bluuebunny/crossref_metadata_2025 huggingface dataset:
TNF Influences Chemokine Expression of Macrophages In Vitro and That of CD11b+ Cells In Vivo during Mycobacterium tuberculosis Infection
Authors: Algood Holly M. Scott , Lin Philana Ling , Yankura David , Jones Alvin , Chan John , Flynn JoAnne L.
Published: April 2014

Granulomas, focal accumulations of immune cells, form in the lung during Mycobacterium tuberculosis infection. Chemokines, chemotactic cytokines, are logical candidates for inducing migration of T lymphocytes and monocytes to and within the lung. TNF influences chemokine expression in some models. TNF-deficient mice infected with M. tuberculosis are highly susceptible to disease, and granuloma formation is inhibited. Through in vitro assays, we demonstrate that neutralization of TNF in M. tuberculosis-infected macrophages led to a reduction in many inflammatory chemokines, such as C-C chemokine ligand 5, CXC ligand 9 (CXCL9), and CXCL10. In TNF-deficient mice, immune cells migrated to the lungs early after infection, but did not organize to form granulomas within the lung. Although chemokine expression, as measured in whole lung tissue, was not different, the expression of chemokines in the CD11b+ subset of cells isolated ex vivo from the lungs of TNF-deficient mice had reduced expression of C-C chemokine ligand 5, CXCL9, and CXCL10 at early time points after TNF neutralization. Local expression of CXCR3-binding chemokines within the lungs, as determined by in situ hybridization, was also affected by TNF. Therefore, TNF affects the expression of chemokines by macrophages in vitro and CD11b+ cells in vivo, which probably influences the local chemokine gradients and granuloma formation.
Bacterial Ghosts Are an Efficient Delivery System for DNA Vaccines
Authors: Ebensen Thomas , Paukner Susanne , Link Claudia , Kudela Pavol , de Domenico Carola , Lubitz Werner , Guzmán Carlos A.
Published: April 2014

Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52–60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for β-galactosidase under the control of the CMV immediate early gene promoter (pCMVβ) stimulates more efficient Ag-specific humoral and cellular (CD4+ and CD8+) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVβ-containing ghosts also resulted in the elicitation of β-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.
Murine Cytomegalovirus Interference with Antigen Presentation Has Little Effect on the Size or the Effector Memory Phenotype of the CD8 T Cell Response
Authors: Gold Marielle C. , Munks Michael W. , Wagner Markus , McMahon Christopher W. , Kelly Ann , Kavanagh Daniel G. , Slifka Mark K. , Koszinowski Ulrich H. , Raulet David H. , Hill Ann B.
Published: April 2014

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV’s three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8lowCD62L−CD11c+NKG2A+) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Δm4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Δm4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.
C3a Enhances Nerve Growth Factor-Induced NFAT Activation and Chemokine Production in a Human Mast Cell Line, HMC-1
Authors: Ahamed Jasimuddin , Venkatesha Rampura T. , Thangam E. Berla , Ali Hydar
Published: April 2014

Activation of cell surface G protein-coupled receptors leads to transphosphorylation and activation of a number of receptor tyrosine kinases. Human mast cells express G protein-coupled receptors for the complement component C3a (C3aR) and high affinity nerve growth factor (NGF) receptor tyrosine kinase, TrkA. To determine whether C3a cross-regulates TrkA signaling and biological responses, we used a human mast cell-line, HMC-1, that natively expresses both receptors. We found that NGF caused tyrosine phosphorylation of TrkA, resulting in a sustained Ca2+ mobilization, NFAT activation, extracellular-signal regulated kinase (ERK) phosphorylation, and chemokine, macrophage inflammatory protein-1β (MIP-1β) production. In contrast, C3a induced a transient Ca2+ mobilization and ERK phosphorylation but failed to stimulate TrkA phosphorylation, NFAT activation, or MIP-1β production. Surprisingly, C3a significantly enhanced NGF-induced NFAT activation, ERK phosphorylation, and MIP-1β production. Pertussis toxin, a Gi/o inhibitor, selectively blocked priming by C3a but had no effect on NGF-induced responses. Mitogen-activated protein/ERK kinase inhibitor U0126 caused ∼30% inhibition of NGF-induced MIP-1β production but had no effect on priming by C3a. However, cyclosporin A, an inhibitor of calcineurin-mediated NFAT activation, caused substantial inhibition of NGF-induced MIP-1β production both in the absence and presence of C3a. These data demonstrate that NGF caused tyrosine phosphorylation of TrkA to induce chemokine production in HMC-1 cells via a pathway that mainly depends on sustained Ca2+ mobilization and NFAT activation. Furthermore, C3a enhances NGF-induced transcription factor activation and chemokine production via a G protein-mediated pathway that does not involve TrkA phosphorylation.
Volume-Sensitive Chloride Channels Do Not Mediate Activation-Induced Chloride Efflux in Human Neutrophils
Authors: Perez-Cornejo Patricia , Arreola Jorge , Law Foon-Yee , Schultz Joanne B. , Knauf Philip A.
Published: April 2014

Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl− efflux. This Cl− efflux and the increase in the number and affinity of β2 integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl− channels (IClvol), suggesting that IClvol mediates the activator-induced Cl− efflux. To test this hypothesis, we used whole-cell patch clamp in hypotonic media to examine the effects of inhibitors of up-regulation on IClvol in neutrophils and promyelocytic leukemic HL-60 cells. Both the channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid and [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating oxonol, inhibited IClvol at concentrations similar to those that inhibit β2 integrin up-regulation. However, ethacrynic acid, at the same concentration that inhibits activator-induced Cl− efflux and up-regulation, had no effect on IClvol and swelling-activated Cl− efflux, providing evidence against the involvement of IClvol in the activator-induced Cl− efflux.
Role of Calcium in Pancreatic Islet Cell Death by IFN-γ/TNF-α
Authors: Chang Inik , Cho Namjoo , Kim Sunshin , Kim Ja Young , Kim Eunshil , Woo Ji-Eun , Nam Joo Hyun , Kim Sung Joon , Lee Myung-Shik
Published: April 2014

We studied the intracellular events associated with pancreatic β cell apoptosis by IFN-γ/TNF-α synergism. IFN-γ/TNF-α treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca2+ currents, while treatment with IFN-γ or TNF-α alone did not. Cytosolic Ca2+ concentration ([Ca2+]c) was also increased by IFN-γ/TNF-α treatment. Blockade of L-type Ca2+ channel by nifedipine abrogated death of insulinoma cells by IFN-γ/TNF-α. Diazoxide that attenuates voltage-activated Ca2+ currents inhibited MIN6N8 cell death by IFN-γ/TNF-α, while glibenclamide that accentuates voltage-activated Ca2+ currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca2+]c by IFN-γ/TNF-α. Following the increase in [Ca2+]c, calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-γ/TNF-α. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-γ/TNF-α. BAD phosphorylation was decreased by IFN-γ/TNF-α because of the increased calcineurin activity, which was reversed by FK506. IFN-γ/TNF-α induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-γ/TNF-α treatment. These results indicate that IFN-γ/TNF-α synergism induces pancreatic β cell apoptosis by Ca2+ channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca2+ modulation in type 1 diabetes.
Distinct Transcriptional Programs Activated by Interleukin-10 with or without Lipopolysaccharide in Dendritic Cells: Induction of the B Cell-Activating Chemokine, CXC Chemokine Ligand 13
Authors: Perrier Patrick , Martinez Fernando O. , Locati Massimo , Bianchi Giancarlo , Nebuloni Manuela , Vago Gianluca , Bazzoni Flavia , Sozzani Silvano , Allavena Paola , Mantovani Alberto
Published: April 2014

To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Affymetrix technology (Santa Clara, CA) in human monocyte-derived DC treated with IL-10, alone or in combination with LPS. The modulation of selected genes was validated by real-time PCR, Northern blot, and protein production. IL-10 regulated in DC the expression of a limited number of genes, including IL-7, the receptors for transferrin and vitamin D3, structural matrix proteins, and signal transduction elements. The combined treatment with LPS plus IL-10 modulated a number of genes comparable to LPS alone, but the expression profiles were distinct. As expected, IL-10 suppressed the expression of several LPS-inducible proinflammatory molecules. Among genes uniquely modulated by the concomitant treatment with LPS plus IL-10, phosphatidylinositol 3-kinase γ was down-regulated while the suppressor of cytokine signaling 3, signaling lymphocytic activation molecule, regulator of G protein signaling 16, and the chemokine, CXC chemokine ligand (CXCL) 13, were up-regulated. Overall, four distinct transcriptional programs were identified, related to: 1) control of immunity and inflammation; 2) tuning of cytokine receptor and G protein-coupled receptor signaling; 3) remodeling of extracellular matrix; and 4) B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrate that IL-10 synergizes with TLR ligands for the production of functionally active B cell-attracting chemokine, CXCL13, in both myeloid and plasmacytoid DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production in APCs of the chemokine, CXCL13, which amplifies B cell recruitment and promotes lymphoid tissue neogenesis.
Type 2 Monocyte and Microglia Differentiation Mediated by Glatiramer Acetate Therapy in Patients with Multiple Sclerosis
Authors: Kim Ho Jin , Ifergan Igal , Antel Jack P. , Seguin Rosanne , Duddy Martin , Lapierre Yves , Jalili Farzaneh , Bar-Or Amit
Published: April 2014

Glatiramer acetate (GA) therapy of patients with multiple sclerosis (MS) represents a unique setting in which in vivo Th2 deviation of T cells is consistently observed and associated with clinical benefit in a human autoimmune disease. We postulated that APCs are important targets of GA therapy and demonstrate that treatment of MS patients with GA reciprocally regulates the IL-10/IL-12 cytokine network of monocytes in vivo. We further show that Th1- or Th2-polarized GA-reactive T cells isolated from untreated or treated MS patients mediate type 1 and 2 APC differentiation of human monocytes, based on their ability to efficiently induce subsequent Th1 and Th2 deviation of naive T cells, respectively. These observations are extended to human microglia, providing the first demonstration of type 2 differentiation of CNS-derived APCs. Finally, we confirm that the fundamental capacity of polarized T cells to reciprocally modulate APC function is not restricted to GA-reactive T cells, thereby defining a novel and dynamic positive feedback loop between human T cell and APC responses. In the context of MS, we propose that GA therapy results in the generation of type 2 APCs, contributing to Th2 deviation both in the periphery and in the CNS of MS patients. In addition to extending insights into the therapeutic mode of action of GA, our findings revisit the concept of bystander suppression and underscore the potential of APCs as attractive targets for therapeutic immune modulation.
T Cell Ig- and Mucin-Domain-Containing Molecule-3 (TIM-3) and TIM-1 Molecules Are Differentially Expressed on Human Th1 and Th2 Cells and in Cerebrospinal Fluid-Derived Mononuclear Cells in Multiple Sclerosis
Authors: Khademi Mohsen , Illés Zsolt , Gielen Alexander W. , Marta Monica , Takazawa Naruhiko , Baecher-Allan Claire , Brundin Lou , Hannerz Jan , Martin Claes , Harris Robert A. , Hafler David A. , Kuchroo Vijay K. , Olsson Tomas , Piehl Fredrik , Wallström Erik
Published: April 2014

T cell Ig- and mucin-domain-containing molecules (TIMs) comprise a recently described family of molecules expressed on T cells. TIM-3 has been shown to be expressed on murine Th1 cell clones and has been implicated in the pathogenesis of Th1-driven experimental autoimmune encephalomyelitis. In contrast, association of TIM-1 polymorphisms to Th2-related airway hyperreactivity has been suggested in mice. The TIM molecules have not been investigated in human Th1- or Th2-mediated diseases. Using real-time (TaqMan) RT-PCR, we show that human Th1 lines expressed higher TIM-3 mRNA levels, while Th2 lines demonstrated a higher expression of TIM-1. Analysis of cerebrospinal fluid mononuclear cells obtained from patients with multiple sclerosis revealed significantly higher mRNA expression of TIM-1 compared with controls. Moreover, higher TIM-1 expression was associated with clinical remissions and low expression of IFN-γ mRNA in cerebrospinal fluid mononuclear cells. In contrast, expression of TIM-3 correlated well with high expression of IFN-γ and TNF-α. These data imply the differential expression of human TIM molecules by Th1 and Th2 cells and may suggest their differential involvement in different phases of a human autoimmune disease.
In a Transgenic Model of Spontaneous Autoimmune Diabetes, Expression of a Protective Class II MHC Molecule Results in Thymic Deletion of Diabetogenic CD8+ T Cells
Authors: Morgan David J. , Nugent C. Thomas , Raveney Benjamin J. E. , Sherman Linda A.
Published: April 2014

H-2d mice expressing both the influenza virus hemagglutinin (HA) as a transgene-encoded protein on pancreatic islet β cells (InsHA), as well as the Clone 4 TCR specific for the dominant H-2Kd-restricted HA epitope, can be protected from the development of spontaneous autoimmune diabetes by expression of the H-2b haplotype. Protection occurs due to the deletion of KdHA-specific CD8+ T cells. This was unexpected as neither the presence of the InsHA transgene nor H-2b, individually, resulted in thymic deletion. Further analyses revealed that thymic deletion required both a hybrid MHC class II molecule, Eβb Eαd, and the Kd molecule presenting the HA epitope, which together synergize to effect deletion of CD4+CD8+ thymocytes. This surprising example of protection from autoimmunity that maps to a class II MHC molecule, yet effects an alteration in the CD8+ T cell repertoire, suggests that selective events in the thymus represent the integrated strength of signal delivered to each cell through recognition of a variety of different MHC-peptide ligands.
Liver Dendritic Cells Are Less Immunogenic Than Spleen Dendritic Cells because of Differences in Subtype Composition
Authors: Pillarisetty Venu G. , Shah Alaap B. , Miller George , Bleier Joshua I. , DeMatteo Ronald P.
Published: April 2014

The unique immunological properties of the liver may be due to the function of hepatic dendritic cells (DC). However, liver DC have not been well characterized because of the difficulty in isolating adequate numbers of cells for analysis. Using immunomagnetic bead and flow cytometric cell sorting, we compared freshly isolated murine liver and spleen CD11c+ DC. We found that liver DC are less mature, capture less Ag, and induce less T cell stimulation than spleen DC. Nevertheless, liver DC were able to generate high levels of IL-12 in response to CpG stimulation. We identified four distinct subtypes of liver DC based on the widely used DC subset markers CD8α and CD11b. Lymphoid (CD8α+CD11b−) and myeloid (CD8α−CD11b+) liver DC activated T cells to a similar degree as did their splenic DC counterparts but comprised only 20% of all liver DC. In contrast, the two more prevalent liver DC subsets were only weakly immunostimulatory. Plasmacytoid DC (B220+) accounted for 19% of liver DC, but only 5% of spleen DC. Our findings support the widely held notion that liver DC are generally weak activators of immunity, although they are capable of producing inflammatory cytokines, and certain subtypes potently activate T cells.
IL-10 Induces Regulatory T Cell Apoptosis by Up-Regulation of the Membrane Form of TNF-α
Authors: Marra Luciano E. , Zhang Zhu X. , Joe Betty , Campbell Jon , Levy Gary A. , Penninger Josef , Zhang Li
Published: April 2014

Numerous studies have demonstrated the role of regulatory T (Treg) cells in peripheral tolerance. Nevertheless, how the survival and death of Treg cells is controlled is largely unknown. In this study, we investigated the mechanisms involved in regulating the homeostasis of a subset of Ag-specific αβTCR+ CD4−CD8− double negative (DN) Treg cells. We demonstrate that DN Treg cells are naturally resistant to TCR cross-linking-induced apoptosis. Administration of exogenous IL-10 renders DN Treg cells susceptible to apoptosis, and abolishes their suppressive function. Furthermore, TCR cross-linking of DN Treg cells in the presence of IL-10 leads to the up-regulation of the membrane-bound but not the soluble form of TNF-α. Interaction of membrane bound TNF-α with TNFR2 sends death signals to DN Treg cells. Blocking their interaction can reverse the effects of IL-10 on DN Treg cells. These results provide insights into the mechanisms that regulate the function and homeostasis of DN Treg cells.
Allelic Variation at theVHaLocus in Natural Populations of Rabbit (Oryctolagus cuniculus, L.)
Authors: Esteves Pedro J. , Lanning Dennis , Ferrand Nuno , Knight Katherine L. , Zhai Shi-Kang , van der Loo Wessel
Published: April 2014

The large interallelic distances between the three rabbit Ig VHa lineages, a1, a2 and a3, suggest that the persistence time of the VHa polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the VH loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with VHa locus-specific alloantisera, showed either cross-reactivity (“a-positive” variants) or no reaction at all (“a-blank”). Using RT-PCR, we determined 120 sequences of rearranged VH genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the VH genes transcribed in a-positive rabbits are closely related to the VH1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use VH genes that, although clearly related to the known VHa genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit VHa residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the VHa4 and the VHa1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit VHa locus exist, one of which seems to be endemic in the Iberian range.
Genomic Ly49A Transgenes: Basis of Variegated Ly49A Gene Expression and Identification of a Critical Regulatory Element
Authors: Tanamachi Dawn M. , Moniot Deborah C. , Cado Dragana , Liu Scot D. , Hsia Jennifer K. , Raulet David H.
Published: April 2014

Several gene families are known in which member genes are expressed in variegated patterns in differentiated cell types. Mechanisms responsible for imposition of a variegated pattern of gene expression are unknown. Members of the closely linked Ly49 inhibitory receptor gene family are expressed in a variegated fashion by NK cells. Variegated expression of these genes results in subsets of NK cells that differ in specificity for MHC class I molecules. To address the mechanisms underlying variegation, a 30-kb genomic fragment containing a single Ly49 gene was used to generate a panel of murine transgenic lines. The results demonstrated that, in almost all of the lines, the isolated Ly49A gene was expressed in a variegated pattern, remarkably similar in nearly all respects to the expression pattern of the endogenous Ly49A gene. Furthermore, the developmental timing of gene expression and regulation by host MHC molecules closely mirrored that of the endogenous Ly49A gene. Therefore, Ly49 variegation does not require competition in cis between different Ly49 genes, and the sequences imposing variegation are located proximally to Ly49 genes. Efforts to define regulatory elements of the Ly49A gene led to the identification of a DNase I hypersensitive site 4.5 kb upstream of the Ly49A gene transcription initiation site, which was shown to be essential for transgene expression. Highly related sequence elements were found upstream of other Ly49 genes, suggesting that a similar regulatory element controls each Ly49 gene.
Polymorphisms in IL-4Rα Correlate with Airways Hyperreactivity, Eosinophilia, and Ym Protein Expression in Allergic IL-13−/− Mice
Authors: Webb Dianne C. , Matthaei Klaus I. , Cai Yeping , McKenzie Andrew N. J. , Foster Paul S.
Published: April 2014

The development of airways hyperreactivity in allergic IL-13−/− mice is controversial and appears to correlate with the number of times that the original 129 × C57BL/6 founder strain has been crossed to the BALB/c background. In this investigation, we compared allergic responses in founder IL-13−/− mice crossed for either 5 (N5) or 10 (N10) generations to BALB/c mice. Whereas allergic N5 IL-13−/− mice developed airways hyperreactivity, tissue eosinophilia, elevated IgE, and pulmonary expression of Ym proteins, these processes were attenuated in N5 IL-13−/− mice treated with an IL-4-neutralizing Ab, and in N10 IL-13−/− mice. These data showed that IL-4 was more effective in regulating allergic responses in N5 IL-13−/− mice than in N10 IL-13−/− mice. To elucidate the mechanism associated with these observations, we show by restriction and sequence analysis that N5 IL-13−/− mice express the C57BL/6 form of IL-4Rα and N10 IL-13−/− mice express the BALB/c form. Despite the near identical predicted molecular mass of these isoforms, IL-4Rα from N5 IL-13−/− mice migrates with a slower electrophoretic mobility than IL-4Rα from N10 IL-13−/− mice, suggesting more extensive posttranslational modification of the N5 form. The Thre49Ile polymorphism in the extracellular domain of BALB/c IL-4Rα has been demonstrated to disrupt N-linked glycosylation of Asn47 and increase the dissociation rate of the IL-4Rα/IL-4 interaction. Collectively, these data show that polymorphisms in IL-4Rα, which have been shown to affect the interaction with IL-4, correlate with the ability of IL-4 to regulate allergic responses in IL-13−/− mice.
T Cytotoxic-1 CD8+ T Cells Are Effector Cells against Pneumocystis in Mice
Authors: Mc Allister Florencia , Steele Chad , Zheng Mingquan , Young Erana , Shellito Judd E. , Marrero Luis , Kolls Jay K.
Published: April 2014

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. A hallmark of HIV infection is Pneumocystis carinii (PC) pneumonia. Recently, CD8+ T cells, which are recruited to the lung in large numbers in response to PC infection, have been associated with some level of host defense as well as contributing to lung injury in BALB/c mice. In this study, we show that CD8+ T cells that have a T cytotoxic-1 response to PC in BALB/c mice, as determined by secretion of IFN-γ, have in vitro killing activity against PC and effect clearance of the organism in adoptive transfer studies. Moreover, non-T cytotoxic-1 CD8+ T cells lacked in vitro effector activity and contributed to lung injury upon adoptive transfer. This dichotomous response in CD8+ T cell response may in part explain the clinical heterogeneity in the severity of PC pneumonia.
IgE Enhances Parasite Clearance and Regulates Mast Cell Responses in Mice Infected with Trichinella spiralis
Authors: Gurish Michael F. , Bryce Paul J. , Tao Hong , Kisselgof Alison B. , Thornton Elizabeth M. , Miller Hugh R. , Friend Daniel S. , Oettgen Hans C.
Published: April 2014

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE−/−) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE−/− mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood eosinophilia and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the small intestine, serum levels of mouse MC protease-1 also were lower in parasite-infected IgE−/− animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE−/− mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo.
The Role of SIGNR1 and the β-Glucan Receptor (Dectin-1) in the Nonopsonic Recognition of Yeast by Specific Macrophages
Authors: Taylor Philip R. , Brown Gordon D. , Herre Jurgen , Williams David L. , Willment Janet A. , Gordon Siamon
Published: April 2014

We recently demonstrated that the β-glucan receptor Dectin-1 (βGR) was the major nonopsonic β-glucan receptor on macrophages (Mφ) for the yeast-derived particle zymosan. However, on resident peritoneal Mφ, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mφ mannose receptor (MR). In this study, we have studied the mannose-binding potential of murine Mφ and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mφ. Both SIGNR1 and βGR cooperated in the nonopsonic recognition of zymosan by these Mφ. When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mφ, it conferred marked zymosan-binding potential on these cells. However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as βGR may play a more dominant role in particle internalization on professional phagocytes. Binding of zymosan to RAW 264.7 Mφ expressing SIGNR1 resulted in TNF-α production. Treatment of RAW 264.7 Mφ expressing SIGNR1, which express low levels of βGR, with β-glucans had little effect on binding or TNF-α production, indicating that there was no absolute requirement for βGR in this process. These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mφ.
The Dendritic Cell Receptor DC-SIGN Discriminates among Species and Life Cycle Forms of Leishmania
Authors: Colmenares María , Corbí Angel L. , Turco Salvatore J. , Rivas Luis
Published: April 2014

Infection of dendritic cells by the human protozoal parasite Leishmania is part of its survival strategy. The dendritic cell receptors for Leishmania have not been established and might differ in their interactions among Leishmania species and infective stages. We present evidence that the surface C-type lectin DC-SIGN (CD 209) is a receptor for promastigote and amastigote infective stages from both visceral (Leishmania infantum) and New World cutaneous (Leishmania pifanoi) Leishmania species, but not for Leishmania major metacyclic promastigotes, an Old World species causing cutaneous leishmaniasis. Leishmania binding to DC-SIGN was found to be independent of lipophosphoglycan, the major glycoconjugate of the promastigote plasma membrane. Our findings emphasize the relevance of DC-SIGN in Leishmania-dendritic cell interactions, an essential link between innate and Leishmania-specific adaptive immune responses, and suggest that DC-SIGN might be a therapeutic target for both visceral and cutaneous leishmaniasis
Cutting Edge: Novel Priming of Tumor-Specific Immunity by NKG2D-Triggered NK Cell-Mediated Tumor Rejection and Th1-Independent CD4+ T Cell Pathway
Authors: Westwood Jennifer A. , Kelly Janice M. , Tanner Jane E. , Kershaw Michael H. , Smyth Mark J. , Hayakawa Yoshihiro
Published: April 2014

NKG2D is an activation receptor on NK cells and has been demonstrated as a primary cytotoxicity receptor for mouse NK cells. Primary rejection of class I-deficient RMA-S lymphoma cells expressing the NKG2D ligand, retinoic acid early inducible-1β, was critically dependent upon NK cell perforin and occurred independently of T cells. NKG2D-triggered NK cell rejection of RMA-S-retinoic acid early inducible-1β tumor primed a secondary tumor-specific T cell response mediated by both CD4+ and CD8+ T cells in the effector phase. Surprisingly, during the priming phase, CD4+ T cells, but not CD8+ T cells, were also required to generate this secondary T cell immunity; however, T cell priming was independent of Th1 cytokines, such as IFN-γ and IL-12. These data imply a novel pathway for priming T cell immunity, that is, stimulated upon NK cell-mediated cytotoxicity of NKG2D ligand-expressing tumor cells, dependent upon CD4+ T cells in the primary phase, and independent of conventional Th1-type immunity.
Enhanced Priming of Antigen-Specific CTLs In Vivo by Embryonic Stem Cell-Derived Dendritic Cells Expressing Chemokine Along with Antigenic Protein: Application to Antitumor Vaccination
Authors: Matsuyoshi Hidetake , Senju Satoru , Hirata Shinya , Yoshitake Yoshihiro , Uemura Yasushi , Nishimura Yasuharu
Published: April 2014

Dendritic cell (DC)-based immunotherapy is regarded as a promising means for anti-cancer therapy. The efficiency of T cell-priming in vivo by transferred DCs should depend on their encounter with T cells. In the present study, we attempted to improve the capacity of DCs to prime T cells in vivo by genetic modification to express chemokine with a T cell-attracting property. For genetic modification of DCs, we used a recently established method to generate DCs from mouse embryonic stem cells. We generated double-transfectant DCs expressing a chemokine along with a model Ag (OVA) by sequential transfection of embryonic stem cells, and then induced differentiation to DCs. We comparatively evaluated the effect of three kinds of chemokines; secondary lymphoid tissue chemokine (SLC), monokine induced by IFN-γ (Mig), and lymphotactin (Lptn). All three types of double transfectant DCs primed OVA-specific CTLs in vivo more efficiently than did DCs expressing only OVA, and the coexpression of SLC or Lptn was more effective than that of Mig. Immunization with DCs expressing OVA plus SLC or Mig provided protection from OVA-expressing tumor cells more potently than did immunization with OVA alone, and SLC was more effective than Mig. In contrast, coexpression of Lptn gave no additive effect on protection from the tumor. Collectively, among the three chemokines, expression of SLC was the most effective in enhancing antitumor immunity by transferred DCs in vivo. The findings provide useful information for the development of a potent DC-based cellular immunotherapy.
CD7 and CD28 Are Required for Murine CD4+CD25+ Regulatory T Cell Homeostasis and Prevention of Thyroiditis
Authors: Sempowski Gregory D. , Cross Scott J. , Heinly Craig S. , Scearce Richard M. , Haynes Barton F.
Published: April 2014

CD7 and CD28 are T cell Ig superfamily molecules that share common signaling mechanisms. To determine roles CD7 and CD28 might play in peripheral lymphocyte development and function, we have generated CD7/CD28-double-deficient mice. CD7- and CD28-single-deficient and CD7/CD28-double-deficient mice had normal levels of CD4 and CD8-single-positive T cells in thymus and spleen. However, CD28-deficient mice had decreased CD4+CD25+ T cells in spleen compared with wild-type mice, and CD7/CD28-double-deficient mice had decreased numbers of CD4+CD25+ T cells in both thymus and spleen compared with both wild-type and CD28-deficient mice. Functional studies demonstrated that CD4+CD25+ T cells from CD28-deficient and CD7/CD28-double-deficient mice could mediate suppression of CD3 mAb activation of CD4+CD25− wild-type T cells, but were less potent than wild-type CD4+CD25+ T regulatory cells. Thyroiditis developed in aged CD7/CD28-double-deficient mice (>1 year) that was not seen in age-matched control mice or single CD7- or CD28-deficient mice, thus suggesting in vivo loss of T regulatory cells allowed for the development of spontaneous thyroiditis. Taken together, these data demonstrated collaborative roles for both CD7 and CD28 in determination of number and function of CD4+CD25+ T regulatory cells in the thymus and peripheral immune sites and in the development of spontaneous thyroiditis.
TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-Linking
Authors: Jung Kyeong Cheon , Park Weon Seo , Kim Hae Jung , Choi Eun Young , Kook Myeong-Cherl , Lee Han-Woong , Bae Youngmee
Published: April 2014

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4−CD8− double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.
Progressive Surface B Cell Antigen Receptor Down-Regulation Accompanies Efficient Development of Antinuclear Antigen B Cells to Mature, Follicular Phenotype
Authors: Heltemes-Harris Lynn , Liu Xiaohe , Manser Tim
Published: April 2014

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of “learned ignorance” could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.
Dendritic Cell Immunization Route Determines CD8+ T Cell Trafficking to Inflamed Skin: Role for Tissue Microenvironment and Dendritic Cells in Establishment of T Cell-Homing Subsets
Authors: Dudda Jan C. , Simon Jan C. , Martin Stefan
Published: April 2014

The effector/memory T cell pool branches in homing subsets selectively trafficking to organs such as gut or skin. Little is known about the critical factors in the generation of skin-homing CD8+ T cells, although they are crucial effectors in skin-restricted immune responses such as contact hypersensitivity and melanoma defense. In this study, we show that intracutaneous, but not i.v. injection of bone marrow-derived dendritic cells induced skin-homing CD8+ T cells with up-regulated E-selectin ligand expression and effector function in contact hypersensitivity. The skin-homing potential and E-selectin ligand expression remained stable in memory phase without further Ag contact. In contrast, i.p. injection induced T cells expressing the gut-homing integrin α4β7. Although differential expression of these adhesion molecules was strictly associated with the immunization route, the postulated skin-homing marker CCR4 was transiently up-regulated in all conditions. Interestingly, dendritic cells from different tissues effectively induced the corresponding homing markers on T cells in vitro. Our results suggest a crucial role for the tissue microenvironment and dendritic cells in the instruction of T cells for tissue-selective homing and demonstrate that Langerhans cells are specialized to target T cells to inflamed skin.
MHC Class II Molecules Play a Role in the Selection of Autoreactive Class I-Restricted CD8 T Cells That Are Essential Contributors to Type 1 Diabetes Development in Nonobese Diabetic Mice
Authors: Serreze David V. , Holl T. Matthew , Marron Michele P. , Graser Robert T. , Johnson Ellis A. , Choisy-Rossi Caroline , Slattery Robyn M. , Lieberman Scott M. , DiLorenzo Teresa P.
Published: April 2014

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2g7 molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2b) or CD8 expression (H2q). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2nb1 MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.
Innate Immune Discrimination of Apoptotic Cells: Repression of Proinflammatory Macrophage Transcription Is Coupled Directly to Specific Recognition
Authors: Cvetanovic Marija , Ucker David S.
Published: April 2014

Physiological cell death is a process the purpose of which is the elimination of functionally inappropriate cells in a manner that does not elicit an inflammatory response. We have shown previously that the ability of apoptotic corpses to be recognized by macrophages and to modulate the proinflammatory responses of those cells represents paradoxically a gain-of-function acquired during the physiological cell death process. Cells that die pathologically (that is, necrotic vs apoptotic corpses) also are recognized by macrophages but do not down-regulate macrophage inflammatory responses; the recognition of these two classes of native dying cells occurs via distinct and noncompeting mechanisms. We have examined the apoptotic modulation of proinflammatory cytokine gene transcription in macrophages (by real-time RT-PCR analysis) and the corresponding modulation of transcriptional activators (by transcriptional reporter analyses). Our data demonstrate that apoptotic cells target the proinflammatory transcriptional machinery of macrophages with which they interact, without apparent effect on proximal steps of Toll-like receptor signaling. The modulatory activity of the corpse is manifest as an immediate-early inhibition of proinflammatory cytokine gene transcription, and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement. Recognition and inflammatory modulation represent key elements of an innate immune response that discriminates live from effete cells, and without regard to self.
Dual Role of CCR2 during Initiation and Progression of Collagen-Induced Arthritis: Evidence for Regulatory Activity of CCR2+ T Cells
Authors: Brühl Hilke , Cihak Josef , Schneider Martin A. , Plachý Jiří , Rupp Tamara , Wenzel Isabell , Shakarami Mehdi , Milz Stefan , Ellwart Joachim W. , Stangassinger Manfred , Schlöndorff Detlef , Mack Matthias
Published: April 2014

Chemokines play an important role in the recruitment of leukocytes and have recently been shown to also attract regulatory T cells. Using blocking mAbs, we analyzed the role of the chemokine receptor CCR2 during initiation and progression of collagen-induced arthritis in mice. Blockade of CCR2 from days 0 to 15 markedly improved clinical signs of arthritis and histological scores measuring leukocyte infiltration, synovial hyperplasia, and bone and cartilage erosion. CCR2 blockade during disease initiation significantly reduced plasma titers of collagen Abs in vivo. In vitro CCR2 blockade also interfered with collagen-specific activation and proliferation of T cells. Surprisingly, CCR2 blockade from days 21 to 36 markedly aggravated clinical and histological signs of arthritis and increased the humoral immune response against collagen. We show that CCR2 is expressed on regulatory T cells. Purified CCR2+ T cells are fully anergic toward polyclonal and collagen-specific activation and potently suppress activation of other T and B cells. The subpopulation of CCR2+ CD25+ regulatory T cells increases ∼5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response.
NKp44 Triggers NK Cell Activation through DAP12 Association That Is Not Influenced by a Putative Cytoplasmic Inhibitory Sequence
Authors: Campbell Kerry S. , Yusa Sei-ichi , Kikuchi-Maki Akiko , Catina Tracey L.
Published: April 2014

NKp44 (NCR2) is a member of the natural cytotoxicity receptor (NCR) family that is expressed on activated human NK cells. We dissected structural attributes of NKp44 to determine their contributions to receptor function. Our results demonstrate that surface expression and NK cell activation by NKp44 is mediated through noncovalent association with the immunoreceptor tyrosine-based activation motif-containing protein, DAP12. Physical linkage to DAP12 requires lysine-183 in the NKp44 transmembrane domain. Intriguingly, the cytoplasmic domain of NKp44 also contains a sequence that matches the immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus. By expressing a chimeric receptor in an NK-like cell line, we found that this ITIM-like motif from NKp44 lacks inhibitory capacity in a redirected cytotoxicity assay. The NKp44 cytoplasmic tyrosine was efficiently phosphorylated in the chimeric receptor upon treating the cells with pervanadate, but it was unable to recruit ITIM-binding negative effector phosphatases. We also generated NK-like cell lines expressing epitope-tagged wild-type or tyrosine to phenylalanine mutant (Y238F) versions of NKp44 and compared their capacities to induce activation marker expression, promote IFN-γ production, or stimulate target cell cytotoxicity. We did not detect any tyrosine-dependent reduction or enhancement of NK cell activation through wild-type vs. Y238F mutant NKp44. Finally, the cytoplasmic tyrosine-based sequence did not provide a docking site for the AP-2 clathrin adaptor, nor did it potentiate receptor internalization. In summary, all activating properties and surface expression of NKp44 are mediated through its association with DAP12, and the putative ITIM in the NKp44 cytoplasmic domain does not appear to attenuate activating function.