bluuebunny/crossref_metadata_2025 huggingface dataset:
New Loci Regulating Rat Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis
Authors: Becanovic Kristina , Wallstrom Erik , Kornek Barbara , Glaser Anna , Broman Karl W. , Dahlman Ingrid , Olofsson Peter , Holmdahl Rikard , Luthman Holger , Lassmann Hans , Olsson Tomas
Published: April 2014

Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease in rats that closely mimics many clinical and histopathological aspects of multiple sclerosis. Non-MHC quantitative trait loci regulating myelin oligodendrocyte glycoprotein-induced EAE have previously been identified in the EAE-permissive strain, DA, on rat chromosomes 4, 10, 15, and 18. To find any additional gene loci in another well-known EAE-permissive strain and thereby to assess any genetic heterogeneity in the regulation of the disease, we have performed a genome-wide linkage analysis in a reciprocal (LEW.1AV1 × PVG.1AV1) male/female F2 population (n = 185). We examined reciprocal crosses, but no parent-of-origin effect was detected. The parental rat strains share the RT1av1 MHC haplotype; thus, non-MHC genes control differences in EAE susceptibility. We identified Eae16 on chromosome 8 and Eae17 on chromosome 13, significantly linked to EAE phenotypes. Two loci, on chromosomes 1 and 17, respectively showed suggestive linkage to clinical and histopathological EAE phenotypes. Eae16 and Eae17 differ from those found in previously studied strain combinations, thus demonstrating genetic heterogeneity of EAE. Furthermore, we detected a locus-specific parent-of-origin effect with suggestive linkage in Eae17. Further genetic and functional dissection of these loci may disclose critical disease-regulating molecular mechanisms.
Chemoattraction of Human T Cells by IL-18
Authors: Komai-Koma Mousa , Gracie J. Alastair , Wei Xiao-qing , Xu Damo , Thomson Neil , McInnes Iain B. , Liew Foo Y.
Published: April 2014

Cell locomotion is crucial to the induction of an effective immune response. We report here the chemoattraction of CD4+ T cells by IL-18, a member of the IL-1 cytokine family. Recombinant IL-18 increased the proportion of T cells in polarized morphology in vitro and stimulated their subsequent invasion into collagen gels in an IL-18 concentration gradient-dependent manner. Immunofluorescent microscopy studies determined that the major cell type responding to IL-18 was IL-18R+CD4+. Importantly, synovial CD4+ T cells from patients with rheumatoid arthritis responded to IL-18, adopting polarized morphology and gel invasion without further activation ex vivo, indicating the physiologic relevance of our observations. Finally, injection of rIL-18 into the footpad of DBA/1 mice led to local accumulation of inflammatory cells. These data therefore demonstrate for the first time lymphocyte chemoattractant properties of a member of the IL-1 cytokine family and its relevance in inflammatory diseases.
Diverse Repertoire of HIV-1 p24-Specific, IFN-γ-Producing CD4+ T Cell Clones Following Immune Reconstitution on Highly Active Antiretroviral Therapy
Authors: Boritz Eli , Palmer Brent E. , Livingston Brian , Sette Alessandro , Wilson Cara C.
Published: April 2014

HIV-1 Ag-specific CD4+ T cell proliferative responses in human subjects with advanced, untreated HIV-1 disease are often weak or undetectable. Conversely, HIV-1-specific CD4+ T cell proliferation is occasionally detected following suppression of HIV-1 replication with highly active antiretroviral therapy (HAART). These observations suggest that unchecked HIV-1 replication may lead to depletion or dysfunction of HIV-1-specific CD4+ T cells, and that these defects may be partially corrected by viral suppression and subsequent immune reconstitution. However, the impact of this immune reconstitution on the repertoire of HIV-1-specific CD4+ T cells has not been thoroughly evaluated. To examine the HIV-1-specific CD4+ T cell repertoire in this clinical setting, we established HIV-1 p24-specific CD4+ T cell clones from a successfully HAART-treated subject whose pretreatment peripheral CD4 count was 0 cells/μl. Eleven different p24-specific CD4+ T cell clonotypes were distinguished among 13 clones obtained. Most clones produced both IFN-γ and IL-4 upon Ag stimulation. Clones targeted eight distinct epitopes that varied in their conservancy among HIV-1 strains, and responses were restricted by one of three MHC II molecules. Clones showed a range of functional avidities for both protein and peptide Ags. Additional studies confirmed that multiple HIV-1 p24-derived epitopes were targeted by IFN-γ-producing CD4+ cells from subjects first treated with HAART during advanced HIV-1 disease (median, 4.5 peptides/subject; range, 3–6). These results suggest that in HAART-treated subjects whose peripheral CD4+ T cell pools were once severely depleted, the HIV-1-specific CD4+ T cell repertoire may include a diverse array of clonotypes targeting multiple HIV-1 epitopes.
Decreased E12 and/or E47 Transcription Factor Activity in the Bone Marrow As Well As in the Spleen of Aged Mice
Authors: Frasca Daniela , Nguyen Diep , Riley Richard L. , Blomberg Bonnie B.
Published: April 2014

The E2A-encoded transcription factors E12 and E47 are key regulators of B cell functions. They bind to the E-box site, found in regulatory regions of B cell-specific genes; promote cell survival of early pre-B cells; help to initiate Ig rearrangements; and are also involved in class switch in mature B cells in the periphery. We have investigated the expression and function of E47 and E12 in IL-7-expanded pro-B/pre-B cell precursors and in unstimulated or LPS-activated splenic B cells from young and old BALB/c mice. Results show that B cell precursors from the bone marrow of old mice exhibit a reduced expression of E2A proteins and a reduced ability to bind DNA, as compared with young mice. In the spleen, E2A protein expression and DNA binding are present in unstimulated B cells from young mice and, to a significantly lesser extent, from old mice. These are both strongly induced by activation in splenic B cells from young mice but only moderately induced in old mice, indicating that aging affects the expression and activity of E2A-encoded genes and also that DNA binding correlates with the amount of protein expression. The levels of E2A DNA binding in the spleen correlate with those in the bone marrow for individual mice. In splenic mature B cells, only E47/E47 complexes bind DNA; whereas in bone marrow B cell precursors, E47/E12 complexes participate in DNA binding. Only nuclear extracts of splenic mature B cells, but both nuclear and cytoplasmic extracts of bone marrow B cell precursors, exhibit DNA binding.
CD4+ T Cells from Lupus-Prone Mice Avoid Antigen-Specific Tolerance Induction In Vivo
Authors: Bouzahzah Farida , Jung Sungsoo , Craft Joseph
Published: April 2014

Activated T cells in spontaneous lupus presumably bypass normal tolerance mechanisms in the periphery, since thymic tolerance appears intact. To determine whether such T cells indeed avoid in vivo peripheral tolerance mechanisms, we assessed their activation and recall responses after in vivo Ag stimulation in the absence of exogenously supplied costimulatory signals. Naive CD4+ AND (transgenic mice bearing rearranged TCR specific for pigeon cytochrome c, peptides 88–104) TCR-transgenic T cells, specific for pigeon cytochrome c, from lupus-prone Fas-intact MRL/Mp+Fas-lpr and from H-2k-matched control CBA/CaJ and B10.BR mice (MRL.AND, CBA.AND, and B10.AND, respectively) were adoptively transferred into (MRL × CBA)F1 or (MRL × B10)F1 recipients transgenically expressing membrane-bound pigeon cytochrome c as a self-Ag. MRL.AND and control CBA.AND and B10.AND-transgenic T cells were activated and divided after transfer, indicating encounter with their cognate Ag; however, T cells from CBA.AND and B10.AND mice were impaired in their ability to proliferate and produce IL-2 after challenge with pigeon cytochrome c in ex vivo recall assays, a typical phenotype of anergized cells. By contrast, MRL.AND T cells proliferated more, and a significantly higher percentage of such cells produced IL-2, compared with control T cells. This observation that MRL T cells avoided anergy induction in vivo was confirmed in an in vitro system where the cells were stimulated with an anti-CD3 in the absence of a costimulatory signal. These experiments provide direct evidence that CD4+ T cells from Fas-intact lupus-prone MRL mice are more resistant than nonautoimmune control cells to anergy induction. Anergy avoidance in the periphery might contribute to the characteristic finding in lupus of inappropriate T cell activation in response to ubiquitous self-Ags.
IL-4 Induces Apoptosis in A549 Lung Adenocarcinoma Cells: Evidence for the Pivotal Role of 15-Hydroxyeicosatetraenoic Acid Binding to Activated Peroxisome Proliferator-Activated Receptor γ Transcription Factor
Authors: Shankaranarayanan Pattabhiraman , Nigam Santosh
Published: April 2014

The proinflammatory cytokine IL-4 is secreted in large amounts during allergic inflammatory response in asthma and plays a pivotal role in the airway inflammation. IL-4 has been shown to up-regulate 15-lipoxygenase and produce 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in A549 cells via the Janus kinase/STAT6 pathway under coactivation of CREB binding protein/p300. IL-4 has also been shown to up-regulate peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptors in macrophages and A549 cells. In this study we demonstrate that 15(S)-HETE binds to PPARγ nuclear receptors and induces apoptosis in A549 cells. Moreover, pretreatment of cells with nordihydroguaiaretic acid, a 15-lipoxygenase inhibitor, prevented PPARγ activation and apoptosis. The latter was accomplished by the interaction of the 15(S)-HETE/PPARγ complex with the adapter protein Fas-associating protein with death domain and caspase-8, as shown by transfection of Fas-associating protein with death domain dominant negative vector and cleavage of caspase 8 to active subunits p41/42 and p18. Whereas IL-4 and PPARγ ligands failed to induce cleavage of Bid and release of cytochrome c from mitochondria, they caused translocation of the proapoptotic protein Bax from cytoplasm to mitochondria with a concomitant decrease in the Bcl-xL level. We therefore believe that in unstimulated cells Bcl-xL and Bax form a heterodimer, in which Bcl-xL dominates and prevents the induction of apoptosis, whereas in IL-4-stimulated cells the 15(S)-HETE/PPARγ complex down-regulates Bcl-xL, and the resulting overweight of Bax commits the cell to apoptosis via caspase-3. However, this pathway does not rule out the direct caspase-8-mediated activation of caspase-3. In conclusion, IL-4-induced apoptosis may contribute to severe loss of alveolar structures and infiltration of eosinophils, mononuclear phagocytes, etc., into the lung tissue of chronic asthma patients.
Lipid Raft-Independent B Cell Receptor-Mediated Antigen Internalization and Intracellular Trafficking
Authors: Putnam Michelle A. , Moquin Amy E. , Merrihew Megan , Outcalt Christopher , Sorge Emily , Caballero Adriana , Gondré-Lewis Timothy A. , Drake James R.
Published: April 2014

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.
Lipid Raft Distribution of CD4 Depends on its Palmitoylation and Association with Lck, and Evidence for CD4-Induced Lipid Raft Aggregation as an Additional Mechanism to Enhance CD3 Signaling
Authors: Fragoso Roben , Ren Dejian , Zhang Xiaoping , Su Michael Wei-Chih , Burakoff Steven J. , Jin Yong-Jiu
Published: April 2014

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys396 and Cys399of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.
Impaired Transporter Associated with Antigen Processing (TAP) Function Attributable to a Single Amino Acid Alteration in the Peptide TAP Subunit TAP1
Authors: Ritz Ulrike , Drexler Ingo , Sutter Dirk , Abele Rupert , Huber Christoph , Seliger Barbara
Published: April 2014

The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1−/− cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu263 was comparable to that of TAP1−/− cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe265 of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu263 of the TAP1 protein, but not the Phe265, is critical for proper TAP function.
A Single Polymorphic Residue Within the Peptide-Binding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence
Authors: Park Boyoun , Lee Sungwook , Kim Euijae , Ahn Kwangseog
Published: April 2014

Different HLA class I alleles display a distinctive dependence on tapasin for surface expression and Ag presentation. In this study, we show that the tapasin dependence of HLA class I alleles correlates to the nature of the amino acid residues present at the naturally polymorphic position 114. The tapasin dependence of HLA class I alleles bearing different residues at position 114 decreases in the order of acidity, with high tapasin dependence for acidic amino acids (aspartic acid and glutamic acid), moderate dependence for neutral amino acids (asparagine and glutamine), and low dependence for basic amino acids (histidine and arginine). A glutamic acid to histidine substitution at position 114 allows the otherwise tapasin-dependent HLA-B4402 alleles to load high-affinity peptides independently of tapasin and to have surface expression levels comparable to the levels seen in the presence of tapasin. The opposite substitution, histidine to glutamic acid at position 114, is sufficient to change the HLA-B2705 allele from the tapasin-independent to the tapasin-dependent phenotype. Furthermore, analysis of point mutants at position 114 reveals that tapasin plays a principal role in transforming the peptide-binding groove into a high-affinity, peptide-receptive conformation. The natural polymorphisms in HLA class I H chains that selectively affect tapasin-dependent peptide loading provide insights into the functional interaction of tapasin with MHC class I molecules.
The GDP Exchange Factor AND-34 Is Expressed in B Cells, Associates With HEF1, and Activates Cdc42
Authors: Cai Dongpo , Felekkis Kyriakos N. , Near Richard I. , O’Neill Geraldine M. , van Seventer Jean Maguire , Golemis Erica A. , Lerner Adam
Published: April 2014

AND-34, a novel GDP exchange factor, is expressed constitutively at significant levels in murine splenic B cells, but not in murine splenic T cells or thymocytes. In B cell lines, anti-IgM treatment up-regulates AND-34 transcript levels. B cell AND-34 associates with both the docking molecules p130Cas and HEF1. AND-34 binds by its GDP exchange factor domain to the C terminus of HEF1, a region of HEF1 previously implicated in apoptotic, adhesion, and cell cycle-regulated signaling. Overexpression of AND-34 in murine B cell lines activates the Rho family GTPase Cdc42, but not Rac, Rho, RalA, or Rap1. Consistent with this, a subpopulation of AND-34 overexpressing B cells have long filamentous actin-containing cellular extensions. AND-34 overexpression augments both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. As previously reported for lymphoid cells transfected with constitutively active Cdc42, AND-34 overexpression inhibits SDF-1α-induced B cell polarization. These studies suggest that p130Cas and HEF1-associated AND-34 may regulate B cell adhesion and motility through a Cdc42-mediated signaling pathway.
A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA
Authors: Kosinski Penelope A. , Laughlin Jennifer , Singh Karnail , Covey Lori R.
Published: April 2014

CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of “regulated instability” of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3′ untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Δ1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Δ1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.
TNF-Related Apoptosis-Inducing Ligand Mediates Tumoricidal Activity of Human Monocytes Stimulated by Newcastle Disease Virus
Authors: Washburn Birgit , Weigand Markus A. , Grosse-Wilde Anne , Janke Markus , Stahl Heiko , Rieser Eva , Sprick Martin R. , Schirrmacher Volker , Walczak Henning
Published: April 2014

The Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently clinically tested in anticancer therapy. However, the tumoricidal mechanisms of NDV tumor therapy are not fully understood. The results presented here demonstrate that NDV-stimulated human monocytes (Mφ) kill various human tumor cell lines and that this tumoricidal activity is mediated by TRAIL. In contrast to soluble TRAIL-R2-Fc, soluble CD95-Fc and TNF-R2-Fc showed only minimal blocking of the antitumor effect. TRAIL expression is induced on human Mφ after stimulation with NDV and UV-inactivated NDV. These results show that TRAIL induction on human Mφ after NDV stimulation is independent from viral replication and that TRAIL mediates the tumoricidal activity of NDV-stimulated human Mφ.
H2-M3-Restricted Memory T Cells: Persistence and Activation Without Expansion
Authors: Kerksiek Kristen M. , Ploss Alexander , Leiner Ingrid , Busch Dirk H. , Pamer Eric G.
Published: April 2014

H2-M3-restricted T cells respond more rapidly to primary Listeria monocytogenes infection than conventional MHC class Ia-restricted T cells. Reinfection with L. monocytogenes, while inducing explosive proliferation of H2-Kd-restricted T cells, does not stimulate significant expansion of H2-M3-restricted CTL. These disparate responses to reinfection are apparent within 5 days of primary L. monocytogenes infection. However, H2-M3-restricted memory T cells are generated, and are indistinguishable from classically restricted T cells in terms of cell surface memory markers and longevity. Early responses of H2-M3- and H2-Kd-restricted memory T cells to reinfection are similar, with increases in size and expression of activation markers. Interestingly, priming of H2-M3-restricted T cells with an L. monocytogenes-derived N-formyl peptide plus anti-CD40 generates memory T cells that expand upon re-exposure to Ag during L. monocytogenes infection. Our data indicate that disparate H2-M3- and MHC class Ia-restricted memory T cell responses reflect intrinsic differences between these T cell populations. Although distinct proliferative programs appear to be hardwired in these populations during primary L. monocytogenes infection, under different inflammatory circumstances M3-restricted T cell populations can maintain the ability to expand upon re-exposure to Ag.
Structure Function Differences in Nonpeptide CCR1 Antagonists for Human and Mouse CCR1
Authors: Onuffer James , McCarrick Margaret A. , Dunning Laura , Liang Meina , Rosser Mary , Wei Guo-Ping , Ng Howard , Horuk Richard
Published: April 2014

A useful strategy for identifying ligand binding domains of G protein-coupled receptors has been the exploitation of species differences in antagonist potencies. We have used this approach for the CCR1 chemokine receptor with a novel series of antagonists, the 4-hydroxypiperidines, which were discovered by high throughput screening of human CCR1 and subsequently optimized. The structure-activity relationships for a number of different 4-hydroxypiperidine antagonists for human and mouse CCR1 were examined by receptor binding and functional assays. These compounds exhibit major differences in their rank order of potency for the human and mouse chemokine receptor CCR1. For example, the initial lead template, BX 510, which was a highly potent functional antagonist for human CCR1 (Ki = 21 nM) was >400-fold less active on mouse CCR1 (Ki = 9150 nM). However, increasing the length of the linker between the piperidine and dibenzothiepine groups by one methylene group generated a compound, BX 511, which was equipotent for both human and mouse CCR1. These and other analogs of the lead template BX 510, which have major differences in potency for human and mouse CCR1, are described, and a model for their interaction with human CCR1 is presented.
The Mitochondrial Network of Human Neutrophils: Role in Chemotaxis, Phagocytosis, Respiratory Burst Activation, and Commitment to Apoptosis
Authors: Fossati Gianluca , Moulding Dale A. , Spiller David G. , Moots Robert J. , White Michael R. H. , Edwards Steven W.
Published: April 2014

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC50 = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 μg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.
Modulation of HLA-A*0201-Restricted T Cell Responses by Natural Polymorphism in the IE1315–324 Epitope of Human Cytomegalovirus
Authors: Prod’homme Virginie , Retière Christelle , Imbert-Marcille Berthe-Marie , Bonneville Marc , Hallet Marie-Martine
Published: April 2014

Cytotoxic T lymphocytes play a central role in the control of persistent human CMV (HCMV) infection and reactivation. In healthy virus carriers, the specific CD8+ CTL response is almost entirely directed against the virion tegument protein pp65 and/or the 72-kDa major immediate early protein, IE1. Studies that included a large panel of HCMV+ donors suggested that immunorelevance of pp65 and IE1 was directly related with individual HLA haplotype difference. Nevertheless, there are no data on the incidence of HCMV natural polymorphism on virus-specific CTL responses. To assess the impact of IE1 polymorphism on CTL response, we have sequenced in 103 clinical isolates the DNA region corresponding to IE1315–324, an immunodominant epitope presented by HLA-A*0201 molecules. Seven peptidic variants were found with extensive difference in their frequencies. The response of four HLA-A*0201-restricted anti-IE1 T lymphocyte clones, which were previously generated from one donor against autologous B lymphoblastoid cells expressing a recombinant clinical variant of IE1, was then evaluated using target cells loaded with mutant synthetic peptides or expressing rIE1 variants. One of four clones, which have been sorted 19 times among 22 clones targeted against IE1315–324, recognized six of the seven tested variant epitopes. All three other clones showed distinct reactivity patterns to target cells loaded with the different mutant peptides or expressing IE1 variants. Therefore, in the HLA-A2 context, clonal expansions of anti-IE1 memory CTLs may confer a protection against HCMV successive infections and reactivations by killing cells presenting most of the naturally occurring IE1315–324 epitope variants.
IL-13 Is Sufficient for Respiratory Syncytial Virus G Glycoprotein-Induced Eosinophilia After Respiratory Syncytial Virus Challenge
Authors: Johnson Teresa R. , Parker Robert A. , Johnson Joyce E. , Graham Barney S.
Published: April 2014

Although well studied in settings of helminth infection and allergen sensitization, the combined contributions of IL-4 and IL-13 and their signaling pathways in models of viral pathogenesis have not been reported. Using a murine model of respiratory syncytial virus (RSV) infection, we evaluated the contribution of IL-13, alone and in conjunction with IL-4, during immunization with recombinant vaccinia virus expressing RSV G glycoprotein (vvGs) or with formalin-inactivated RSV (FI-RSV). We showed that both IL-4 and IL-13 activity must be inhibited to modulate G-specific responses resulting in severe RSV-induced disease. Inhibition of IL-4 or IL-13 activity alone had minimal impact on disease in vvGs-immunized mice. However, treatment of IL-4-deficient mice with IL-13Ra during vvGs immunization reduced IL-5, IL-13, and eotaxin production and pulmonary eosinophilia after RSV challenge. In contrast, FI-RSV-induced immune responses were diminished when either IL-4 or IL-13 activity was blocked. After RSV challenge, these type 2 T cell responses were also diminished in vvGs-primed IL-4Rα-deficient mice. Our data suggest that secreted vvGs uses mechanisms requiring signaling through the IL-4Rα-chain by either IL-4 or IL-13 for induction of eosinophilia and is the first description of the relative contributions of IL-4, IL-13, and their receptors in viral pathogenesis.
Persistence and Turnover of Antigen-Specific CD4 T Cells During Chronic Tuberculosis Infection in the Mouse
Authors: Winslow Gary M. , Roberts Alan D. , Blackman Marcia A. , Woodland David L.
Published: April 2014

CD4 T cells are critical for resistance to Mycobacterium tuberculosis infection, but how effective T cell responses are maintained during chronic infection is not well understood. To address this question we examined the CD4 T cell response to a peptide from ESAT-6 during tuberculosis infection in the mouse. The ESAT-61–20/IAb-specific CD4 T cell response in the lungs, mediastinal lymph nodes, and spleen reached maxima 3–4 wk postinfection, when the bacteria came under the control of the immune response. Once chronic infection was established, the relative frequencies of Ag-specific CD4 T cells were maintained at nearly constant levels for at least 160 days. ESAT-61–20/IAb-specific CD4 T cells that responded in vitro expressed activation markers characteristic of chronically activated effector cells and used a limited Vβ repertoire that was clonally stable in vivo for at least 12 wk. 5-Bromo-2-deoxyuridine incorporation studies indicated a relatively high rate of cell division among both total CD4 and ESAT-61–20/IAb-specific CD4 T cells during acute infection, but the degree of 5-bromo-2-deoxyuridine incorporation by both the CD4 T cells and the Ag-specific cells declined at least 3-fold during chronic infection. The data indicate that the peripheral ESAT-61–20/IAb-specific CD4 T cell response to M. tuberculosis is characterized during the acute phase of infection by a period of extensive proliferation, but once bacterial control is achieved, this is followed during chronic infection by an extended containment phase that is associated with a persistent response of activated, yet more slowly proliferating, T cells.
Poly(ADP-Ribose) Polymerase-1 Regulates Activation of Activator Protein-1 in Murine Fibroblasts
Authors: Andreone Teresa L. , O’Connor Michael , Denenberg Alvin , Hake Paul W. , Zingarelli Basilia
Published: April 2014

Poly(ADP-ribose) polymerase (PARP)-1 is activated in response to DNA injury in the nucleus of eukaryotic cells and has been implicated in cell dysfunction in inflammation. We investigated the role of PARP-1 on the AP-1 pathway, which is involved in the signal transduction of the inflammatory process. In murine wild-type fibroblasts, oxidative challenge by peroxynitrite and hydrogen peroxide or immunological challenge by IL-1 and 20% FCS induced phosphorylation of the mitogen-activated protein kinase kinase-4, activation of c-Jun N-terminal kinase (JNK), and DNA binding of AP-1. In comparative experiments, peroxynitrite induced DNA binding of heat shock factor-1. Pretreatment of wild-type cells with 5-iodo-6-amino-1,2-benzopyrone, a PARP-1 inhibitor, inhibited JNK activation and DNA binding of AP-1. In parallel experiments in PARP-1-deficient fibroblasts, DNA binding of AP-1 was completely abolished. Activation of JNK was significantly elevated at basal condition, but it exhibited a lesser increase after oxidative or immunological challenge than in wild-type fibroblasts. Nuclear content of phosphorylated mitogen-activated protein kinase kinase-4 was observed in PARP-1-deficient cells after peroxynitrite challenge only. Western blotting analysis for AP-1 subunits indicated that c-Fos was similarly expressed in wild-type and PARP-1-deficient cells. Phosphorylated c-Jun was expressed after oxidative or immunological challenge, but not in basal condition, in wild-type cells; however, it was significantly elevated at basal condition and further enhanced after oxidative or immunological challenge in PARP-1-deficient cells. No DNA binding of heat shock factor-1 was observed in PARP-1-deficient cells. These data demonstrate that PARP-1 plays a pivotal role in the modulation of transcription.
Survival and Tumor Localization of Adoptively Transferred Melan-A-Specific T Cells in Melanoma Patients
Authors: Meidenbauer Norbert , Marienhagen Joerg , Laumer Monika , Vogl Sandra , Heymann Jana , Andreesen Reinhard , Mackensen Andreas
Published: April 2014

Adoptive T cell therapy has been successfully used for treatment of viral and malignant diseases. However, little is known about the fate and trafficking of transferred Ag-specific T cells. Using the tetramer (TM) technology which allows for detection and quantification of Ag-specific CTL, we assessed the frequency of circulating Melan-A-specific CTL in advanced melanoma patients during adoptive T cell therapy. Melan-A-specific CTL were generated from HLA-A2.1+ patients by in vitro stimulation of CD8+ T cells with dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. Eight patients received three infusions of 0.25–11 × 108 Melan-A-specific CTL i.v. at 2-wk intervals along with low-dose IL-2. The transferred T cell product contained a mean of 42.1% Melan-A-TM+ CTL. Before therapy, the frequencies of Melan-A-specific CTL in patients’ circulating CD8+ T cells ranged from 0.01 to 0.07%. Characterization of the TM frequencies before and at different time points after transfer revealed an increase of circulating Melan-A-specific CTL up to 2%, correlating well with the number of transferred CTL. An elevated frequency of TM+ T cells was demonstrated up to 14 days after transfer, suggesting long-term survival and/or proliferation of transferred CTL. Combining TM analysis with a flow cytometry-based cytokine secretion assay, unimpaired production of IFN-γ was demonstrated in vivo for at least 24 h after transfer. Indium-111 labeling of Melan-A-specific CTL demonstrated localization of transferred CTL to metastatic sites as early as 48 h after injection. Overall, the results suggest that in vitro-generated Melan-A-specific CTL survive intact in vivo for several weeks and localize preferentially to tumor.
Microsatellite Instability and Suppressed DNA Repair Enzyme Expression in Rheumatoid Arthritis
Authors: Lee Sang-Heon , Chang Dong Kyung , Goel Ajay , Boland C. Richard , Bugbee William , Boyle David L. , Firestein Gary S.
Published: April 2014

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSα (hMSH2 and hMSH6) and MutSβ (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.
Cutting Edge: Molecular Cloning of a Killer Cell Ig-Like Receptor in the Mouse and Rat
Authors: Hoelsbrekken Sigurd E. , Nylenna Øyvind , Saether Per C. , Slettedal Imer Ö. , Ryan James C. , Fossum Sigbjørn , Dissen Erik
Published: April 2014

We report the molecular cloning of a KIR3DL1 receptor in the mouse and the rat, between 37.4 and 45.4% identical with primate killer cell Ig-like receptors (KIRs/CD158). Both mouse and rat molecules contain a pair of immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic regions, suggesting an inhibitory function. Southern blot analysis indicated a single KIR gene in the rat, whereas the mouse genome contains more than one KIR-related element. The rat Kir3dl1 locus was mapped to the leukocyte receptor gene complex on chromosome 1, whereas mouse Kir3dl1 was localized to the X chromosome. RT-PCR demonstrated that KIR3DL1 was selectively expressed by NK cells in both rat and mouse. An epitope-tagged expression construct of mouse KIR3DL1 transfected into 293T cells induced expression of a ∼55-kDa protein. Our data indicate that KIR receptors may contribute to the NK cell receptor repertoire in rodents, alongside the Ly-49 family.
Cutting Edge: Scavenging of Inflammatory CC Chemokines by the Promiscuous Putatively Silent Chemokine Receptor D6
Authors: Fra Anna M. , Locati Massimo , Otero Karel , Sironi Marina , Signorelli Paola , Massardi Maria L. , Gobbi Marco , Vecchi Annunciata , Sozzani Silvano , Mantovani Alberto
Published: April 2014

In an effort to define the actual function of the promiscuous putatively silent chemokine receptor D6, transfectants were generated in different cell types. Engagement of D6 by inflammatory CC chemokines elicited no calcium response nor chemotaxis, but resulted in efficient agonist internalization and degradation. Also in lymphatic endothelium, where this receptor is expressed in vivo, D6 did not elicit cellular responses other than ligand internalization and degradation. In particular, no evidence was obtained for D6-mediated transcytosis of chemokines in the apical-to-basal or basal-to-apical directions. These results indicate that D6 acts as an inflammatory chemokine scavenging nonactivatory decoy receptors and suggest that in lymphatic vessels D6 may function as a gatekeeper for inflammatory CC chemokines, by clearing them and preventing excessive diffusion via afferent lymphatics to lymph nodes.
Oligodeoxynucleotides Without CpG Motifs Work as Adjuvant for the Induction of Th2 Differentiation in a Sequence-Independent Manner
Authors: Sano Kunio , Shirota Hidekazu , Terui Tadashi , Hattori Toshio , Tamura Gen
Published: April 2014

The outcomes of immune responses are regulated by various parameters including how Ags are handled by APCs. In this study, we describe the intrinsic immunomodulatory characteristics of oligodeoxynucleotides (ODNs) that improve the Ag presentation by APCs. ODNs (20-mer) containing CpG motifs induced strong Th1-skewed responses. In contrast, those without CpG motifs enhanced cytokine production by effector Th cells without particular skewing toward Th1 responses or induced the differentiation of unprimed CD4+ T cells toward Th2 cells. These functional features were prominently envisaged when ODNs were conjugated to the Ag, and were underlain by the facilitated binding of ODN-conjugated Ag to Ia+ cells. Despite the functional differences between ODNs with CpG motifs and those without CpG motifs, both ODNs bound to Ia+ cells with similar affinity and kinetics. Immunoenhancing activities of the ODNs were not sequence-dependent; the characteristics, including the facilitation of Ag capture, enhancement of effector Th cell responses, and induction of Th2 cells, were shared by randomly synthesized ODNs conjugated to Ag. This is the first study suggesting that ODNs, independent of the sequences, enhance immune responses through the promoted capture of ODN-conjugated Ag by APCs.
Polycyclic Aromatic Hydrocarbons Inhibit Differentiation of Human Monocytes into Macrophages
Authors: van Grevenynghe Julien , Rion Sophie , Le Ferrec Eric , Le Vee Marc , Amiot Laurence , Fauchet Renée , Fardel Olivier
Published: April 2014

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are ubiquitous environmental carcinogenic contaminants exerting deleterious effects toward cells acting in the immune defense such as monocytic cells. To investigate the cellular basis involved, we have examined the consequences of PAH exposure on macrophagic differentiation of human blood monocytes. Treatment by BP markedly inhibited the formation of adherent macrophagic cells deriving from monocytes upon the action of either GM-CSF or M-CSF. Moreover, it reduced expression of macrophagic phenotypic markers such as CD71 and CD64 in GM-CSF-treated monocytic cells, without altering cell viability or inducing an apoptotic process. Exposure to BP also strongly altered functional properties characterizing macrophagic cells such as endocytosis, phagocytosis, LPS-triggered production of TNF-α and stimulation of allogeneic lymphocyte proliferation. Moreover, formation of adherent macrophagic cells was decreased in response to PAHs distinct from BP such as dimethylbenz(a)anthracene and 3-methylcholanthrene, which interact, like BP, with the arylhydrocarbon receptor (AhR) known to mediate many PAH effects. In contrast, benzo(e)pyrene, a PAH not activating AhR, had no effect. In addition, AhR was demonstrated to be present and functional in cultured monocytic cells, and the use of its antagonist α-naphtoflavone counteracted inhibitory effects of BP toward macrophagic differentiation. Overall, these data demonstrate that exposure to PAHs inhibits functional in vitro differentiation of blood monocytes into macrophages, likely through an AhR-dependent mechanism. Such an effect may contribute to the immunotoxicity of these environmental carcinogens owing to the crucial role played by macrophages in the immune defense.
Primary and Secondary Immunocompetence in Mixed Allogeneic Chimeras
Authors: Williams Matthew A. , Adams Andrew B. , Walsh Melody B. , Shirasugi Nozomu , Onami Thandi M. , Pearson Thomas C. , Ahmed Rafi , Larsen Christian P.
Published: April 2014

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.
Memory CD8+ T Cells Provide an Early Source of IFN-γ
Authors: Kambayashi Taku , Assarsson Erika , Lukacher Aron E. , Ljunggren Hans-Gustaf , Jensen Peter E.
Published: April 2014

During the non-Ag-specific early phase of infection, IFN-γ is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-γ release, IFN-γ-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-γ was also detected in a significant fraction of CD8+ T cells. CD8+ T cells represented the second major population of IFN-γ-producing cells in the spleen (∼30%) and the majority of IFN-γ+ cells in the lymph nodes (∼70%). LPS-induced IFN-γ production by CD8+ T cells was MHC class I independent and was restricted to CD44high (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8+ T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-α/β, IL-12, and IL-18. IFN-γ was also detected in memory CD8+ T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-γ.
The 19-kDa Mycobacterium tuberculosis Protein Induces Macrophage Apoptosis Through Toll-Like Receptor-2
Authors: López Martín , Sly Laura M. , Luu Yvonne , Young Douglas , Cooper Howard , Reiner Neil E.
Published: April 2014

Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis. Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection. The 19-kDa M. tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor. We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis. The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis. The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent. Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19. Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2. The viability of M. tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection. Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.