bluuebunny/crossref_metadata_2025 huggingface dataset:
Enrichment of Anti-Glomerular Antigen Antibody-Producing Cells in the Kidneys of MRL/MpJ-Faslpr Mice
Authors: Sekine Hideharu , Watanabe Hiroshi , Gilkeson Gary S.
Published: April 2014

Lupus nephritis is characterized by immune complex deposition and infiltration of inflammatory cells into the kidney including Ab-producing cells (AbPCs). Although AbPCs play a central role in the pathogenesis of immune complex glomerulonephritis in lupus, the specificity and pathogenic role of AbPCs infiltrating into the kidneys in lupus are poorly understood. To characterize AbPCs present in lupus kidneys, we isolated AbPCs from diseased MRL/MpJ-Faslpr (MRL/lpr) mouse kidneys. ELISPOT assays, using glomerular Ag (GA) extracts as Ag, demonstrated significant enhancement of anti-GA AbPCs in the kidneys as compared in peripheral blood or spleen of the same mouse. We isolated hybridomas with anti-GA specificity from MRL/lpr mouse kidneys. All the anti-GA mAbs had polyreactive binding to ssDNA, dsDNA, and IgG (i.e., rheumatoid factor), but not to histones or Sm. Sequence analysis of anti-GA Abs suggested the occurrence of somatic mutations and amino acid replacement in complementarity-determining regions with a high replacement to silent ratio resulting in charged amino acids. Intravenous administration of the monoclonal anti-GA Abs into BALB/c mice resulted in graded deposition in glomeruli paralleling their ELISA anti-GA reactivity. These results suggest that AbPCs infiltrating the kidneys in MRL/lpr mice accumulate as a result of Ag selection and likely play a pathologic role in lupus nephritis.
Cloned Human TCR from Patients with Autoimmune Disease Can Respond to Two Structurally Distinct Autoantigens
Authors: De Silva-Udawatta Mihiri , Kumar Senthil R. , Greidinger Eric L. , Hoffman Robert W.
Published: April 2014

There is increasing evidence that the TCR can have significant plasticity in the range of Ags that a single receptor can recognize. Although it has been proposed that such TCR plasticity might contribute to autoimmunity, there have been few studies examining this possibility in either animal models or human disease. In the present study, we examined human T cell clones that were generated against two structurally dissimilar proteins, U1-70 kDa and Smith-B, that are physically associated in the U1-small nuclear ribonucleoprotein complex and that are frequent targets of autoantibodies and T cells in the same lupus patient. We found that the TCR from all clones isolated had substantial sequence homology within their complementarity-determining region 3. We molecularly cloned and expressed individual TCR/A and TCR/B genes in a TCR-negative human cell line J.RT3-T3.5. We then examined the interaction between the TCR and U1-70 kDa and Smith-B antigenic peptides. We found that there was plasticity or degeneracy of the TCR reactive with these lupus autoantigens in that two structurally dissimilar lupus autoantigenic peptides could stimulate a single TCR. These studies support an important role of plasticity of the TCR in the development of human autoimmunity.
HLA-DQ8-Associated T Cell Responses to the Diabetes Autoantigen Phogrin (IA-2β) in Human Prediabetes
Authors: Kelemen Katalin , Gottlieb Peter A. , Putnam Amy L. , Davidson Howard W. , Wegmann Dale R. , Hutton John C.
Published: April 2014

Susceptibility to type 1A autoimmune diabetes is linked to expression of particular MHC class II molecules, notably HLA-DQ8 in man and the orthologous I-Ag7 in the nonobese diabetic mouse. In the present study, we analyzed two peptide epitopes (peptides 2 and 7) from the diabetes autoantigen phogrin (IA-2β), in the context of their presentation by the I-Ag7 and HLA-DQ8 molecules and their role as potential T cell antigenic epitopes in human diabetes. Both of these peptides are targets of diabetogenic CD4+ T cell clones in the nonobese diabetic mouse. Transgenic mice expressing HLA-DQ8 as the sole class II molecule generated a robust T cell-proliferative response when primed with peptide 2 or peptide 7 in CFA. Analysis of the IL-2 secretion from peptide 2-reactive T cell hybridomas stimulated with alanine-substituted peptides identified three residues that were crucial to the response. Among 41 islet cell Ag-positive prediabetic human subjects, 36.5% showed PBMC-proliferative responses to peptide 7, 17.1% to peptide 2, and 17.1% to both peptides; no response was seen among 20 matched healthy controls. Stratification of the data based upon HLA haplotype suggested that peptide 7 could be presented by at least one HLA-DR molecule in addition to HLA-DQ8, a finding that was supported by blocking studies with monomorphic mAbs. The results indicate that common phogrin peptides are targeted by autoreactive T cells in human and murine type 1A diabetes, and that the responses may in part be associated with the similar peptide-binding specificities of I-Ag7 and HLA-DQ8.
Protection from Fluorescein Isothiocyanate-Induced Fibrosis in IL-13-Deficient, but Not IL-4-Deficient, Mice Results from Impaired Collagen Synthesis by Fibroblasts
Authors: Kolodsick Jill E. , Toews Galen B. , Jakubzick Claudia , Hogaboam Cory , Moore Thomas A. , McKenzie Andrew , Wilke Carol A. , Chrisman Cara J. , Moore Bethany B.
Published: April 2014

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4−/−), IL-13 (IL-13−/−), or IL-4 and IL-13 combined (IL-4/13−/−). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4−/− mice developed fibrosis, whereas the IL-13−/− and IL-4/13−/− mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4−/−, IL-13−/−, and IL-4/13−/− mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13−/− mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-β1 production was not reduced in IL-13−/− mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Rα1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.
Mapping of Epitopes for Autoantibodies to the Type 1 Diabetes Autoantigen IA-2 by Peptide Phage Display and Molecular Modeling: Overlap of Antibody and T Cell Determinants
Authors: Dromey James A. , Weenink Sarah M. , Peters Günther H. , Endl Josef , Tighe Patrick J. , Todd Ian , Christie Michael R.
Published: April 2014

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787–817 and 841–869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn858, Glu836, and Trp799 reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.
Ligation of B7-1/B7-2 by Human CD4+ T Cells Triggers Indoleamine 2,3-Dioxygenase Activity in Dendritic Cells
Authors: Munn David H. , Sharma Madhav D. , Mellor Andrew L.
Published: April 2014

Human monocyte-derived dendritic cells (DCs) are capable of expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which allows them to suppress Ag-driven proliferation of T cells in vitro. In DCs that express IDO, the activity of the enzyme is tightly regulated, with the protein being constitutively expressed, but functional activity requiring an additional set of triggering signals supplied during Ag presentation. We now show that triggering of functional IDO obligately requires ligation of B7-1/B7-2 molecules on the DCs by CTLA4/CD28 expressed on T cells. When this interaction was disrupted, IDO remained in the inactive state, and the DCs were unable to inhibit T cell proliferation. Inhibition could be fully restored by direct Ab-mediated cross-linking of B7-1/B7-2. Although both CD4+ and CD8+ T cells were susceptible to inhibition once IDO was induced, the ability to trigger functionally active IDO was strictly confined to the CD4+ subset. Thus, the ability of CD4+ T cells to induce IDO activity in DCs allowed the CD4+ population to dominantly inhibit proliferation of the CD8+ population via the bridge of a conditioned DC. We hypothesize that IDO activation via engagement of B7-1/B7-2 molecules on DCs, specifically, engagement by CTLA4 expressed on regulatory CD4+ T cells, may function as a physiologic regulator of T cell responses in vivo.
Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center
Authors: Mirnics Zeljka Korade , Caudell Eva , Gao YanHua , Kuwahara Kazuhiko , Sakaguchi Nobuo , Kurosaki Tomohiro , Burnside Joan , Mirnics Károly , Corey Seth J.
Published: April 2014

Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-κB, elongation factor 1α, CD79b, octamer binding factor 1, Ig H chain, stathmin, and γ-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn→PU.1→GANP.
Effect of Decreasing the Affinity of the Class II-Associated Invariant Chain Peptide on the MHC Class II Peptide Repertoire in the Presence or Absence of H-2M1
Authors: Honey Karen , Forbush Katherine , Jensen Peter E. , Rudensky Alexander Y.
Published: April 2014

The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-Ab, this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.
Glucose-Regulated Protein 94/Glycoprotein 96 Elicits Bystander Activation of CD4+ T Cell Th1 Cytokine Production In Vivo
Authors: Baker-LePain Julie C. , Sarzotti Marcella , Nicchitta Christopher V.
Published: April 2014

Glucose-regulated protein 94 (GRP94/gp96), the endoplasmic reticulum heat shock protein 90 paralog, elicits both innate and adaptive immune responses. Regarding the former, GRP94/gp96 stimulates APC cytokine expression and dendritic cell maturation. The adaptive component of GRP94/gp96 function reflects a proposed peptide-binding activity and, consequently, a role for native GRP94/gp96-peptide complexes in cross-presentation. It is by this mechanism that tumor-derived GRP94/gp96 is thought to suppress tumor growth and metastasis. Recent data have demonstrated that GRP94/gp96-elicited innate immune responses can be sufficient to suppress tumor growth and metastasis. However, the immunological processes activated in response to tumor Ag-negative sources of GRP94/gp96 are currently unknown. We have examined the in vivo immunological response to nontumor sources of GRP94/gp96 and report that administration of syngeneic GRP94/gp96- or GRP94/gp96-N-terminal domain-secreting KBALB fibroblasts to BALB/c mice stimulates CD11b+ and CD11c+ APC function and promotes bystander activation of CD4+ T cell Th1 cytokine production. Only modest activation of CD8+ T cell or NK cell cytolytic function was observed. The GRP94/gp96-dependent induction of CD4+ T cell cytokine production was markedly inhibited by carrageenan, indicating an essential role for APC in this response. These results identify the bystander activation of CD4+ T lymphocytes as a previously unappreciated immunological consequence of GRP94/gp96 administration and demonstrate that GRP94/gp96-elicited alterations in the in vivo cytokine environment influence the development of CD4+ T cell effector functions, independently of its proposed function as a peptide chaperone.
Maintenance, Loss, and Resurgence of T Cell Responses During Acute, Protracted, and Chronic Viral Infections
Authors: Fuller Michael J. , Khanolkar Aaruni , Tebo Anne E. , Zajac Allan J.
Published: April 2014

The acute phase of many viral infections is associated with the induction of a pronounced CD8 T cell response which plays a principle role in clearing the infection. By contrast, certain infections are not as readily controlled. In this study, we have used the well-defined system of lymphocytic choriomeningitis virus (LCMV) infection of mice to determine quantitative and qualitative changes in virus-specific CD8 T cell responses that rapidly resolve acute infections, more slowly control protracted infections, or fail to clear chronic infections. Acute LCMV infection elicits potent, functional, multi-epitope-specific CD8 T cell responses. Virus-specific CD8 T cells also expand, albeit to a lesser extent, during protracted LCMV infection. Under these conditions, there is a progressive diminution in the capacity to produce IL-2, TNF-α, and IFN-γ. Changes in cytotoxic activities are also detectable but differ depending upon the specificity of the responding cells. As the infection is slowly resolved, a resurgence of cytokine production by virus-specific CD8 T cells is observed. CD4-deficient mice cannot control infection with certain strains of LCMV, but do mount multi-epitope-specific CD8 T cell responses that also lose effector capabilities; however, they are not maintained indefinitely in an unresponsive state as these cells become deleted over time. Overall, our findings suggest that constant high viral loads result in the progressive diminution of T cell effector functions and subsequent physical loss of the responding cells, whereas if the viral load is brought under control a partial restoration of CD8 T cell functions can occur.
Defining Th1 and Th2 Immune Responses in a Reciprocal Cytokine Environment In Vivo
Authors: Chitnis Tanuja , Salama Alan D. , Grusby Michael J. , Sayegh Mohamed H. , Khoury Samia J.
Published: April 2014

The ability of committed Th1 and Th2 cells to function in altered cytokine environments is a central issue in autoimmune and immune-mediated diseases. Therefore, it is of interest to study the ability of Th1 or Th2 cells to expand and produce cytokine reciprocal environments in vivo. Using STAT4- and STAT6-deficient mice, we studied the expansion and cytokine production of Ag-specific Th1 or Th2 cells after transfer into Th1, Th2, or wild-type recipients. Our data show that these Th1 or Th2 cells proliferated and clonally expanded normally, regardless of the in vivo cytokine environment. These data have implications for the treatment of immune-mediated diseases by immunomodulatory agents that alter the cytokine milieu in vivo.
Recombinogenic Phenotype of Human Activation-Induced Cytosine Deaminase
Authors: Poltoratsky Vladimir P. , Wilson Samuel H. , Kunkel Thomas A. , Pavlov Youri I.
Published: April 2014

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.
Depletion of Cellular Cholesterol and Lipid Rafts Increases Shedding of CD30
Authors: von Tresckow Bastian , Kallen Karl-Josef , von Strandmann Elke Pogge , Borchmann Peter , Lange Hans , Engert Andreas , Hansen Hinrich P.
Published: April 2014

CD30, a lymphoid activation marker, is shed into the cell environment after endoproteolytic cleavage of its ectodomain. Soluble (s)CD30 is able to suppress the Th1-type immune response. Because high serum levels of sCD30 and cholesterol-lowering drugs seem to be beneficial in some Th1-type autoimmune diseases, we focused on a link between CD30 shedding and the amount of cellular cholesterol. Cholesterol depletion of human Hodgkin lymphoma- and non-Hodgkin lymphoma-derived cell lines by methyl-β-cyclodextrin led to a down-regulation of membrane-bound CD30 and increased release of sCD30. Additionally, the cholesterol-interfering drugs lovastatin, cholesterol oxidase, and filipin increased CD30 shedding. Both the down-regulation of membrane-anchored CD30 and the release of sCD30 were dependent on metalloproteinases. Using specific inhibitors, we detected TNF-α converting enzyme (TACE) as the leading enzyme responsible for cholesterol-dependent CD30 shedding. A Triton X-100-based method for lipid raft isolation revealed that CD30 was partially present in lipid rafts, whereas TACE was localized in the nonraft fractions. Disintegration of lipid rafts by cholesterol depletion might therefore lead to dynamic interactions of CD30 with TACE, resulting in enhanced shedding of CD30. Our results suggest a possible role of cholesterol-dependent shedding of CD30 in the pathogenesis of immune diseases.
TNF and Phorbol Esters Induce Lymphotoxin-β Expression through Distinct Pathways Involving Ets and NF-κB Family Members
Authors: Voon Dominic C. , Subrata Lily S. , Karimi Mahdad , Ulgiati Daniela , Abraham Lawrence J.
Published: April 2014

Lymphotoxin-β (LT-β) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-α on the cell surface to form the heterotrimeric LTα1,β2 complex, which binds the LT-β receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer’s patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-β promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-β promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-κB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-β promoter. TNF inducibility was localized to the NF-κB site positioned at −83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-β in response to different immunological or inflammatory stimuli.
Cloning, Expression, Cellular Distribution, and Role in Chemotaxis of a C5a Receptor in Rainbow Trout: The First Identification of a C5a Receptor in a Nonmammalian Species
Authors: Boshra Hani , Li Jun , Peters Rodney , Hansen John , Matlapudi Anjan , Sunyer J. Oriol
Published: April 2014

C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.
Signaling through Toll-Like Receptors Triggers HIV-1 Replication in Latently Infected Mast Cells
Authors: Sundstrom J. Bruce , Little Dawn M. , Villinger Francois , Ellis Jane E. , Ansari Aftab A.
Published: April 2014

Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcεRIα+ cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34+ cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2–3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in (∼34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.
Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response
Authors: Sunderkötter Cord , Nikolic Tatjana , Dillon Marilyn J. , van Rooijen Nico , Stehling Martin , Drevets Douglas A. , Leenen Pieter J. M.
Published: April 2014

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6Chigh subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6Chigh monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6Chigh monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6Cmed-high monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.
HIV-Specific CD8+ T Cells Exhibit Markedly Reduced Levels of Bcl-2 and Bcl-xL
Authors: Petrovas Constantinos , Mueller Yvonne M. , Dimitriou Ioannis D. , Bojczuk Paul M. , Mounzer Karam C. , Witek James , Altman John D. , Katsikis Peter D.
Published: April 2014

Human immunodeficiency virus-specific CD8+ T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-xL in HIV-specific CD8+ T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8+ T cells compared with CMV-specific and total CD8+ T cells from HIV-infected individuals as well as total CD8+ T cells from healthy donors. CD8+ T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8+ T cells from HIV-infected individuals. HIV-specific CD8+ T cells also had significantly lower levels of Bcl-xL compared with CMV-specific CD8+ T cells. Finally, IL-15 induces both Bcl-2 and Bcl-xL expression in HIV-specific and total CD8+ T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-xL may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8+ T cells and suggest a possible mechanism by which IL-15 increases their survival.
Macrophage Interactions with Neutrophils RegulateLeishmania majorInfection
Authors: Ribeiro-Gomes Flávia L. , Otero Ana C. , Gomes Nitza A. , Moniz-de-Souza Maria Carolina A. , Cysne-Finkelstein Lea , Arnholdt Andrea C. , Calich Vera L. , Coutinho Sergio G. , Lopes Marcela F. , DosReis George A.
Published: April 2014

Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE2 and TGF-β production by macrophages, while parasite killing depended on neutrophil elastase and TNF-α production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.
O-Methylated Catechins from Tea Leaves Inhibit Multiple Protein Kinases in Mast Cells
Authors: Maeda-Yamamoto Mari , Inagaki Naoki , Kitaura Jiro , Chikumoto Takao , Kawahara Hiroharu , Kawakami Yuko , Sano Mitsuaki , Miyase Toshio , Tachibana Hirofumi , Nagai Hiroichi , Kawakami Toshiaki
Published: April 2014

Tea contains a variety of bioactive compounds. In this study, we show that two O-methylated catechins, (-)-epigallocatechin-3-O-(3-O-methyl) gallate and (-)-epigallocatechin-3-O-(4-O-methyl) gallate, inhibit in vivo mast cell-dependent allergic reactions more potently than their nonmethylated form, (-)-epigallocatechin-3-O-gallate. Consistent with this, these O-methylated catechins inhibit IgE/Ag-induced activation of mouse mast cells: histamine release, leukotriene release, and cytokine production and secretion were all inhibited. As a molecular basis for the catechin-mediated inhibition of mast cell activation, Lyn, Syk, and Bruton’s tyrosine kinase, the protein tyrosine kinases, known to be critical for early activation events, are shown to be inhibited by the O-methylated catechins. In vitro kinase assays using purified proteins show that the O-methylated catechins can directly inhibit the above protein tyrosine kinases. These catechins inhibit IgE/Ag-induced calcium response as well as the activation of downstream serine/threonine kinases such as Akt and c-Jun N-terminal kinase. These observations for the first time have revealed the molecular mechanisms of antiallergic effects of tea-derived catechins.
Immunization with Glucose-6-Phosphate Isomerase Induces T Cell-Dependent Peripheral Polyarthritis in Genetically Unaltered Mice
Authors: Schubert David , Maier Bert , Morawietz Lars , Krenn Veit , Kamradt Thomas
Published: April 2014

Rheumatoid arthritis is a chronic inflammatory disease primarily affecting the joints. The search for arthritogenic autoantigens that trigger autoimmune responses in rheumatoid arthritis has largely focused on cartilage- or joint-specific Ags. In this study, we show that immunization with the ubiquitously expressed glycolytic enzyme glucose-6-phosphate isomerase (G6PI) induces severe peripheral symmetric polyarthritis in normal mice. In genetically unaltered mice, T cells are indispensable for both the induction and the effector phase of G6PI-induced arthritis. Arthritis is cured by depletion of CD4+ cells. In contrast, Abs and FcγR+ effector cells are necessary but not sufficient for G6PI-induced arthritis in genetically unaltered mice. Thus, the complex pathogenesis of G6PI-induced arthritis in normal mice differs strongly from the spontaneously occurring arthritis in the transgenic K/B × N model where Abs against G6PI alone suffice to induce the disease. G6PI-induced arthritis demonstrates for the first time the induction of organ-specific disease by systemic autoimmunity in genetically unaltered mice. Both the induction and effector phase of arthritis induced by a systemic autoimmune response can be dissected and preventive and therapeutic strategies evaluated in this model.
Deletion of the Mouse Meprin β Metalloprotease Gene Diminishes the Ability of Leukocytes to Disseminate through Extracellular Matrix
Authors: Crisman Jacqueline M. , Zhang Binzhi , Norman Lourdes P. , Bond Judith S.
Published: April 2014

Meprins are metalloendopeptidases expressed by leukocytes in the lamina propria of the human inflamed bowel, that degrade extracellular matrix proteins in vitro implicating them in leukocyte transmigration events. The aims of these studies were to 1) examine the expression of meprins in the mouse mesenteric lymph node, 2) determine whether macrophages express meprins, and 3) determine whether deletion of the meprin β gene (Mep-1β) mitigated the ability of leukocytes to disseminate through extracellular matrix in vitro. These studies show that meprin α and β are expressed in leukocytes of the mouse mesenteric lymph node, and meprin α, but not β, decreased during intestinal inflammation. Deletion of Mep-1β gene decreased the ability of leukocytes to migrate through matrigel compared with wild-type leukocytes. Meprin β, but not α, was detected in cortical and medullary macrophages of the lymph node. Thus overall, meprin β is expressed by leukocytes in the draining lymph node of the intestine, regardless of the inflammatory status of the animal, and is likely to contribute to leukocyte transmigration events important to intestinal immune responses. Thus, the expression of meprins by leukocytes of the intestinal immune system may have important implications for diseases such as inflammatory bowel diseases, which are aggravated by leukocyte infiltration.
IL-4-Dependent Th2 Collateral Priming to Inhaled Antigens Independent of Toll-Like Receptor 4 and Myeloid Differentiation Factor 88
Authors: Eisenbarth Stephanie C. , Zhadkevich Alex , Ranney Patricia , Herrick Christina A. , Bottomly Kim
Published: April 2014

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed “collateral priming,” in which naive CD4+ T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.
Multiepitope Trojan Antigen Peptide Vaccines for the Induction of Antitumor CTL and Th Immune Responses
Authors: Lu Jun , Higashimoto Yuichiro , Appella Ettore , Celis Esteban
Published: April 2014

We describe in this study a strategy to produce synthetic vaccines based on a single polypeptide capable of eliciting strong immune responses to a combination CTL and Th epitopes with the purpose of treating malignancies or preventing infectious diseases. This strategy is based on the capacity of Trojan Ags to deliver exogenous Ags into the intracellular compartments, where processing into MHC-binding peptides takes place. Our previous work demonstrated that Trojan Ags containing a CTL epitope localized to intracellular compartments, where MHC class I-binding peptides were generated in a TAP-independent fashion by the action of various exopeptidases and the endopeptidase furin. In this study, we report that Trojan Ags containing several CTL epitopes joined via furin-sensitive linkers generated all of the corresponding MHC class I-binding peptides, which were recognized by CTL. However, Trojan Ags prepared with furin-resistant linkers failed to produce the MHC class I-binding peptides. We also present data indicating that Trojan Ags bearing both CTL and Th epitopes can generate the corresponding MHC class I- and II-binding peptides, which are capable of stimulating T cell responses. Most significantly, in vivo vaccination of mice with a single injection of multiepitope Trojan Ags resulted in strong CTL and Th responses that translated into significant antitumor responses in a model of malignant melanoma. The overall results indicate that Trojan Ags prepared with furin-sensitive linkers are ideal candidates for producing synthetic multiepitope vaccines for the induction of CTL and Th responses that could be used against a variety of diseases, including cancer.
Pregnancy-Specific Down-Regulation of NF-κB Expression in T Cells in Humans Is Essential for the Maintenance of the Cytokine Profile Required for Pregnancy Success
Authors: McCracken Sharon A. , Gallery Eileen , Morris Jonathan M.
Published: April 2014

It is accepted that human pregnancy is associated with a shift away from Th1 type and a bias toward Th2-type immune responses. The molecular mechanisms that regulate this shift are as yet unknown. We assessed the expression and activity of NF-κB, a transcription factor that plays a central role in regulating immune responses. We isolated T cells from PBMCs from nonpregnant and pregnant females and demonstrated that the NF-κB/IκB signaling pathway is down-regulated in T cells in pregnancy. Using Western blotting, high levels of NF-κB (p65) were detected in all nuclear fractions of T cells from nonpregnant females. In contrast, low levels of p65 were detected in nuclear fractions from T cells from pregnant females. Levels of IκBα and -β were also higher in cytoplasmic fractions from T cells from nonpregnant than from pregnant females. The reduction in p65 levels in pregnancy was reflected in the activity of NF-κB in EMSA; T cells from pregnant females contain less active NF-κB than from nonpregnant females. Stimulation of T cells from nonpregnant females with PMA/ionomycin resulted in IκBα degradation, p65 translocation, and subsequent production of the Th1 cytokines IFN-γ and IL-2. In contrast, PMA stimulation had no effect on NF-κB activity in T cells from pregnant females, and this was reflected in reduced Th1 cytokine production. Using the inhibitor of NF-κB activity, SN50, we were able to show that NF-κB activity was essential for the production of Th1 cytokines, suggesting that specific down-regulation of NF-κB in T cells throughout gestation is paramount to pregnancy success through specific regulation of cytokine production.
Mycobacterium tuberculosis Resides in Nonacidified Vacuoles in Endocytically Competent Alveolar Macrophages from Patients with Tuberculosis and HIV Infection
Authors: Mwandumba Henry C. , Russell David G. , Nyirenda Mukanthu H. , Anderson Jennifer , White Sarah A. , Molyneux Malcolm E. , Squire S. Bertel
Published: April 2014

Alveolar macrophages (AM) are the first professional phagocytes encountered by aerosols containing infections in the lungs, and their phagocytic capacity may be affected by these infections or environmental particles. The aim of this study was to evaluate the innate endocytic and phagocytic properties of human AM obtained from patients with pulmonary tuberculosis and to characterize the vacuoles in which Mycobacterium tuberculosis bacilli reside in vivo. AM were obtained by bronchoalveolar lavage from patients with suspected tuberculosis and from asymptomatic volunteers (controls). Clinical case definitions were based on mycobacterial culture of respiratory specimens and HIV serology. To assess phagocytosis, endocytosis, and acidification of the endosomal system, AM were cultured with IgG-coated polystyrene beads, dextran, and a pH-sensitive reporter (3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine) and were evaluated by light and immunoelectron microscopy. Cells from 89 patients and 10 controls were studied. We found no significant difference between the two groups in the ability of AM either to ingest beads and dextran or to deliver them to acidified lysosomes. In AM from patients with tuberculosis, the bacilli were located in vacuoles that failed to accumulate endocytosed material and were not acidified. We concluded that AM from patients with tuberculosis and HIV infections were competent to endocytose and phagocytose material and to deliver the material to functional, acidified lysosomes. M. tuberculosis residing in these AM arrests the progression of their phagosomes, which fail to fuse with acidified lysosomes. This confirms, for the first time in humans with tuberculosis and HIV, the conclusions from previous animal and in vitro studies.
Treatment of Melanoma with 5-Fluorouracil or Dacarbazine In Vitro Sensitizes Cells to Antigen-Specific CTL Lysis through Perforin/Granzyme- and Fas-Mediated Pathways
Authors: Yang Sixun , Haluska Frank G.
Published: April 2014

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.
Angiogenesis-Inflammation Cross-Talk: Vascular Endothelial Growth Factor Is Secreted by Activated T Cells and Induces Th1 Polarization
Authors: Mor Felix , Quintana Francisco J. , Cohen Irun R.
Published: April 2014

Vascular endothelial growth factor (VEGF) and its receptors are critical in angiogenesis. The main player in the secretion and response to VEGF is the endothelial cell. We initiated this study to test whether T cells can secrete VEGF and are able to respond to it. Here we show that VEGF is secreted by T cells on stimulation by specific Ag or by IL-2 and by hypoxia; thus, activated T cells might enhance angiogenesis. Hypoxia also induced the expression in T cells of VEGFR2, suggesting that T cells might also respond to VEGF. Indeed, VEGF augmented IFN-γ and inhibited IL-10 secretion by T cells responding to mitogen or Ag; thus, VEGF can enhance a Th1 phenotype. Encephalitogenic T cells stimulated in the presence of VEGF caused more severe and prolonged encephalomyelitis. Thus, T cells can play a role in angiogenesis by delivering VEGF to inflammatory sites, and VEGF can augment proinflammatory T cell differentiation.
Cutting Edge: The Ontogeny and Function of Va14Ja18 Natural T Lymphocytes Require Signal Processing by Protein Kinase Cθ and NF-κB
Authors: Stanic Aleksandar K. , Bezbradica Jelena S. , Park Jang-June , Van Kaer Luc , Boothby Mark R. , Joyce Sebastian
Published: April 2014

The rapid and robust immunoregulatory cytokine response of Va14Ja18 natural T (iNKT) cells to glycolipid Ags determines their diverse functions. Unlike conventional T cells, iNKT lymphocyte ontogeny absolutely requires NF-κB signaling. However, the precise role of NF-κB in iNKT cell function and the identity of upstream signals that activate NF-κB in this T cell subset remain unknown. Using mice in which iNKT cell ontogeny has been rescued despite inhibition of NF-κB signaling, we demonstrate that iNKT cell function requires NF-κB in a lymphocyte-intrinsic manner. Furthermore, the ontogeny of functional iNKT cells requires signaling through protein kinase Cθ, which is dispensable for conventional T lymphocyte development. The unique requirement of protein kinase Cθ implies that signals emanating from the TCR activate NF-κB during iNKT cell development and function. Thus, we conclude that NF-κB signaling plays a crucial role at distinct levels of iNKT cell biology.