ligands. We have investigated TLR responses during acute experimental P. falciparum (P.f.) infection in 15 malaria-naive volunteers. TLR-4 responses in whole blood ex vivo stimulations were characterized by significantly (p < 0.01) up-regulated proinflammatory cytokine production during infection compared with baseline, whereas TLR-2/TLR-1 responses demonstrated increases in both proinflammatory and anti-inflammatory cytokine production. Responses through other TLRs were less obviously modified by malaria infection. The degree to which proinflammatory TLR responses were boosted early in infection was partially prognostic of clinical inflammatory parameters during the subsequent clinical course. Although simultaneous costimulation of human PBMC with P.f. lysate and specific TLR stimuli in vitro did not induce synergistic effects on cytokine synthesis, PBMC started to respond to subsequent TLR-4 and TLR-2 stimulation with significantly (p < 0.05) increased TNF-α and reduced IL-10 production following increasing periods of preincubation with P.f. Ag. In contrast, preincubation with preparations derived from other parasitic, bacterial, and fungal pathogens strongly suppressed subsequent TLR responses. Taken together, P.f. primes human TLR responses toward a more proinflammatory cytokine profile both in vitro and in vivo, a characteristic exceptional among microorganisms.
IκB Kinase 2/β Deficiency Controls Expansion of Autoreactive T Cells and Suppresses Experimental Autoimmune Encephalomyelitis
Authors: Greve Bernhard , Weissert Robert , Hamdi Nada , Bettelli Estelle , Sobel Raymond A. , Coyle Anthony , Kuchroo Vijay K. , Rajewsky Klaus , Schmidt-Supprian Marc
Published: April 2014

The NF-κB family of transcription factors plays a pivotal role in T cell activation and survival during (auto) immune responses. IκB kinase 2/β (IKK2) is part of the IκB kinase complex, a central component of the intracellular signaling pathway mediating NF-κB activation. We studied the role of IKK2 in autoantigen-specific T cell activation and induction of autoimmune disease using mice that lack this kinase specifically in T cells (IKK2ΔT cell mice). We found highly impaired myelin-oligodendrocyte-glycoprotein (MOG)35–55-specific T cell activation in vitro and complete resistance to MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) in IKK2ΔT cell C57BL/6 mice in vivo. By contrast, transgenic expression of a pathogenic MOG35–55-specific TCR (2D2 TCR) rendered IKK2ΔT cell mice susceptible to MOG35–55-induced EAE and restored in vitro MOG35–55-specific T cell responses, indicating an expansion defect in IKK2-deficient T cells. Treatment with the IKK2-inhibitory compound PS-1145 reduced MOG35–55-specific proliferation and cytokine production of 2D2 transgenic spleen cells in vitro and diminished clinical signs of EAE in vivo. Our data underscore the potential of therapeutic IKK inhibition in autoimmune diseases.
Synergism between NF-κB1/p50 and Notch2 during the Development of Marginal Zone B Lymphocytes
Authors: Moran Stewart T. , Cariappa Annaiah , Liu Haoyuan , Muir Beth , Sgroi Dennis , Boboila Cristian , Pillai Shiv
Published: April 2014

NF-κB1 and Notch2 are both required for the development of marginal zone (MZ) B cells. Analysis of B lymphocyte development in mice that are doubly heterozygous at the Notch2 and NF-κB1 loci revealed synergism between Notch2 and NF-κB1 during MZ B cell development. Two known transcriptional targets of the Notch pathway, Hes-5 and Deltex-1, were found to be preferentially expressed in MZ B cells and regulated by NF-κB1. These studies provide in vivo evidence for a genetic interaction between the Notch and NF-κB pathways.
Early Intrahepatic Accumulation of CD8+ T Cells Provides a Source of Effectors for Nonhepatic Immune Responses
Authors: Polakos Noelle K. , Klein Ingo , Richter Martin V. , Zaiss Dietmar M. , Giannandrea Matthew , Crispe Ian N. , Topham David J.
Published: April 2014

Interactions between the liver and CD8+ T cells can lead to tolerance, due in part to CD8+ T cell death. To test whether this was the case in an extrahepatic infection, we investigated the fate and effector capacity of intrahepatic CD8+ T cells during lung-restricted influenza infection in mice. Virus-specific T cells accumulated in livers without detectable intrahepatic presentation of viral Ags, and this accumulation was not restricted to the contraction phase, but was apparent as early as day 5. Intrahepatic influenza-specific cells were functionally similar to those recovered from the bronchioalveolar lavage, based on ex vivo cytokine production and specific target lysis. Both adoptive transfer of liver lymphocytes and orthotopic liver transplant of organs containing accumulated effector T cells revealed that activated CD8s from the liver were viable, expanded during reinfection, and generated a memory population that trafficked to lymphoid organs. Thus, intrahepatic CD8+ T cells re-enter circulation and generate functional memory, indicating that the liver does not uniformly incapacitate activated CD8+ T cells. Instead, it constitutes a substantial reservoir of usable Ag-specific effector CD8+ T cells involved in both acute and recall immune responses.
A Reduced Antigen Load In Vivo, Rather Than Weak Inflammation, Causes a Substantial Delay in CD8+ T Cell Priming againstMycobacterium bovis(Bacillus Calmette-Guérin)
Authors: Russell Marsha S. , Iskandar Monica , Mykytczuk Oksana L. , Nash John H. E. , Krishnan Lakshmi , Sad Subash
Published: April 2014

Regardless of the dose of Ag, Ag presentation occurs rapidly within the first few days which results in rapid expansion of the CD8+ T cell response that peaks at day 7. However, we have previously shown that this rapid priming of CD8+ T cells is absent during infection of mice with Mycobacterium bovis (bacillus Calmette-Guérin (BCG)). In this study, we have evaluated the mechanisms responsible for the delayed CD8+ T cell priming. Because BCG replicates poorly and survives within phagosomes we considered whether 1) generation of reduced amounts of Ag or 2) weaker activation by pathogen-associated molecular patterns (PAMPs) during BCG infection is responsible for the delay in CD8+ T cell priming. Using rOVA-expressing bacteria, our results indicate that infection of mice with BCG-OVA generates greatly reduced levels of OVA, which are 70-fold lower in comparison to the levels generated during infection of mice with Listeria monocytogenes-expressing OVA. Furthermore, increasing the dose of OVA, but not PAMP signaling during BCG-OVA infection resulted in rapid Ag presentation and consequent expansion of the CD8+ T cell response, indicating that the generation of reduced Ag levels, not lack of PAMP-associated inflammation, was responsible for delayed priming of CD8+ T cells. There was a strong correlation between the relative timing of Ag presentation and the increase in the level of OVA in vivo. Taken together, these results reveal that some slowly replicating pathogens, such as mycobacteria, may facilitate their chronicity by generating reduced Ag levels which causes a substantial delay in the development of acquired immune responses.
A New Subset of Human Naive CD8+ T Cells Defined by Low Expression of IL-7Rα
Authors: Alves Nuno L. , van Leeuwen Ester M. M. , Remmerswaal Ester B. M. , Vrisekoop Nienke , Tesselaar Kiki , Roosnek Eddy , ten Berge Ineke J. M. , van Lier René A. W.
Published: April 2014

Concomitant with an increased number of memory-type cells, the amount of naive T cells steadily declines with age. Although the regulatory mechanisms behind this conversion are not fully understood, the suggestion is that both alterations in thymic output and homeostatic signals mold the naive T cell pool. In this study, we identify a new subset of circulating CD27highCD45RAhigh CD8+ T cells characterized by low IL-7Rα message and protein expression. Analysis of TCR repertoire and TCR excision circle content together with ex vivo recovery of IL-7Rα expression indicated that these cells should be placed into the naive T cell pool. Compared with conventional IL-7Rαhigh naive T cells, this subset displayed significantly lower levels of CD28 and higher levels of HLA-DR. Proliferative responses to anti-CD3/CD28 mAbs were indistinguishable from conventional naive T cells, but the responsiveness to IL-7 was limited. Strikingly, IL-7Rαlow naive T cells were particularly increased in circumstances of naive CD8+ T cells shortage, as in the elderly, in patients early after hemopoietic stem cell transplantation, and in HIV-infected individuals. As common γ chain cytokines induce rapid down-regulation of IL-7Rα, we propose that this new subset of naive T cells may encompass cells that have recently received homeostatic signals.
B Cell Receptor Cross-Talk: Exposure to Lipopolysaccharide Induces an Alternate Pathway for B Cell Receptor-Induced ERK Phosphorylation and NF-κB Activation
Authors: Dye John R. , Palvanov Arkadiy , Guo Benchang , Rothstein Thomas L.
Published: April 2014

BCR signaling in naive B cells depends on the function of signalosome mediators; however, prior engagement of CD40 or of IL-4R produces an alternate signaling pathway in which Bruton’s tyrosine kinase, PI3K, phospholipase Cγ2, and protein kinase Cβ are no longer required for BCR-induced downstream events. To explore the range of mediators capable of producing such an alternate pathway for BCR signaling, we examined the TLR4 agonist, LPS. B cell treatment with LPS at relatively low doses altered subsequent BCR signaling such that ERK phosphorylation and NF-κB activation occurred in a PI3K-independent manner. This effect of LPS extended to MEK phosphorylation and IκBα degradation, and it developed slowly over a period of 16–24 h. The involvement of TLRs is suggested by similar effects observed with a structurally distinct TLR agonist, PAM3CSK4 and by the need for MyD88 for induction of alternate BCR signaling by LPS. Thus, LPS-mediated TLR engagement produces an alternate pathway for BCR-triggered signal propagation that differs from the classical, signalosome-dependent pathway.
Aryl Hydrocarbon Receptor Activation during Influenza Virus Infection Unveils a Novel Pathway of IFN-γ Production by Phagocytic Cells
Authors: Neff-LaFord Haley , Teske Sabine , Bushnell Timothy P. , Lawrence B. Paige
Published: April 2014

The contribution of environmental factors is important as we consider reasons that underlie differential susceptibility to influenza virus. Aryl hydrocarbon receptor (AhR) activation by the pollutant dioxin during influenza virus infection decreases survival, which correlates with a 4-fold increase in pulmonary IFN-γ levels. We report here that the majority of IFN-γ-producing cells in the lung are neutrophils and macrophages not lymphocytes, and elevated IFN-γ is associated with increased pulmonary inducible NO synthase (iNOS) levels. Moreover, we show that even in the absence of dioxin, infection with influenza virus elicits IFN-γ production by B cells, γδ T cells, CD11c+ cells, macrophages and neutrophils, as well as CD3+ and NK1.1+ cells in the lung. Bone marrow chimeric mice reveal that AhR-mediated events external to hemopoietic cells direct dioxin-enhanced IFN-γ production. We also show that AhR-mediated increases in IFN-γ are dependent upon iNOS, but elevated iNOS in lung epithelial cells is not driven by AhR-dependent signals from bone marrow-derived cells. Thus, the lung contains important targets of AhR regulation, which likely influence a novel iNOS-mediated mechanism that controls IFN-γ production by phagocytic cells. This suggests that AhR activation changes the response of lung parenchymal cells, such that regulatory pathways in the lung are cued to respond inappropriately during infection. These findings also imply that environmental factors may contribute to differential susceptibility to influenza virus and other respiratory pathogens.
The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway
Authors: Marcet-Palacios Marcelo , Ulanova Marina , Duta Florentina , Puttagunta Lakshmi , Munoz Samira , Gibbings Derrick , Radomski Marek , Cameron Lisa , Mayers Irvin , Befus A. Dean
Published: April 2014

Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5′ flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-γ resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.
Cutting Edge: Autoantigen Ro52 Is an Interferon Inducible E3 Ligase That Ubiquitinates IRF-8 and Enhances Cytokine Expression in Macrophages
Authors: Kong Hee Jeong , Anderson D. Eric , Lee Chang Hoon , Jang Moon Kyoo , Tamura Tomohiko , Tailor Prafullakumar , Cho Hyun Kook , Cheong JaeHun , Xiong Huabao , Morse Herbert C. , Ozato Keiko
Published: April 2014

IFN regulatory factor (IRF)-8 is a transcription factor important for the development and function of macrophages. It plays a critical role in the induction of cytokine genes, including IL-12p40. Immunopurification and mass spectrometry analysis found that IRF-8 interacted with Ro52 in murine macrophages upon IFN-γ and TLR stimulation. Ro52 is an IFN-inducible protein of the tripartite motif (TRIM) family and is an autoantigen present in patients with Sjögren’s syndrome and systemic lupus erythematosus. Ro52 has a RING motif and is capable of ubiquitinating itself. We show that IRF-8 is ubiquitinated by Ro52 both in vivo and in vitro. Ectopic expression of Ro52 enhanced IL-12p40 expression in IFN-γ/TLR-stimulated macrophages in an IRF-8-dependent manner. Together, Ro52 is an E3 ligase for IRF-8 that acts in a non-degradation pathway of ubiquitination, and contributes to the elicitation of innate immunity in macrophages.
Receptor Activator of NF-κB Ligand Inhibition Suppresses Bone Resorption and Hypercalcemia but Does Not Affect Host Immune Responses to Influenza Infection
Authors: Miller Robert E. , Branstetter Daniel , Armstrong Allison , Kennedy Bryan , Jones Jon , Cowan Laine , Bussiere Jeanine , Dougall William C.
Published: April 2014

Receptor activator of NF-κB (RANK) and its ligand (RANKL) are essential for osteoclast formation, function, and survival. Osteoprotegerin (OPG) inhibits RANK signaling by sequestering RANKL. This study evaluated the antiosteoclast and immunoregulatory effects of mouse rRANK-Fc, which, similar to OPG, can bind RANKL. The effect of RANKL inhibition by RANK-Fc on osteoclast function was determined by inhibition of vitamin D3 (1,25(OH)2D3)-induced hypercalcemia. Mice were injected with a single dose of 0, 10, 100, 500, or 1000 μg of RANK-Fc; 100 μg of OPG-Fc; or 5 μg of zoledronate 2 h before 1,25(OH)2D3 challenge on day 0, and sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20. RANK-Fc doses of 100 or 500 μg were tested in a mouse respiratory influenza virus host-resistance model. A single dose of RANK-Fc ≥100 μg suppressed elevation of serum calcium levels and suppressed the bone turnover marker serum pyridinoline at day 4 and later time points, similar to those observed with OPG-Fc and zoledronate (p ≤ 0.01 vs controls). By day 6, both immature and mature osteoclasts were depleted by high doses of RANK-Fc (500 and 1000 μg) or 100 μg of OPG-Fc. RANK-Fc doses of 100 or 500 μg had no detectable effect on immune responses to influenza infection, as measured by activation of cytotoxic T cell activity, influenza-specific IgG response, and virus clearance. RANK-Fc inhibition of RANKL has antiosteoclast activity at doses that have no detectable immunoregulatory activity, suggesting that RANKL inhibitors be further studied for their potential to treat excess bone loss.
A Key Role for Poly(ADP-Ribose) Polymerase-1 Activity during Human Dendritic Cell Maturation
Authors: Aldinucci Alessandra , Gerlini Gianni , Fossati Silvia , Cipriani Giulia , Ballerini Clara , Biagioli Tiziana , Pimpinelli Nicola , Borgognoni Lorenzo , Massacesi Luca , Moroni Flavio , Chiarugi Alberto
Published: April 2014

Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-κB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-κB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.
Psychological Stress Compromises CD8+ T Cell Control of Latent Herpes Simplex Virus Type 1 Infections
Authors: Freeman Michael L. , Sheridan Brian S. , Bonneau Robert H. , Hendricks Robert L.
Published: April 2014

Recurrent HSV-1 ocular disease results from reactivation of latent virus in trigeminal ganglia, often following immunosuppression or exposure to a variety of psychological or physical stressors. HSV-specific CD8+ T cells can block HSV-1 reactivation from latency in ex vivo trigeminal ganglia cultures through production of IFN-γ. In this study, we establish that either CD8+ T cell depletion or exposure to restraint stress permit HSV-1 to transiently escape from latency in vivo. Restraint stress caused a reduction of TG-resident HSV-specific CD8+ T cells and a functional compromise of those cells that survive. Together, these effects of stress resulted in an approximate 65% reduction of cells capable of producing IFN-γ in response to reactivating virus. Our findings demonstrate persistent in vivo regulation of latent HSV-1 by CD8+ T cells, and strongly support the concept that stress induces HSV-1 reactivation from latency at least in part by compromising CD8+ T cell surveillance of latently infected neurons.
Potential of Transfected Muscle Cells to Contribute to DNA Vaccine Immunogenicity
Authors: Shirota Hidekazu , Petrenko Lev , Hong Choongman , Klinman Dennis M.
Published: April 2014

The mechanism(s) by which DNA vaccines trigger the activation of Ag-specific T cells is incompletely understood. A series of in vivo and in vitro experiments indicates plasmid transfection stimulates muscle cells to up-regulate expression of MHC class I and costimulatory molecules and to produce multiple cytokines and chemokines. Transfected muscle cells gain the ability to directly present Ag to CD8 T cells through an IFN-regulatory factor 3-dependent process. These findings suggest that transfected muscle cells at the site of DNA vaccination may contribute to the magnitude and/or duration of the immune response initiated by professional APCs.
Intimate Cell Conjugate Formation and Exchange of Membrane Lipids Precede Apoptosis Induction in Target Cells during Antibody-Dependent, Granulocyte-Mediated Cytotoxicity
Authors: Horner Heike , Frank Carola , Dechant Claudia , Repp Roland , Glennie Martin , Herrmann Martin , Stockmeyer Bernhard
Published: April 2014

Ab-dependent polymorphonuclear granulocyte (PMN)-mediated cytotoxicity may play an important role in the control of malignant diseases. However, little is known as to which particular pathways are used for the killing of malignant cells by PMN. The production of reactive oxygen intermediates (ROI) has been observed to occur during Ab-dependent, cell-mediated cytotoxicity (ADCC). However, PMN from a patient with chronic granulomatous disease demonstrated strong ADCC against malignant lymphoma cells. Furthermore, the inhibition of ROI production in PMN from healthy donors had no significant effect on ADCC. Therefore, ROI production by the NADPH oxidase of PMN does not appear to be mandatory for PMN-mediated ADCC. Recent data suggest a role for perforins in PMN-mediated cytotoxicity. However, in our assays concanamycin A, an inhibitor of perforin-mediated ADCC by mononuclear cells, had no inhibitory effect on PMN-mediated ADCC. Using electron microscopy we observed that PMN and their target cells intimately interact with the formation of interdigitating membrane protrusions. During PMN and target cell contact there was a mutual exchange of fluorescent membrane lipid dyes that was strongly increased in the presence of tumor-targeting Abs. This observation may be closely related to the recently described process of trogocytosis by lymphocytes. The presence of transient PMN-tumor cell aggregates and the accumulation of PMN with tumor cell-derived membrane lipids and vice versa were associated with effective ADCC as measured by chromium-release or apoptosis induction.
Abrogation of Prostaglandin E2/EP4 Signaling Impairs the Development of rag1+ Lymphoid Precursors in the Thymus of Zebrafish Embryos
Authors: Villablanca Eduardo J. , Pistocchi Anna , Court Felipe A. , Cotelli Franco , Bordignon Claudio , Allende Miguel L. , Traversari Catia , Russo Vincenzo
Published: April 2014

PGE2 is involved in a wide variety of physiological and pathological processes; however, deciphering its role in early mammalian development has been difficult due to the maternal contribution of PGE2. To overcome this limitation we have investigated the role of PGE2 during T cell development in zebrafish. In this study, we show that zebrafish ep4a, a PGE2 receptor isoform of EP4, is expressed at 26 h postfertilization in the dorsal aorta-posterior cardinal vein joint region, which has a high homology with the mammal aorta-gonad-mesonephros area and where definitive hemopoiesis arises. Furthermore, it is expressed in the presumptive thymus rudiment by 48 h postfertilization. Supplementation of PGE2 results in a strong increase in rag1 levels and cell proliferation in the thymus. In contrast, the inhibition of PGE2 production, as well as EP4 blockade, abrogates the expression of rag1 in the thymus and that of the lymphoid precursor marker ikaros, not only in the dorsal aorta-posterior cardinal vein joint region but also in the newly identified caudal hemopoietic tissue without affecting early hemopoietic (scl, gata2) and erythropoietic (gata1) markers. These results identify ep4a as the earliest thymus marker and define a novel role for the PGE2/EP4 pathway in controlling T cell precursor development in zebrafish.
IL-4-STAT6 Signal Transduction-Dependent Induction of the Clinical Phase of Sjögren’s Syndrome-Like Disease of the Nonobese Diabetic Mouse
Authors: Nguyen Cuong Q. , Gao Jue-hua , Kim Hyuna , Saban Daniel R. , Cornelius Janet G. , Peck Ammon B.
Published: April 2014

NOD.B10-H2b and NOD/LtJ mice manifest, respectively, many features of primary and secondary Sjögren’s syndrome (SjS), an autoimmune disease affecting primarily the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). B lymphocytes play a central role in the onset of SjS with clinical manifestations dependent on the appearance of autoantibodies reactive to multiple components of acinar cells. Previous studies with NOD.IL4−/− and NOD.B10-H2b.IL4−/− mice suggest that the Th2 cytokine, IL-4, plays a vital role in the development and onset of SjS-like disease in the NOD mouse model. To investigate the molecular mechanisms by which IL-4 controls SjS development, a Stat6 gene knockout mouse, NOD.B10-H2b.C-Stat6−/−, was constructed and its disease profile was defined and compared with that of NOD.B10-H2b.C-Stat6+/+ mice. As the NOD.B10-H2b.C-Stat6−/− mice aged from 4 to 24 wk, they exhibited leukocyte infiltration of the exocrine glands, produced anti-nuclear autoantibodies, and showed loss and gain of saliva-associated proteolytic enzymes, similar to NOD.B10-H2b.C-Stat6+/+ mice. In contrast, NOD.B10-H2b.C-Stat6−/− mice failed to develop glandular dysfunction, maintaining normal saliva flow rates. NOD.B10-H2b.C-Stat6−/− mice were found to lack IgG1 isotype-specific anti-muscarinic acetylcholine type-3 receptor autoantibodies. Furthermore, the IgG fractions from NOD.B10-H2b.C-Stat6−/− sera were unable to induce glandular dysfunction when injected into naive recipient C57BL/6 mice. NOD.B10-H2b.C-Stat6−/− mice, like NOD.B10-H2b.IL4−/− mice, are unable to synthesize IgG1 Abs, an observation that correlates with an inability to develop end-stage clinical SjS-like disease. These data imply a requirement for the IL-4/STAT6-pathway for onset of the clinical phase of SjS-like disease in the NOD mouse model.
Sequential Activation of CD8+ T Cells in the Draining Lymph Nodes in Response to Pulmonary Virus Infection
Authors: Yoon Heesik , Legge Kevin L. , Sung Sun-sang J. , Braciale Thomas J.
Published: April 2014

We have used a TCR-transgenic CD8+ T cell adoptive transfer model to examine the tempo of T cell activation and proliferation in the draining lymph nodes (DLN) in response to respiratory virus infection. The T cell response in the DLN differed for mice infected with different type A influenza strains with the onset of T cell activation/proliferation to the A/JAPAN virus infection preceding the A/PR8 response by 12–24 h. This difference in T cell activation/proliferation correlated with the tempo of accelerated respiratory DC (RDC) migration from the infected lungs to the DLN in response to influenza virus infection, with the migrant RDC responding to the A/JAPAN infection exhibiting a more rapid accumulation in the lymph nodes (i.e., peak migration for A/JAPAN at 18 h, A/PR8 at 24–36 h). Furthermore, in vivo administration of blocking anti-CD62L Ab at various time points before/after infection revealed that the virus-specific CD8+ T cells entered the DLN and activated in a sequential “conveyor belt”-like fashion. These results indicate that the tempo of CD8+ T cell activation/proliferation after viral infection is dependent on the tempo of RDC migration to the DLN and that T cell activation occurs in an ordered sequential fashion.
Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains
Authors: Chentouf Myriam , Ghannam Soufiane , Bès Cédric , Troadec Samuel , Cérutti Martine , Chardès Thierry
Published: April 2014

The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-κB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3ζ, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Cθ, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cγ1, and p95vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10–30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-κB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects.
Impaired Trafficking of Gnai2+/− and Gnai2−/− T Lymphocytes: Implications for T Cell Movement within Lymph Nodes
Authors: Hwang Il-Young , Park Chung , Kehrl John H.
Published: April 2014

Signals generated by the engagement of chemoattractants with their cognate receptors orchestrate lymphocyte movements into and out of lymphoid organs and sites of inflammation. Yet, the role of chemokines in organizing lymphocyte movements in lymphoid organs is controversial. Recent evidence suggests that the extensive network of fibroblastic reticular cells within the T cell areas helps guide T cells. The expression of adhesion molecules and chemokines by fibroblastic reticular cells most likely facilitates their influence on T cell movements. Consistent with this hypothesis, CD4 T cells with defective chemokine receptor signaling move very differently within lymph nodes than do normal cells. For the imaging studies, we used CD4 T cells prepared from Gnai2−/− mice, which lack Gαi2 expression. We first demonstrate that CD4 as well as CD8 T cells from these mice are markedly defective in chemokine receptor signaling. Gnai2−/− T cells have profound defects in chemokine-induced intracellular calcium mobilization, chemotaxis, and homing, whereas Gnai2+/− T cells exhibit modest defects. Intravital imaging revealed that within the inguinal lymph nodes Gnai2−/− CD4 T accumulate at the cortical ridge, poorly accessing the lymph node paracortex. They also lack the customary amoeboid-like cell movements and active membrane projections observed with normal CD4 T cells. These results demonstrate the importance of Gαi2 for T lymphocyte chemokine receptor signaling and argue that local chemoattractants regulate the movement of CD4 T cells in lymph nodes.
Developmental Stage-Specific Regulation of TCR-α-Chain Gene Assembly by Intrinsic Features of the TEA Promoter
Authors: Huang Ching-Yu , Sleckman Barry P.
Published: April 2014

The TCR δ- and α-chain genes lie in a single complex locus, the TCRα/δ locus. TCRδ-chain genes are assembled in CD4−CD8− (double negative (DN)) thymocytes and TCRα-chain genes are assembled in CD4+CD8+ (double positive) thymocytes due, in part, to the developmental stage-specific activities of the TCRδ and TCRα enhancers (Eδ and Eα), respectively. Eδ functions with TCRδ promoters to mediate TCRδ-chain gene assembly in DN thymocytes. However, Eδ is unable to function with TCRα promoters such as the TEA promoter to drive TCRα-chain gene assembly in these cells. This is important, because the premature assembly of TCRα-chain genes in DN thymocytes would disrupt αβ and γδ T cell development. The basis for TEA inactivity in DN thymocytes is unclear, because Eδ can activate the Vδ5 gene segment promoter that lies only 4 kb upstream of TEA promoter. In this study, we use gene targeting to construct a modified TCRα/δ locus (TCRα/δ5ΔT) in which the TEA promoter lies in the same location as the Vδ5 gene segment on the wild-type TCRα/δ allele. Remarkably, the TEA promoter on this allele exhibits normal developmental stage-specific regulation, being active in double positive thymocytes but not in DN thymocytes as is the case with the Vδ5 promoter. Thus, the inactivity of the TEA promoter in DN thymocytes is due primarily to intrinsic developmental stage-specific features of the promoter itself and not to its location relative to other cis-acting elements in the locus, such as Eδ.
Peptide Vaccines of the HER-2/neu Dimerization Loop Are Effective in Inhibiting Mammary Tumor Growth In Vivo
Authors: Allen Stephanie D. , Garrett Joan T. , Rawale Sharad V. , Jones Audra L. , Phillips Gary , Forni Guido , Morris John C. , Oshima Robert G. , Kaumaya Pravin T. P.
Published: April 2014

Human epidermal growth factor receptor-2 (HER-2)/neu (ErbB2), a member of the epidermal growth factor family of receptors, is overexpressed in 20–30% of breast cancers. It is an attractive target for receptor-directed antitumor therapy using mAbs. Unlike other epidermal growth factor receptor family members, HER-2/neu does not bind a high-affinity ligand, but rather functions as the preferred dimerization partner. Pertuzumab (Omnitarg) is a humanized mAb directed against the HER-2/neu dimerization domain that inhibits receptor signaling. The recent definition of the crystal structure of the HER-2/neu-pertuzumab complex demonstrated that the receptor dimerization region encompassed residues 266–333. Based on the three-dimensional structure of the complex, we have designed three conformational peptide constructs (sequences 266–296, 298–333, and 315–333) to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo antitumor efficacy. All the constructs elicited high-affinity peptide Abs that inhibited multiple signaling pathways including HER-2/neu-specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. All the peptide Abs showed Ab-dependent cellular cytotoxicity to varying degrees with the 266–296 constructs being equally effective as compared with Herceptin. The 266–296 peptide vaccine had statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF+/−Neu2–5+/−). The 266–296 construct represents the most promising candidate for antitumor vaccination and could also be used to treat a variety of cancers with either normal or elevated expression of HER-2 including breast, lung, ovarian, and prostate.
Nod1/RICK and TLR Signaling Regulate Chemokine and Antimicrobial Innate Immune Responses in Mesothelial Cells
Authors: Park Jong-Hwan , Kim Yun-Gi , Shaw Michael , Kanneganti Thirumala-Devi , Fujimoto Yukari , Fukase Koichi , Inohara Naohiro , Núñez Gabriel
Published: April 2014

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-γ and cooperated with IFN-γ in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.
The Hypervirulent Mycobacterium tuberculosis Strain HN878 Induces a Potent TH1 Response followed by Rapid Down-Regulation
Authors: Ordway Diane , Henao-Tamayo Marcela , Harton Marisa , Palanisamy Gopinath , Troudt Jolynn , Shanley Crystal , Basaraba Randall J. , Orme Ian M.
Published: April 2014

The HN878 strain of Mycobacterium tuberculosis is regarded as “hypervirulent” due to its rapid growth and reduced survival of infected mice when compared with other clinical isolates. This property has been ascribed due to an early increase in type I IFNs and a failure to generate TH1-mediated immunity, induced by a response to an unusual cell wall phenolic glycolipid expressed by the HN878 isolate. We show, however, that although type I IFN does play an inhibitory role, this response was most apparent during the chronic disease stage and was common to all M. tuberculosis strains tested. In addition, we further demonstrate that the HN878 infection was associated with a potent TH1 response, characterized by the emergence of both CD4 and CD8 T cell subsets secreting IFN-γ. However, where HN878 differed to the other strains tested was a subsequent reduction in TH1 immunity, which was temporally associated with the rapid emergence of a CD4+CD25+FoxP3+CD223+IL-10+ regulatory T cell population. This association may explain the paradoxical initial emergence of a TH1 response in these mice but their relatively short time of survival.
Prophylactic and Therapeutic Use of Antibodies for Protection against Respiratory Infection with Francisella tularensis
Authors: Kirimanjeswara Girish S. , Golden Jacqueline M. , Bakshi Chandra Shekhar , Metzger Dennis W.
Published: April 2014

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcγR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-γ. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-γ. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-γ. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24–48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.
Genome-Wide Expression Analysis of Placental Malaria Reveals Features of Lymphoid Neogenesis during Chronic Infection
Authors: Muehlenbachs Atis , Fried Michal , Lachowitzer Jeff , Mutabingwa Theonest K. , Duffy Patrick E.
Published: April 2014

Chronic inflammation during placental malaria (PM) is most frequent in first time mothers and is associated with poor maternal and fetal outcomes. In the first genome-wide analysis of the local human response to sequestered malaria parasites, we identified genes associated with chronic PM and then localized the corresponding proteins and immune cell subsets in placental cryosections. B cell-related genes were among the most highly up-regulated transcripts in inflamed tissue. The B cell chemoattractant CXCL13 was up-regulated >1,000-fold, and B cell-activating factor was also detected. Both proteins were expressed by intervillous macrophages. Ig L and H chain transcription increased significantly, and heavy depositions of IgG3 and IgM were observed in intervillous spaces. The B cell phenotype was heterogenous, including naive (CD27-negative), mature (CD138-positive), and cycling (Ki-67-positive) cells. B cells expressed T-bet but not Bcl-6, suggesting T cell-independent activation without germinal center formation. Genes for the Fc binding proteins FcγRIa, FcγRIIIa, and C1q were highly up-regulated, and the proteins localized to intervillous macrophages. Birth weight was inversely correlated with transcript levels of CXCL13, IgG H chain, and IgM H chain. The iron regulatory peptide hepcidin was also expressed but was not associated with maternal anemia. The results suggest that B cells and macrophages contribute to chronic PM in a process resembling lymphoid neogenesis. We propose a model where the production of Ig during chronic malaria may enhance inflammation by attracting and activating macrophages that, in turn, recruit B cells to further produce Ig in the intervillous spaces.